CBP/p300 is critical for the expansion and maintenance of functional pancreatic α cell mass

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: major_revision

Abstract
Pancreatic α cell senses amino acid availability to adjust secretion function and proliferation, yet the underlying molecular mechanisms remain unclear. Here, α cell-specific deletion of CBP/p300 in mice leads to hypoglucagonemia and hyperaminoacidemia, along with decreased functional pancreatic α cell mass due to impaired cell proliferation, dedifferentiation, and cell loss. The knockout of CBP/p300 blocks glucagon receptor antibody-stimulated α cell proliferation and mTORC1 signaling in mice. In CBP/p300 deficient α cells, single-cell RNA sequencing reveals upregulated autophagy-related genes and downregulated α cell identity genes and amino acid transporters, including Slc7a2. Slc7a2 is involved in regulating α cell identity gene expression through lysine-facilitated H3K27 acetylation. In addition, Slc7a2 downregulation compromises arginine-stimulated mTORC1 signaling, thereby suppressing α cell proliferation and triggering autophagy. Collectively, our findings uncover CBP/p300 as central regulators of functional α cell mass partially by orchestrating Slc7a2-mediated amino acid sensing.

Introduction
As structural elements and energy sources for normal cell growth and function, amino acids are essential to human health1. The disturbance of amino acid homeostasis has been closely linked to metabolic diseases. Branched-chain amino acids and their metabolites have been identified as the strongest biomarkers of obesity, insulin resistance, and type 2 diabetes mellitus (T2DM)2. 2-Aminoadipic acid (2-AAA), a catabolic metabolite of lysine, could even predict the risk of developing T2DM3. It is widely recognized that impaired pancreatic β-cell function is the core problem in T2DM4. However, accumulating evidence suggests that α cell dysfunction, characterized by inappropriate glucagon secretion, also contributes to hyperglycemia in diabetes5. Notably, α cell function and number is tightly coupled to changes in plasma amino acid levels6. However, the response mechanism of α cells to amino acids remains largely unclear.

Pancreatic α cells are essential to maintaining glucose homeostasis primarily through secreting glucagon, which promotes hepatic glucose production during fasting or hypoglycemic states7. Glucagon binds to the glucagon receptor (GCGR), activating PKA-CREB signaling cascades that stimulate hepatic gluconeogenesis and glycogenolysis8. In addition, glucagon facilitates hepatic amino acid uptake and catabolism, thereby supplying substrates for glucose production9. Thus, glucagon secreted from α cell plays a critical role in the turnover of amino acids to glucose in hepatocytes, resulting in decreased circulating amino acid concentration and increased blood glucose level. In turn, changes in plasma glucose and amino acid levels modulate glucagon secretion in an opposite direction6. In T2DM, α cell dysregulation is characterized by excessive basal glucagon secretion that occurs independent of glycemic status, contributing to hyperglucagonemia and increased hepatic glucose output10,11.

Recent studies have highlighted the role of α cells as key amino acid sensors12. In humans, the ingestion of whey protein induces a significant rise in circulating amino acids and glucagon levels13. Several amino acids, including arginine, glutamine, alanine, and glycine, have been shown to acutely stimulate glucagon secretion14. Prolonged elevations of amino acids also drive excessive α cell proliferation, leading to its hyperplasia and hyperglucagonemia15-18. Increased circulating glutamine has been shown to be causative to amino acid-induced α cell hyperplasia in GCGR signaling interference models, involving the upregulation of SLC38A5, a transporter of glutamine, and stimulation of mTORC1 pathway. However, whether or how α cells sense other types of amino acids, especially those related to T2DM, remains elusive.

It has been demonstrated that amino acids provide substrates for several protein post-translational modifications such as acetylation and crotonylation, which link amino acids catabolism to the transcriptional regulation of genes involved in cell function or proliferation19,20. Besides cell number, the maintenance of α cell identity is also critical for their function21,22. Epigenetics plays a crucial role in the maintenance of cell type-specific features and lineage stability22,23. ATAC-Seq analysis shows that α cells possess more poised and accessible chromatin at genes related to identity and diabetes risk22. Histone acetylation is an important epigenetic modification involved in regulating cell type-specific gene expression patterns and determining cell fate24. The lysine acetyltransferases type 3 (KAT3) family members CBP (Crebbp) and p300 (Ep300) are key transcriptional co-activators with similar structure and function23. Of interest, acetylation of H3 lysine 27 (H3K27Ac) mediated by CBP and p300 is reported to be a biomarker of cell type-specific promoters and enhancers related to cell fate25. However, the role of CBP/p300 in controlling α cell identity remains poorly under stood.

In this study, we generated α cell-specific Crebbp/Ep300 knockout mice to explore the role of two acetyltransferases in maintaining α cell identity. Dual deletion of CBP and p300 led to a significant reduction in functional α cell mass and glucagon secretion due to impaired α cell proliferation, α cell dedifferentiation, and cell loss. Single-cell RNA sequencing and in-vitro experiments revealed the involvement of SLC7A2 in CBP/p300-governed α cell identity, proliferation, and autophagy through lysine-promoted histone H3K27 acetylation and arginine-stimulated mTORC1 activation. These findings uncover a critical role for CBP/p300 in linking amino acid sensing with the maintenance of α cell identity and function.

Results
Pancreatic α cell-specific double knockout of Crebbp and Ep300 mice exhibit low plasma glucagon level and high amino acid level

To investigate the role of CBP and p300 in pancreatic α cells, we generated α cell-specific CBP/p300 knockout (CBP/p300αDKO) mice by crossing Gcg-Cre mice with Crebbpflox/flox and Ep300flox/flox mice (Fig. 1a). The knockdown efficiency was confirmed by immunofluorescence staining within 1 day after birth, showing negative CBP and p300 staining in more than 90% Gcg-positive α cells of CBP/p300αDKO mice (Fig. 1b, c). The potential off-target effects of Gcg-Cre in intestinal L-cells and brain were also evaluated. Intestinal co-expression of glucagon-like peptide-1 (GLP-1) and tdTomato in Gcg-Cre-driven tdTomato lineage-tracing wild-type (WT) and CBP/p300αDKO mice confirmed that Gcg-Cre was indeed active in the intestinal L-cells (Supplementary Fig. 1a). However, neither the average number of GLP-1+ cell per intestinal section nor circulating level of active GLP-1 differed between control and CBP/p300αDKO mice (Supplementary Fig. 1b, c). In addition, CBP/p300αDKO mice exhibited normal oral glucose tolerance (Supplementary Fig. 1d), further indicating preserved L-cell function. We also confirmed Gcg-Cre-mediated recombination in neurons of the nucleus tractus solitarius (NTS) (Supplementary Fig. 1e). The preproglucagon neurons of NTS have been reported to regulate appetite and energy metabolism26,27, but no significant alterations were observed in food intake or energy expenditure in CBP/p300αDKO mice (Supplementary Fig. 1f, g), suggesting minimal impact on the central nervous system.

Interestingly, fasting blood glucagon level was significantly reduced in 8-week-old CBP/p300αDKO mice, but not in single-gene knockout (CBLPαKO, p300αKO) or triple-allele knockout (CBPαKO; p300HE, CBPHE; p300αKO) mice compared to wild-type (WT) controls (Fig. 1d). However, fasting blood glucose and plasma insulin levels remained unchanged in 8-week-old CBP/p300αDKO mice (Fig. 1e, Supplementary Fig. 1h), accompanied by normal hepatic glucose output (Fig. 1f) and mRNA levels of key gluconeogenic genes, including Pck1, Fbp1, and G6pc (Fig. 1g). This was the case for the levels of PEPCK protein and phosphorylated CREB (Fig. 1h). In contrast, total plasma amino acid level was significantly increased in fasted CBP/p300αDKO mice compared to WT controls (Fig. 1i). We further performed metabolomic assays on plasma samples from 8-week-old WT and CBP/p300αDKO mice. A total of 193 metabolites were identified, with univariate analysis revealing 53 upregulated and 2 downregulated metabolites in CBP/p300αDKO mice (P < 0.05) (Fig. 1j). Pathway enrichment analysis highlighted that differential metabolites were mainly associated with amino acid metabolism, including arginine and proline metabolism, as well as alanine, aspartate, glutamate, glycine, serine, and threonine metabolism (Fig. 1k). Of the 20 basic amino acids examined, 16 were significantly increased, including several glucogenic amino acids such as alanine, glutamine, and serine (Fig. 1l). These results suggest that CBP and p300 deletion in α cells causes hypoglucagonemia and significant disturbance in amino acid homeostasis, without affecting plasma glucose levels.

CBP/p300 is necessary for the expansion and maintenance of postnatal α cell number

During murine islet endocrine cell development, glucagon (Gcg)-positive cells first appeared at embryonic day 9.528. We next examined whether CBP/p300 deletion-mediated hypoglucagonemia is due to impaired development or postnatal maintenance of Gcg-positive α cells. Morphometric analysis showed that Gcg-positive cell number in CBP/p300αDKO mice was normal at birth (0 w) (Fig. 2a, b, Supplementary Fig. 2a), indicating that CBP/p300 are not essential for α cell development. With the elongation of postnatal time, significant difference was observed in Gcg-positive cell number between WT and CBP/p300αDKO mice. In WT mice, α cell numbers increased rapidly during the first 2 weeks, with the fastest growth occurring between 1 and 2 weeks. Afterward, the rate of α cell expansion slowed and stabilized from 4 weeks onward (Fig. 2b). Whereas, α cell number was decrease by 37% at 2 weeks and 70% at 8 weeks in CBP/p300αDKO mice compared to WT mice (Fig. 2b, c). We further analyzed additional morphometric indicators. At 2 weeks, pancreatic weight and the average number of islets per section were similar between two groups (Supplementary Fig. 2b). However, the α cell area as a percentage of the total islet area was significantly reduced in CBP/p300αDKO mice (Fig. 2d), with similar α cell size between two groups (Fig. 2e). Notably, the number of Gcg-positive α cells per islet was dramatically decreased in CBP/p300αDKO mice (Fig. 2f), while the number of β, δ, and PP cells remained unchanged (Supplementary Fig. 2c).

We also examined α cell proliferation during the neonatal period, which plays a key role in postnatal α cell mass establishment29. Ki67 staining revealed a significant decrease in α cell proliferation in 1-week-old CBP/p300αDKO mice (Fig. 2g, h), consistent with the reduced α cell expansion observed in these mice (Fig. 2i). However, the apoptosis rates of α cell were comparable in 4 and 8 week-old WT and CBP/p300αDKO mice as shown by cleaved-Caspase 3 staining (Supplementary Fig. 2d), indicating that other forms of cell loss may contribute to the decline in α-cell numbers. At 8 weeks, although pancreatic weight and the average number of islets per section were similar between WT and CBP/p300αDKO mice (Supplementary Fig. 2e), the α cell area as a percentage of total islet area was even further reduced in CBP/p300αDKO mice (Fig. 2j), with no significant change in α cell size (Fig. 2k). The number of Gcg-positive α cells per islet remained significantly lower in CBP/p300αDKO mice (Fig. 2l), while the β cell area was still unaffected (Supplementary Fig. 2f). Together, these results demonstrate that CBP/p300 are essential for the postnatal proliferation and survival of α cells.

Double deficiency of CBP and p300 leads to α-cell identity loss and population reduction

Building on our previous findings that HAT activity of CBP/p300 are critical for the transcription of β cell identity genes30, we next investigated their role in α cell fate maintenance using a lineage-tracing system based on ROSA-LSL-tdTomato reporter mice crossed with Gcg-Cre to label glucagon-expressing (Gcg+) α cells (Fig. 3a). The labeling efficiency was confirmed in 2-week-old ROSAfl/wt;Gcg-Cre mice, in which over 90% of Gcg+ cells co-expressed tdTomato (Supplementary Fig. S3a, b). We then examined islet sections at postnatal weeks 1, 2, 4, and 8, and found that nearly all tdTomato+ cells consistently co-expressed Gcg in WT mice (Fig. 3b, Supplementary Fig. 3c). However, CBP/p300αDKO mice showed a progressive and significant reduction in the number of tdTomato+ cells per islet section starting from 2 weeks of age (Fig. 3c), indicating tdTomato+ cell loss. Although the percentage of tdTomato+Gcg+/tdTomato+ cells in the knockout mice was similar to WT at 1 week, it dropped to 79% at 2 weeks, 55% at 4 weeks, and 32% at 8 weeks (Fig. 3d), indicating a gradual loss of α cell identity. Immunofluorescence staining revealed no significant transdifferentiation into β or δ cells in CBP/p300αDKO mice when α cell lost its identity. However, the ratio of tdTomato+Ppy+ cells was significantly reduced in CBP/p300αDKO mice compared to WT mice (Fig. 3e, f).

To further investigate the role of CBP/p300 in adult α cells, we generated tamoxifen-induced α cell-specific CBP/p300 knockout mice (iCBP/p300αDKO) by crossing Gcg-CreERT2 with Crebbpflox/flox/Ep300flox/flox mice, and ROSA-LSL-tdTomato reporter mice (Fig. 3g). Tamoxifen administration in 8-week-old mice resulted in efficient tdTomato labeling of Gcg+ cells (> 90%) (Fig. 3h, Supplementary Fig. 3d). In line with findings from CBP/p300αDKO mice, iCBP/p300αDKO mice exhibited decreased fasting plasma glucagon level and elevated plasma amino acid level, without changes in fasting blood glucose level (Supplementary Fig. 3e-g). Histological analysis revealed that iCBP/p300αDKO mice had significantly fewer tdTomato+ cells and Gcg+ α cells compared to WT mice (Fig. 3i, j), as well as a reduced proportion of tdTomato+Gcg+/tdTomato+ cells (Fig. 3k), further supporting the occurrence of α cell loss and dedifferentiation. Similar to the developmental model, no transdifferentiation of α cells into β or δ cells was observed, but tdTomato+ Ppy+ cells were also diminished (Fig. 3l, m). Together, these results confirm that CBP/p300 are indispensable for maintaining α cell survival and identity throughout the postnatal life.

Expressions of α cell identity genes are compromised in CBP/p300αDKO mice

To elucidate the molecular mechanisms underlying α cell identity loss in CBP/p300αDKO mice, we performed single-cell RNA sequencing (scRNA-seq) on islet cells from 4-week-old WT and CBP/p300αDKO mice using tdTomato as a lineage tracer (Fig. 4a). Intact islets were isolated from two groups of mice, dissociated into single-cell suspensions, and subjected to scRNA-seq analysis. After filtering out low-quality cells and doublets, 11,797 high-quality cells, including 4,993 from WT mice and 6,804 from CBP/p300αDKO mice were retained for downstream analysis. Dimensionality reduction and clustering analysis identified seven major cell types, including β, α, δ, and PP cells, as well as acinar, endothelial, and immune cells, based on canonical markers (Fig. 4b, c). Lineage tracing showed that approximately 90% of Gcg+ cells in WT mice expressed tdTomato, validating efficient Cre recombination (Fig. 4d, e).

To characterize α-cell state transitions in the absence of CBP/p300, we performed pseudotime trajectory analysis on α cells from CBP/p300αDKO mice due to tdTomato marking biologically distinct α-cell states in WT versus CBP/p300αDKO mice, which would otherwise confound joint trajectory interference. The trajectory showed a temporal progression from tdTomato-negative (tdTomato-) to tdTomato-positive (tdTomato+) α cells, with tdTomato- cells mainly located at early to intermediate stages, while tdTomato+ cells enriched at intermediate to late stages, suggestive of a differentiation trajectory toward identity loss following Cre-mediated CBP/p300 deletion (Fig. 4f, g). Gene expression dynamics along pseudotime identified four co-regulated modules with temporal expression patterns, including a decline in α cell identity genes (e.g., Gcg, MafB, and Ttr) and a concurrent upregulation of autophagy-related genes (e.g., Ctsf, Ctsl, and Tmem59) (Fig. 4h). Consistent with the downregulation of α-cell identity genes, islets isolated from CBP/p300αDKO mice exhibited markedly impaired ex vivo glucagon secretion in response to basal glucose, low glucose, and arginine, indicating functional impairment of α cells following CBP/p300 ablation (Supplementary Fig. 4a).

We next investigated the transcriptional disruptions in α cells by performing UMAP co-visualization of Gcg and tdTomato expression in α cells from CBP/p300αDKO mice. This revealed two major tdTomato+ subsets. One retained high Gcg expression and another lacked Gcg expression, indicative of heterogeneous α cell states (Fig. 4i, j). Based on the expression levels of Gcg and tdTomato, four α cell subpopulations were defined: (i) Gcg+tdTomato+ cells (Both, 42%), representing functional α cells; (ii) Gcg- tdTomato+ cells (tdTomato, 39%), denoting identity-compromised α cells; (iii) Gcg+tdTomato- cells (GCG, 14%), likely α cells escaping recombination; and (iv) Gcg- tdTomato- cells (NONE, 5%), potentially immature α cells (Fig. 4k).

To further dissect molecular changes across these α cell subpopulations, we analyzed the expression of key markers associated with known markers, identity, autophagy, transdifferentiation, dedifferentiation, glucose-responsiveness and amino acid metabolism (Fig 4l, Supplementary Fig. 4b). Gcg and MafB expressions were diminished in the tdTomato group, confirming identity erosion. Autophagy-related genes, such as Map1lc3a, Map1lc3b, Lamp1, Lamp2, Atp6v0c and Ctsf, were upregulated in the tdTomato group, while the expressions of Ins1, Mafa and Ucn3 remained unchanged, excluding transdifferentiation into β cells. In the tdTomato group, Ngn3 was undetectable and other progenitor-related genes showed no significant changes, arguing against persistence of progenitor-like cells. Several glucose-responsive genes, such as Gck and G6pc2 were significantly downregulated in the tdTomato group. Notably, expressions of amino acid transporter genes (Slc16a10, Slc38a5, Slc38a4, and Slc7a2) were profoundly altered in identity-lost tdTomato group, implicating impaired amino acid sensing in the progression of α cell dysfunction.

SLC7A2 is involved in the regulation of α cell identity gene expression via lysine-facilitated histone acetylation modification

Based on the transcriptional disruptions observed in α cells of CBP/p300αDKO mice from scRNA-seq, we sought to identify key metabolic genes involved in the maintenance of α cell identity. Differential expression analysis highlighted Slc7a2, encoding a cationic amino acid transporter for lysine and arginine20,31, as one of the most significantly downregulated genes in α cells from CBP/p300αDKO mice compared to WT controls (Log2 Fold Change = -1.0578, P value = 1.0074E-140) (Supplementary Fig. 5a). Violin plots confirmed that this downregulation was specific to α cells, with minimal changes observed in other islet cell types (Fig. 5a). Pseudotime analysis revealed a progressive decline in Slc7a2 expression along the α cell differentiation trajectory in CBP/p300αDKO mice (Fig. 5b). Correlation analysis further showed that Slc7a2 expression positively correlated with key α cell identity markers (Gcg, MafB, and Pcsk2) and negatively with autophagy-related genes (Ctsf and Ctsl) (Fig. 5c), suggesting a potential association with α cell dysfunction and stress responses. Immunofluorescence staining confirmed reduced SLC7A2 protein level (Fig. 5d, Supplementary Fig. 5b), accompanied by a significant decrease in MafB expression in α cells of CBP/p300αDKO mice (Fig. 5e, Supplementary Fig. 5c).

CBP/p300 are known to regulate lineage-specific gene expression through histone H3K27 acetylation30,32. Consistent with this, H3K27Ac level was significantly reduced in α cells of CBP/p300αDKO mice (Fig. 5f, Supplementary Fig. 5d). Structurally, CBP/p300 possess a histone acetyltransferase (HAT) domain that catalyzes acetylation of lysine residues and a bromodomain that recognizes acetylated lysines to facilitates chromatin targeting33. To determine the contribution of each domain to transcriptional regulation of α cell genes, we treated primary islets from WT mice with either I-CBP112 (a bromodomain inhibitor34, 5 μM) or A-485 (a selective HAT inhibitor35, 3 μM). Only A-485 markedly suppressed the expressions of Gcg, Mafb and Slc7a2 (Fig. 5g), highlighting the crucial role of CBP/p300 HAT activity in maintaining α cell identity. This was further validated in α-TC1-6 cells, where A-485 treatment led to similar decreases in the expressions of these genes (Fig. 5h). ChIP-qPCR analysis revealed that A-485 markedly lowered H3K27Ac enrichment at their promoter regions (Fig. 5i-k), a finding consistent with our previous H3K27Ac ChIP-seq data from A-485-treated rat islets (Supplementary Fig. 5e).

Amino acid metabolism has been implicated as a key regulator of α cell identity and function36. In this study, we found that amino acid starvation conditions (HBSS) significantly reduced the mRNA expressions of Gcg, MafB, Pcsk2, and Slc7a2 in α-TC1-6 cells (Fig. 5l). Similarly, Slc7a2 knockdown produced a similar effect, supporting the essential role of Slc7a2-mediated amino acid transport in maintaining α cell identity. On the contrary, lysine supplementation significantly upregulated the expression of these identity genes and partially rescued the inhibitory effects of Slc7a2 knockdown (Fig. 5m), suggesting that Slc7a2-mediated lysine uptake supports α cell function. Ex vivo islet secretion assays further demonstrated that lysine pretreatment enhanced both basal and arginine-stimulated glucagon secretion (Supplementary Fig. 5f). Mechanistically, lysine catabolism yields acetyl-CoA, which serves as a substrate for histone acetylation37. Supporting this, ChIP-qPCR analysis showed that lysine treatment significantly increased H3K27Ac levels at the promoters of Gcg and MafB (Fig. 5n, o). We further tested whether the catabolite of lysine 2-AAA, a known biomarker of T2DM, elicits similar effects. Indeed, 2-AAA treatment robustly elicited the expression of Gcg, Mafb, and Pcsk2 (Fig. 5p), with elevated H3K27Ac levels at their promoters (Fig. 5q, r), linking amino acid metabolism to CBP/p300-dependent epigenetic regulation of α cell identity genes.

Together, these results suggest that Slc7a2-mediated lysine uptake fuels acetyl-CoA production, thereby promoting CBP/p300-dependent histone acetylation and expression of α cell identity genes (Fig. 5s).

Both CBP/p300 inhibition and Slc7a2 silencing trigger amino acids-regulated α cell autophagy

Autophagy is essential for maintaining cellular homeostasis38. It is specifically activated in response to amino acid starvation through two key signaling cascades: the mammalian target of rapamycin complex 1 (mTORC1) and general control nonderepressible 2 (GCN2) pathway39,40. To assess autophagic activity in CBP/p300αDKO mice, we performed single-cell gene set enrichment analysis (scGSEA), which revealed significant upregulation of lysosome-related genes in α cells (Fig. 6a, b). Immunofluorescence staining showed a higher percentage of tdTomato+ α cells with LC3 puncta, a hallmark of autophagosome formation41, in knockout mice (Fig. 6c, d). Consistently, higher accumulation of autophagosomes was observed in α cells from CBP/p300αDKO mice compared to WT mice as shown by transmission electron microscopy (Fig. 6e, f, Supplementary Fig. 6a), confirming increased autophagic flux in the absence of CBP/p300.

To determine whether amino acid availability regulates autophagic activity in α cells, we treated α-TC1-6 cells with HBSS. This treatment significantly upregulated the mRNA expression of autophagy-related genes (Fig. 6g). Importantly, knockdown of Slc7a2 in α-TC1-6 cells also increased the expression of autophagy markers (Fig. 6h), suggesting that Slc7a2 modulates autophagic activity. CBP/p300 inhibition by A-485 elicited a similar response (Fig. 6i). Furthermore, Slc7a2 knockdown in α-TC1-6 cells led to elevated level of LC3-II, a marker of autophagosome accumulation (Fig. 6j, Supplementary Fig. 6b). This was the case for A-485 treatment (Fig. 6k, Supplementary Fig. 6c), further supporting the roles of Slc7a2 and CBP/p300 in amino acids-regulated autophagy.

CBP/p300 deficiency blocks glucagon receptor antibody-stimulated α cell proliferation

Arginine activates the mTORC1 signaling pathway to regulates cell growth and proliferation42. Since Slc7a2 mediates arginine uptake, it is reasonable to suppose that Slc7a2 influences α cell proliferation by activating mTORC1. To test this, isolated mouse islets were cultured with DMEM (control), HBSS (starvation), and arginine (Fig. 7a). Under starvation condition, mTORC1 activation in islets α-cells was significantly reduced as shown by low phosphorylation of S6 (pS6), a key downstream target of mTORC143. Conversely, arginine supplementation enhanced mTORC1 activation (Fig. 7b, Supplementary Fig. 7a). Slc7a2 knockdown blunted the ability of arginine to activate S6 phosphorylation in α-TC1-6 cells (Fig. 7c, Supplementary Fig. 7b), whereas Slc7a2 overexpression potentiated mTORC1 activation in response to arginine (Fig. 7d, Supplementary Fig. 7c), indicating that Slc7a2 is indeed a key arginine sensor in α cells. To examine whether CBP/p300 inhibition affects mTORC1 signaling through Slc7a2 regulation, we treated α-TC1-6 cells with A-485. This treatment significantly diminished arginine-stimulated mTORC1 activation (Fig. 7e, Supplementary Fig. 7d).

The link between hyperaminoacidemia and α cell proliferation has been well established in models with disrupted glucagon signaling44. To assess the impact of CBP/p300 loss on α cell responses to amino acids, WT and CBP/p300 knockout mice were treated with a monoclonal antibody (REMD 2.59) targeting GCGR (Fig. 7f). Immunofluorescence analysis showed that GCGR antibody treatment strongly increased the number of tdTomato+ α cells in WT mice, whereas this effect was abolished in CBP/p300αDKO mice (Fig. 7g). Quantification confirmed a significant expansion of tdTomato+ cells per islet in WT mice following GCGR signaling blockade, without significant change in knockout mice (Fig. 7h). Proliferative activity was further assessed by co-staining for Ki67. In WT mice, GCGR mAb robustly increased the proportion of Ki67+ tdTomato+ α cells, while CBP/p300-deficient mice failed to mount a proliferative response (Fig. 7i), indicating that CBP/p300 are required for α cell proliferation in response to glucagon receptor inhibition. We next examined mTORC1 signaling by assessing phosphorylation of S6. GCGR antibody significantly increased the proportion of pS6⁺ tdTomato⁺ cells among tdTomato⁺ cells in WT islets, but this induction was markedly blunted in CBP/p300αDKO mice (Fig. 7j, k), indicating impaired mTORC1 activation.

To validate these findings in an inducible model, we administered GCGR antibody to iCBP/p300αDKO mice. Similarly, GCGR signaling blockade promoted α cell expansion in WT mice, but failed to increase tdTomato+ cell numbers in iCBP/p300αDKO mice (Fig. 7l, m). Ki67 staining also showed enhanced α cell proliferation in WT mice, but not in knockout mice (Fig. 7l, n). pS6 staining was significantly lower in iCBP/p300αDKO mice compared to WT controls (Fig. 7o, p). Collectively, these findings suggest that CBP/p300 are necessary for α cell proliferation and proper mTORC1 signaling in response to glucagon receptor blockage.

Methods
Mice

Crebbp-Loxp (Crebbpflox/flox) and Ep300-Loxp (Ep300flox/flox) mice were generated by Gempharmatech CO. using the CRISP-Cas9 system to insert LoxP sites flanking exon 2 of the Crebbp or Ep300 genes. Gcg-Cre knock-in (Gcg-Cre) mice (Stock No. 030663) were acquired from the Jackson Laboratory, while Gcg-IRES-CreERT2 knock-in (Gcg-CreERT2) mice were generated via CRISPR-Cas9 by Gempharmatech CO. ROSA26-tdTomato reporter mice (Stock No. 007909) were purchased from the Jackson Laboratory. For generating conditional Crebbp and Ep300 knockout mice, Crebbp/Ep300flox/flox mice were crossed with Gcg-Cre mice. The resulting F1 progeny (Crebbpflox/wt/Ep300flox/wt; Gcg-cre) were interbred with Crebbpflox/wt/Ep300flox/wt mice. This breeding strategy yielded the following specific genotypes: Crebbpflox/flox/Ep300wt/wt; Gcg-cre (CBPαKO) mice, Crebbpwt/wt/Ep300flox/flox; Gcg-cre (p300αKO) mice, Crebbpflox/flox/Ep300flox/wt; Gcg-cre (CBPαKO/p300HE) mice, Crebbpflox/wt/Ep300flox/flox; Gcg-cre (CBPHE/p300αKO) mice and Crebbpflox/flox/Ep300flox/flox; Gcg-cre (CBP/p300αDKO) mice. Cre-negative littermates from each mating combination were used as wild-type (WT) controls. To generate inducible α-cell-specific Crebbp and Ep300 double knockout (iCBP/p300αDKO) mice, Gcg-CreERT2 mice were crossed with Crebbpflox/flox/Ep300flox/flox mice. For induction of Cre-mediated recombination, mice aged 6-8 weeks received three intraperitoneal injections of tamoxifen (Sigma-Aldrich #T5648) at 100 µg/g body weight, with each injection spaced 24 hours apart. For lineage tracing studies, ROSA26-tdTomato reporter mice were further bred with CBP/p300αDKO or iCBP/P300αDKO mice to facilitate tracking of Crebbp or Ep300 deletion in specific cells. Both male and female mice were used in this study. All mice were kept on a C57BL/6J background and housed under specific pathogen-free conditions, following a standard 12-hour light/dark cycle with unrestricted access to food (normal chow) and water. Animal protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Model Organisms Center, Inc., Shanghai, China.

Genomic PCR for mouse genotyping

Mouse genotype was detected with genomic DNA prepared from tail using One Step Mouse Genotyping Kit (Vazyme, PD101-01). Multiple pairs of primers were used to identify different genetic modifications. For Crebbp/Ep300 floxed alleles, Crebbp-F1/Crebbp-R1 and Ep300-F1/Ep300-R1 primer pairs were used. The Crebbp-F1 and Crebbp-R1 primers were used to distinguish WT (147bp) or floxed Crebbp allele (245bp), whereas the Ep300-F1 and Ep300-R1 primers were used to distinguish WT (416bp) or floxed Ep300 allele (521bp). For Cre recombinase detection, three different Cre systems were genotyped: Gcg-Cre was detected using Gcg-Cre-P1, Gcg-Cre-P2, and Gcg-Cre-P3 primers; Gcg:CreERT2 was identified using Gcg:CreERT2-F1 and Gcg:CreERT2-R1 primers; and Rosa26 reporter alleles were genotyped using Rosa-P1, Rosa-P2, Rosa-P3, and Rosa-P4 primers to distinguish between different Rosa26 allelic states. The primer sequences used for genotyping mice are provided in Supplementary Table 1.

Metabolic studies

Random and 16 h-fasting blood glucose concentrations were measured using a glucometer (Accu-Chek, Roche, Mannheim, Germany) by tail vein sampling. For pyruvate tolerance test, the mice in an overnight-fasting state received an i.p. injection of sodium pyruvate (2 g/kg body weight, Sigma Aldrich), blood glucose concentration was measured before and 15, 30, 60, and 120 min after pyruvate injection. Fasting plasma glucagon level was quantitated using homogenous time-resolved FRET (HTRF) assays (Cisbio/PerkinElmer). Fasting plasma insulin level was detected by mouse insulin Elisa kit (Mercodia). The level of total fasting plasma amino acids was measured using a commercially available Total Amino Acid assay kit (Nanjing Jiancheng Bioengineering Institute).

We performed the metabolomics analysis by Q300 Kit according to a detailed procedure (Metabo-Profile, Shanghai, China). A ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) was used to quantitate all targeted metabolites in this study by Metabo-Profile Biotechnology (Shanghai) Co., Ltd. Principal component analysis (PCA) was performed. The volcano plot integrated the log2 (Fold Change) and P-value. VIP (variable importance in projection) was obtained based on the OPLS-DA model. Differentially expressed metabolites (DEMs) were selected with VIP ≥ 1 and P-value < 0.05. The Z-score indicates standard deviations concerning the mean of the control group. The raw data are provided in Supplementary Data 1.

For GCGR mAb treatments, the animals were treated weekly with 5 mg/kg of a humanized monoclonal anti-GCGR antibody (REMD 2.59) intraperitoneally for 4 weeks via intraperitoneal administration as previously reported67.

Immunofluorescence staining and image analysis

Mouse whole pancreas (including head, body, and tail) was dissected, fixed in 4% formalin and processed for paraffin embedding. For the immunostaining analysis, entire pancreatic tissues were continuously sectioned at 5-μm thickness. The primary antibodies were added and incubated overnight at 4 ℃. Then tissue sections were treated with fluorescence-labeled secondary antibody and stained with DAPI. For cell quantification in the immunofluorescent staining, six to eight equally spaced sections (which covered the entire pancreas) per pancreas were imaged using a TissueGnostics microscope or a ZEISS LSM880 confocal microscope, and the total numbers of positive staining cells from three to five mice per group were counted manually. The α cell area from the glucagon antibody-stained sections and β cell area from the insulin antibody-stained sections were measured using HistoQuest software (TissueGnostics).

Ex vivo islet assays

Mouse pancreatic islets were isolated as described in our previous study68. For amino acid starvation experiment, islets from 4 weeks old C57BL/6J mice were overnight recovered in RPMI-1640 containing 16.7 mM glucose and 10% FBS. They were then cultured in RPMI-1640 containing 16.7 mM glucose and 0.2% BSA or Hanks' balanced salt solution (HBSS) containing 16.7 mM glucose and 0.2% BSA for 50 min. For arginine re-stimulation experiment, starved islets were then stimulated with 400 μM arginine for 10 min. At the end of the culture, these islets were fixed in 4% formalin for 24 h and subsequently blocked in 1% agarose gel and embedded in paraffin. These islets paraffin blocks were continuously sectioned at 5-μm thickness. Then immunofluorescence staining was performed on the tissue section to detect the phosphorylated level of S6 in Gcg+ cells.

Single-cell RNA-sequencing analysis

For the single-cell RNA-seq, islets were pooled from three Gcg-Cre;ROSAflox/+ mice and three CBP/p300αDKO;ROSAflox/+ mice at 4 weeks of age. Each group contained 300-400 islets, which were digested with 0.25% Trypsin-EDTA (Sigma-Aldrich) for 5 min at 37 ℃. Digestion was terminated with an equal volume of ice-cold RPMI-1640 containing 10% FBS and the sample was centrifuged at 500 g for 3 min. The suspension was then sieved and the pellet was resuspended in ice cold PBS containing 0.2% BSA. Cell viability and number were assessed by Trypan blue staining and the Eve automatic cell counter respectively. Approximately 10,000 - 15,000 cells for each sample were loaded into the 10× Chromium Chip B according to the 10× Genomics Single Cell 30 Reagent Kit v3 GEM protocol. The sequencing strategy used was the Illumina NovaSeq PE150. The scRNAseq data were subsequently processed using Cell Ranger v. 5.0.1 (10× Genomics) to make fastq files for each sample. Cell Ranger Count aligned reads to the mm10 reference genome via STAR. To enable mapping of tdTomato-derived transcripts, the sequence for tdTomato was added to the reference genome and GTF file. Cell filtration was performed with Seurat v4.0.0, removing low quality sequencing data (parameters: nFeature > 200, nUMI > 1000, log10GenesPerUMI > 0.7, mt < 30%, percent of rbc < 0.05) and potential doublets were removed by DoubleFinder69. Count matrices were normalized and scaled using the NormalizeData function with standard parameters. Dimensionality reduction was performed via principal-component analysis (PCA) to enable cell clustering. Cell clustering was done using the FindClusters method with variable-feature 2000 and resolution 0.4. Uniform manifold approximation and projection (u-MAP) was performed. Gene Set Enrichment Analysis (GSEA)70 was used to complete Gene Ontology (GO) and KEGG term enrichment analysis with the Molecular Signatures Database (MSigDB) C5 GO gene sets and C2 KEGG gene sets (version 7.2) separately. Slingshot71 could provide the lineage structure and pseudotime variables inferences for cells along each lineage. The slingshot analysis was performed by slingshot R package (version 1.8.0). Initially, the dimensionally reduced Seurat object was transformed into a SingleCellExperiment object by as.SingleCellExperiment function. Pancreatic α cells from WT mice were selected as a starting cluster for the trajectory inference. Then, FitGAM function in tradeSeq package72 (version 1.4.0) was used to fit the nonlinear function between gene expression and pseudotime using negative binomial generalized additive model (NB-GAM). By using the associationTest function, the top 100 genes whose expression levels significantly differed from pseudotime were chosen to create a heatmap.

To classify hormone-positive and hormone-negative cells, Gcg+ and Gcg- cells were defined using the Pareto density-based cutoff strategy described by Perez-Frances et al73. Normalized Gcg expression values were fitted with Pareto density functions, and cells above the Pareto-derived threshold were classified as Gcg+, whereas those below were classified as Gcg-. The same approach was applied to define tdTomato+ and tdTomato- cells. The cutoff values used for classification were Gcg: 4.384858 and tdTomato: 2.281258.

Transmission electron microscopy

For ultrastructure observation, islets from 3-4 weeks old WT and CBP/p300αDKO mice were immediately hand-picked without filtration after digestion. Each group contained 200 islets. Then the purified islets were chemically fixed with 2.5% glutaraldehyde in phosphate buffer at real temperature for 2 h. After washing, specimens were osmicated with 1% osmium tetraoxide in the same buffer. Then, specimens were washed and dehydrated in ethanol, embedded into Epon 812. After polymerization, 50-100 nm sections were cut and stained. Micrographs were taken by Transmission Electron Microscope (Hitachi, Japan).

Cell culture and treatment

α-TC1-6 cells (a mouse glucagonoma cell line) were cultured in DMEM (Gibco, #10567-014) supplemented with 10% (v/v) FBS, 10 mM HEPES and 100 IU/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. Empty plasmids and shRNA interference plasmids targeted Slc7a2 were designed and synthesized from GeneChem (Shanghai, China). Plasmid transfection was carried out by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For inhibiting CBP/p300 HAT activity, 3 μM A-485 was used to treat α-TC1-6 cells. After infection or A-485 treatment, cells were harvested for RNA isolation or protein extraction and further analysis. For amino acid starvation and arginine re-stimulation experiments, α-TC1-6 cells were incubated with HBSS buffer containing 1 g/L glucose and 0.2% BSA for 50 min (amino acid starvation) and then treated with 400 μM arginine for 10 min. The target sequences of shRNA plasmids are provided in Supplementary Table 2.

qPCR and Western blotting analysis

For qPCR, total RNA was extracted from α-TC1-6 cells using TRIzol reagent (Vazyme). cDNA was synthesized using a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). Real-time quantitative PCR (qPCR) was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme). The relative mRNA level of the target gene was calculated using the 2-△△Ct method. The △Ct was computed with normalization to 18s rRNA or beta-actin, which served as the internal control. Primer sequences can be obtained in Supplementary Table 3.

α-TC1-6 cells were treated with RIPA lysis buffer (Aksmics, Shanghai, China) for total protein extraction. Histone was extracted using the EpiQuik Total Histone Extraction Kit (EpiGentek, NY, USA) according to the manufacturer’s instructions. Primary antibodies are listed as following: rabbit anti-S6 (1:1000; Cell Signaling Technology), rabbit anti-pS6Ser240/244 (1:1000; Cell Signaling Technology), rabbit anti-CREB (1:1000; Cell Signaling Technology), rabbit anti-pCREB (1:1000; Cell Signaling Technology), mouse anti-HSP90 (1:1000; Santa Cruz Biotechnology). Rabbit anti-H3K9Ac, rabbit anti-H3K18Ac, rabbit anti-H3K27Ac, rabbit anti-H4K12Ac, rabbit anti-H4K8Ac, and rabbit anti-Histone H4 were all purchased from PTM Bio (1:1000 dilution). HSP90 or Histone H4 was used as an internal control to normalize band intensity. Horseradish peroxidase-coupled goat-anti-rabbit IgG or goat-anti-mouse IgG (1:2000; Cell Signaling Technology, USA) were used as a secondary antibody. The primary antibodies used in the experiments are provided in Supplementary Table 4.

ChIP-qPCR

The ChIP assay was performed using an EZ-ChIP Chromatin Immunoprecipitation kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. In brief, α-TC1-6 cells were fixed using the formaldehyde with 1% formaldehyde and homogenized in cell lysis buffer.

DNA was sheared to fragments at 100–1000 bp using sonication. Chromatins were incubated and precipitated with anti-H3K27Ac (Cell Signaling Technology) or IgG (negative background control) antibodies. Precipitated DNA was analyzed by real-time qPCR, using primers listed in Supplementary Table 5.

Statistics and Reproducibility

Each experiment was replicated at least three times for each condition. Statistical analyses were performed using GraphPad Prism 9.0. We used two-tailed Student’s test for comparison between groups, and one-way ANOVA followed by Fisher’s LSD for comparisons among three or more groups. P < 0.05 is used to declare statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001. All data were presented as means ± SEM (standard error). Data from representative experiments, including micrographs and Western blots, are confirmed in at least three independent biological replicates with consistent and reproducible outcomes.

Data availability

The Crebbpflox/flox and Ep300flox/flox mice are available upon request. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. The islets Single-cell RNA sequencing data have been deposited in the GEO database under the accession codes (GSE324884). All data generated in this study are included in the main text or Supplementary Information. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In new study titled “CBP/p300-Slc7a2 axis is critical for the expansion and maintenance of functional pancreatic α cell mass”, Wang et al. generated tissue-specific knockout mice to examine the importance of histone acetyltransferase p300/CBP in α cells, they meticulously analyzed the phenotype and report new findings. Specifically, they report that α-cell-specific double knockout mice for p300/CBP exhibit hypoglucagonemia and hyperaminoacidemia due to reduced α-cell mass. Furthermore, they explore multiple potential mechanisms and conclude that CBP/p300 is a critical regulator of functional α-cell mass by modulating amino acid sensing via Slc7a2. The research was conducted under a highly rigorous design, yielding robust results. While several concerns are noted below, the overall quality of the study appears exceptionally high.\n1. Since the mouse model used Gcg-Cre, Cre recombinase is expected to cause CBP and p300 gene deficiency in Gcg-expressing cells other than α-cells. For example, it should be examined whether there are effects on intestinal L-cells.\n2. The discovery that α-cell mass increases during the early juvenile stage from 1 to 2 weeks of age is highly intriguing. Since α-cell mass in CBP/p300αDKO mice is normal at 1 week of age, this suggests that CBP/p300 is dispensable for α-cell development during the fetal period. However, the subsequent impairment in α-cell growth suggests that the physiological increase in α-cell mass during the early postnatal period is a CBP/p300-dependent response, although the driver for this response has not been addressed. Could the physiological increase in α-cell mass during the early postnatal period be explained by a mechanism similar to that observed with GcgR antibody administration?\nMinor point\n3. Could experiments using the HAT inhibitor A-485 be further examined using mutant CBP and p300 that have lost HAT activity?", "reviewer_2": "Wang et al generated conditional CBP/p300 double KO in alpha-cells on alpha-cell expansion and mass, differentiation and function. Pancreatic alpha-cell-specific deletion of CBP/p300 induced hypoglucagonemia and hyperaminoacidemia, without affecting fasting blood glucose. Alpha-cell CBP/p300 dKO impaired postnatal alpha-cell expansion and differentiation, resulting in decreased alpha-cell mass and to development of hypoglucagonemia. In addition, CBP/p300 deficiency prevented glucagon receptor antibody-stimulated alpha-cell proliferation. Mechanistically, the authors describe a novel CBP/p300-Slc7A2 axis, regulating lysine-induced histone acetylation, including that of the glucagon (Gcg), Mafb and Pcsk2 genes. CBP/p300 KO induces autophagic cell death through attenuation of SLC7A2/arginine-stimulated mTORC1 signaling. Collectively, the authors conclude that in alpha-cells, CBP/p300 are central regulators of alpha-cell mass and function by orchestrating SLC7A2-mediated amino acid sensing. In general, the study design is sound, the findings are interesting, and the manuscript is well-written. However, CBP and p300 are ubiquitous nuclear proteins that play a central role in regulating histone acetyl-transferase activity; therefore, it is not surprising that knocking out both genes impair transcription and cellular growth, differentiation and function. The role of this pathway in regulating alpha-cell function in physiology, e.g. under conditions of amino acid deprivation or overload is not clear. It is also not clear whether this pathway is perturbed in diabetes and/or obesity. New experimental data are required to strengthen the core mechanistic components of the proposed model and to demonstrate its relevance to disease pathophysiology.\nMajor Comments\n1. Are there any alterations in CBP, p300 and/or Slc7a2 expression in alpha-cells in animal models of diabetes and/or obesity/metabolic associated liver dysfuncton(MASLD) and even more important in human subjects? Of note, alpha-cell dysfunction was present only under conditions of complete ablation of both CBP and p300, suggesting that the activity of these proteins is partially redundant and dispensable, therefore it is unlikely that partial inhibition of these genes in obesity or diabetes (if exist) is sufficient to induce alpha-cell dysfunction, arguing against the suggestion that this pathway is involved in disease pathophysiology.\n2. Glucagon deficiency affected amino acids degradation, but not hepatic glucose production, evident by similar fasting glucose, pyruvate tolerance and expression of gluconeogenic enzymes. Are mouse models of glucagon deficiency relevant for studying the role of glucagon in human physiology? Please explain.\n3. The authors argue that SLC7A2/arginine, rather than SLC38A5/glutamine regulates mTORC1 and autophagy. Previous studies showed that GCGR antagonism induces alpha-cell proliferation through glutamine transport via SLC38A5, thereby stimulating mTORC1. The authors found that the expression of both Slc7a2 and Slc38a5 decreased in CBP/p300 dKO mice. They should add control experiments of Slc38a5 knockdown and/or over-expression and study the effects on mTORC1 activity and autophagy in alpha-cells to convincingly show that the SLC7A2 transporter is indeed the main regulator of mTORC1-autophagy signaling in alpha-cells. The authors over-expressed Slc7a2 in islets and claim that this enhanced arginine stimulation of mTORC1 (Fig. 7d). This statement is not entirely supported by the data: Slc7a2 over-expression increased mTORC1 activity in absence of arginine, while the response to arginine was in fact abrogated. Fig. 7b-e lack quantifications, and the number of repeated experiments is not mentioned, therefore it is difficult to interpret the findings.\n4. The data on autophagy are not convincing. The few autophagy genes shown in Figure 4i are involved in regulating lysosome functon(cathepsins) and Golgi-localized transmembrane localization (Temen59); these are not \"classical\" regulators of autophagy. The same is true for many of the genes shown in Fig. 6g-i. A previous study suggested that in alpha-cells inhibiting mTORC1 induces crinophagy, rather than autophagy. Does aCBP/p300 dKO affect crinophagy? Prompt assessment of autophagic flux requires analysis of LC3   conversion to LC   in cells treated with or without a lysosome hydrolases inhibitor, such as chloroquine (as in Fig. 6k) and/or testing the protein level of P62/SQTM1. In Fig. 6j (Slc7a2 knockdown experiment), the authors don't show the effects of Slc7a2 knockdown on autophagic flux. Fig. 6j-k lack quantifications, and the number of repeated experiments is not indicated.\n5. mTORC1 regulates autophagy via phosphorylation of ULK1 and TFEB. Does treatment with arginine, Slc7a2 knockdown and inhibition of CBP/p300 affect ULK1 and TFEB phosphorylation. This may strengthen the conclusion that CBP/p300-SLC7a2 signaling regulates autophagy via mTORC1.\n6. The authors suggest that aCBP/p300 dKO induces autophagic death, however no data are shown throughout the manuscript to support this hypothesis. Autophagy might be an adaptive response to stress and doesn't necessarily induce cell death.\n7. In the morphometric and lineage trace experiments shown in Figs. 2-3, at the age of 8 weeks, the alpha-cell number in CBP/p300 dKO mice was decreased by ~90%, whereas alpha-cell dedifferentiation (tdT tomato+Gcg+/TdT+ cells) occured in ~66% of the cells. The residual alpha-cell loss may be explained by reduced proliferation, which was observed during the early neonatal period and/or alpha-cell death. I would suggest assessing alpha-cell proliferation and apoptosis at the age of 4 weeks and 8 weeks to quantify the contribution of alpha-cell dedifferentiation, decreased proliferation and cell death to alpha-cell loss in aCBP/p300 dKO mice.\n8. In Fig. 3f, ~10% of control alpha-cells are stained positive for glucagon and PPY; this seems to be exaggerated. In the aCBP/p300 dKO, the percentage of Ppy+/tdT tomato+ cells was markedly decreased. These findings are puzzling. The colocalization images (Fig. 3e-f) are not convincing; quantification of colocalization on z-stack images would be valuable.\n9. The authors suggest that hypoglucagonemia is mainly driven by alpha-cell deficiency. It is possible that CBP/p300 deficiency also affects the machinery of glucagon secretion. Ex vivo static incubations testing the glucagon response of aCBP/P300 dKO islets to secretagogues, such as amino acids would clarify this point.\n10. In Figure 5, the authors show that downregulation of Slc7a2 was associated with loss of alpha-cell identity genes and that pharmacological inhibition of histone acetyl transferase recapitulate these findings, suggesting that CBP/p300-driven H3K27 acetylation at the promoter of key alpha-cell genes mediate alpha-cell dysfunction. The expression of several amino acid transporters, including Slc7a2, Slc38a4 and Slc385 was decreased – are these alterations also mediated via CBP/P300 binding to promoters? Add quantifications to Fig. 5d–f.\n11. The authors suggest that lysine and its metabolite acetyl CoA mediates SLC7A2 epigenetic regulation, whereas arginine mediates the effects on mTORC1 activity and autophagy. However, it has been shown that mTORC1 is also regulated by acetyl CoA through Raptor acetylation. Does lysine affect mTORC1 activity and autophagy?\nMinor comments\n1. In Fig. 1b-g-Please specify the age of the animals that were studied.\n2. In Fig. 4c, Ins1/Ins2 gene expression appears high in delta-cells, which is unexpected. Please explain.\n3. In fig 6e, the yellow arrow annotation may point to an insulin granule, rather than an autophagosome, raising questions regarding the correct identification of alpha-cells vs beta-cells. Please annotate cells and organelles with reference to https://doi.org/10.1369/0022155415583535.\n4. Line 302: specify mTORC1 reduction is in alpha-cells.\n5. Line 303: “low phosphorylation of S6 by S6K,” not phosphorylation of S6K.\n6. Fig. 7o–p: text mentions intensity, graph shows % positive cells.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "Wang et al use Gcg-Cre to delete the histone acetyltransferases p300 and CBP in pancreatic α cells. The KO shows a partial reduction in circulating glucagon and modest increases in several plasma amino acids, which the authors attribute to loss of α cell identity, reduced α cell mass and proliferation, arising from diminished α-cell gene expression. They nominate Slc7a2 as a mediator, and that the reduced Slc7a2 leads to reduced H3K27ac at the Gcg and Mafb loci and their gene downregulation, and a failure to mount α-cell hyperplasia after anti-Gcgr treatment.\nThe findings on α-cell identity and proliferation overlap with prior Neurog3-Cre genetics showing that p300/CBP are required for islet endocrine cell mass and proliferation. Please cite and discuss this study, since the overlap limits the incremental advance. A significant issue is that most data shown are underpowered, with experiments from Figures 1c, 2, 3, 5, and 6 presented at n = 3 and no justification of sample size.\nFor the core narrative, the authors argue that loss of p300/CBP downregulates Slc7a2, which then maintains α-cell identity through H3K27ac at Gcg and Mafb, and amino acid-driven proliferation. This interpretation does not fully acknowledge that p300/CBP are fundamental drivers of tissue-specific gene expression that establish endocrine identity. It is therefore equally plausible that Slc7a2 downregulation is a downstream consequence of identity erosion rather than the primary cause. To support causality, one would need in vivo evidence that Slc7a2 loss is sufficient to reduce H3K27ac at Gcg and Mafb with concordant transcriptional downregulation. A recent Slc7a2-/- report does not show loss of α-cell identity. For proliferation, α cells lacking p300/CBP already show impaired proliferation, so the lack of an amino acid response may reflect a general failure to traverse the cell cycle, in addition to any defect in amino acid sensing.\nSpecific comments\n1. The Gcg-Cre driver is active in pancreatic α cells, intestinal L cells, and preproglucagon neurons. Did p300/CBP loss in gut and brain influence oral glucose tolerance or energy metabolism?\n2. Did the authors measure glucagon secretion from intact islets, and is insulin secretion altered in vivo or ex vivo?\n3. For Figures 2b and 3c, please report absolute values in addition to fold change.\n4. The cell death data regarding tdTomato+ cell loss should be presented as supplementary data. The authors cannot claim cell death without direct experimental evidence.\n5. Line 195 states that ‘islets with high tdTomato expression in α cells were isolated… and subjected to scRNA-seq,’ yet the dataset is majority Ins+ cells. Please clarify whether single tdTomato+ cells were fluorescence-activated cell sorted after dissociation. If so, please discuss why the single cell data does not show enriched α cells.\n6. Please explain why pseudotime was not computed on a single integrated dataset containing both WT and KO tdTomato+ cells. Figures 4b and 4d appear to analyze only the WT cells, whereas the pseudotime analysis was performed only on the knockout cells. Joint integration followed by trajectory inference is a standard way to place both genotypes on the same manifold so that differences can be attributed to biology.\n7. In Figure 4g, the inferred trajectory appears to terminate in the cluster that shows high Gcg expression in Figure 4i, yet in Figure 4h the Gcg signal does not peak at the end of the trajectory. In addition, Figure 4i appears to combine wild type and knockout cells, whereas Figures 4g and 4h do not. Please clarify.\n8. Please define the thresholds used to call ‘Gcg+’ and ‘Gcg-’ in the single-cell data (for example unique molecular identifier cutoffs, scaled expression, or module scores), and state where this is documented in the Results.\n9. In Figure 4i, z-score dot plots can exaggerate differences across genes with narrow dynamic ranges. Please use violin plots or dot plots of normalized expression so readers can see absolute distributions. This information should be presented as early as possible, since as of now it is obscure to interpret the effect size of the α cell gene downregulation.\n10. Is Arx differentially regulated in the KO α cells?\n11. Figures 5k and 5l: the conclusion that Slc7a2-mediated lysine uptake drives H3K27ac would be stronger with lysine-rescue experiments in shRNA-Slc7a2 cells.\n12. Figure 7d: can the authors comment on why overexpression of Slc7a2 increases basal pS6?\n13. Figure 7e: the p300/CBP inhibitor reduces pS6 irrespective of arginine, so it is impossible to conclude that blocking p300/CBP specifically suppresses arginine-mediated pS6 induction.\n14. Line 445: “These results indicate that chronic amino acid exposure could stimulate α cell function through epigenetic modification-mediated gene expression” is not supported by the data. The reported effects are chiefly on alpha cell proliferation rather than function, and while they may involve p300/CBP, they do not establish a requirement for epigenetic modification, since these proteins have numerous non-epigenetic functions.", "reviewer_5": "In this paper, the authors demonstrated elegantly that CBP/p300 plays an essential role in pancreatic α cell function and expansion in the postnatal period. They also found cationic amino acid transporter Slc7a2 as a key mediator of the maintenance of αcells.\n1) Slc7A2 has been well studied in a lot of cell types including immune cells and cancer cells and its role in cell proliferation already established. The evidence of the essential role of Slc7A2 in this study should be proved by transgenic overexpression of Slc7a2 in the αcells of DKO mice.\n2) One of the previous studies of glucagon signal blockade by Coate et al(Mol Metab2024) demonstrated the increase of non-alpha cells (mainly delta or beta cells) in a Slc7a2-mTORC1 dependent manner, while in this study, Ppy-positive glucagon cells were increased. Please discuss the apparent discrepancy.\n3) The scRNA-seq revealed four subgroups of alpha cells (Fig4K), which I think are important findings. It should be noted that tdTomato+ Gcg- (tdTomato) cells, consisted of 39%. They were considered as denoting identity-compromised αcells. However, they may have been caused by either impaired differentiation of progenitor cells or dedifferentiation of mature αcells. Please discuss this point by analyzing the expression profiles. As for pancreatic beta cells, the impairment of healthy differentiation resulted in the expression of “disallowed genes” such as alpha cell genes. In this sense, the expression of Mafa, Ngn3, or Pdx1 should be examined.\n4) Related to the point above, tdTomato+Gcg+(Both) cells were considered as escaping the recombination, but the evidence (expression of either CBP or p300) were not provided. If these are the intact alpha cells, then high levels of amino acids should have induced the expansion of these ‘Both cells’. To examine whether the ‘Both cells ‘ were ‘healthy’, nutrient-induced glucagon secretion, either from isolated islets or alpha cells, should be tested.\n5) The transcriptional regulation of Slc7a2 is of great significance. However, it is not clear whether CBP/p300 directly regulate transcription of Slc7a2. Please demonstrate the CBP/p300 location on the promoter/enhancer sites of Slc7a2 genes, and their absence in the DKO mice."}, "author_response": "We sincerely thank the reviewers for their constructive and insightful comments on our manuscript entitled “CBP/p300-Slc7a2 axis is critical for the expansion and maintenance of functional pancreatic α cell mass”. We greatly appreciate the reviewers who contribute their time and expertise to evaluate our work. In response, we have carefully addressed each point raised and incorporated new experimental data and expanded discussion where appropriate. These revisions have strengthened the manuscript. Below, we provide a detailed point-by-point response to all comments. Newly added data are included in the revised figures and text as indicated.\nReviewer #1 (Remarks to the Author):\nIn new study titled “CBP/p300-Slc7a2 axis is critical for the expansion and maintenance of functional pancreatic α cell mass”, Wang et al. generated tissue-specific knockout mice to examine the importance of histone acetyltransferase p300/CBP in α cells, they meticulously analyzed the phenotype and report new findings. Specifically, they report that α-cell-specific double knockout mice for p300/CBP exhibit hypoglucagonemia and hyperaminoacidemia due to reduced α-cell mass. Furthermore, they explore multiple potential mechanisms and conclude that CBP/p300 is a critical regulator of functional α-cell mass by modulating amino acid sensing via Slc7a2. The research was conducted under a highly rigorous design, yielding robust results. While several concerns are noted below, the overall quality of the study appears exceptionally high.\n1. Since the mouse model used Gcg-Cre, Cre recombinase is expected to cause CBP and p300 gene deficiency in Gcg-expressing cells other than α-cells. For example, it should be examined whether there are effects on intestinal L-cells.\nResponse: We appreciate your constructive comment. We fully agree that potential off-target effects of Gcg-Cre in intestinal L-cells should be evaluated to ensure that the observed phenotypes are primarily α-cell specific. To address this issue, we performed additional experiments. Intestinal co-expression of GLP-1 and the tdTomato in Gcg-Cre-driven tdTomato lineage-tracing WT and CBP/p300αDKO mice confirmed that Gcg-Cre was indeed active in the intestinal L-cells (Extended Data Fig.1a). However, neither the average number of GLP-1+ cell per intestinal section nor plasma level of active GLP-1 differed between control and CBP/p300αDKO mice (Extended Data Fig.1b-c). In addition, CBP/p300αDKO mice exhibited normal glucose tolerance (Extended Data Fig.1d), further indicating preserved L-cell function. Together, these results suggest that intestinal GLP-1-producing enteroendocrine cells remain largely intact in CBP/p300αDKO mice, perhaps due to the robust regenerative capacity of L-cells reported by Shiota et al [1]. The new data have been incorporated into Supplementary Figure 1 and described in the Results section.\nExtended Data Fig. 1. Effects of Gcg-Cre recombination in the intestinal L-cells.\n(a) Representative immunofluorescence images of intestinal L-cells from WT and CBP/p300αDKO mice. Co-staining for tdTomato (red) and GLP-1 (green), with nuclei labeled by DAPI (blue). Yellow arrows indicate double-positive cells. Scale bar, 10 μm.\n(b) Quantification of GLP-1+ cells per tissue section in WT and CBP/p300αDKO mice (n = 3). (c) The plasma levels of active GLP-1 in 8-week-old WT and CBP/p300αDKO mice (n = 5). (d) Oral glucose tolerance test (OGTT) in WT and CBP/p300αDKO mice (n = 5), with blood glucose measured at indicated time points. Data are presented as mean ± SEM. (Supplementary Fig.1a-d)\n2. The discovery that α-cell mass increases during the early juvenile stage from 1 to 2 weeks of age is highly intriguing. Since α-cell mass in CBP/p300αDKO mice is normal at 1 week of age, this suggests that CBP/p300 is dispensable for α-cell development during the fetal period. However, the subsequent impairment in α-cell growth suggests that the physiological increase in α-cell mass during the early postnatal period is a CBP/p300-dependent response, although the driver for this response has not been addressed. Could the physiological increase in α-cell mass during the early postnatal period be explained by a mechanism similar to that observed with GcgR antibody administration?\nResponse: We appreciate your insightful suggestion. In various models of GCGR blockade, elevating circulating amino acids have been identified as key secretory signals that drive excessive α-cell proliferation through mTOR-dependent pathways [2,3,4]. Notably, Solloway et al. [5] reported that nearly all proteinogenic amino acids and ornithine are markedly elevated in neonatal Gcgr+/+ mice (P7–P14), with levels progressively decreasing toward adulthood. This decline coincides with the cessation of postnatal α-cell proliferation in wild-type animals. Consistently, Bozadjieva et al. [6] showed that mTORC1 signaling was essential for α cell–mass maintenance during the nutritional transition from a milk-based to a chow-based diet, as α cell-specific mTORC1 regulator Raptor (αRaptorKO) knockout mice exhibited impaired α-cell expansion during this period. Taken together, these findings support the idea that early postnatal increase in α-cell mass is driven by elevated serum amino acids and activation of the mTORC1 pathway during lactation.\nMinor point\n3. Could experiments using the HAT inhibitor A-485 be further examined using mutant CBP and p300 that have lost HAT activity?\nResponse: Thank you for your valuable suggestion. Although time constraints prevented us from completing this experiment, we sought to further validate the role of HAT activity by treating α cell with C646 (a well-characterized inhibitor targeting the CBP/p300 HAT domain) and I-CBP112 (a bromodomain inhibitor), following the approach described by Weinert et al [7]. Like A-485, C646 also suppressed the expression of α-cell functional genes and upregulated autophagy-related genes. However, I-CBP112 showed no such effect (Extended Data Fig.2a-b). These results reinforce the conclusion that the phenotypes observed upon CBP/p300 inhibition are predominantly HAT-dependent.\nExtended Data Fig. 2. Effects of C646 and I-CBP112 on the expression of α-cell identity and autophagy-related genes. (a) Relative mRNA expression levels of identity genes in α-TC1-6 cells treated with 30 μM C646 or 5 μM I-CBP112 (n = 3). (b) Relative mRNA expression levels of autophagy-related genes in α-TC1-6 cells treated with 30 μM C646 or 5 μM I-CBP112 (n = 3). Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD test, compared with CON: *P< 0.05, **P< 0.01, ***P< 0.001.\nReviewer #2 (Remarks to the Author):\nWang et al. generated conditional CBP/p300 double KO in alpha-cells on alpha-cell expansion and mass, differentiation and function. Pancreatic alpha-cell-specific deletion of CBP/p300 induced hypoglucagonemia and hyperaminoacidemia, without affecting fasting blood glucose. Alpha-cell CBP/p300 dKO impaired postnatal alpha-cell expansion and differentiation, resulting in decreased alpha-cell mass and to development of hypoglucagonemia. In addition, CBP/p300 deficiency prevented glucagon receptor antibody-stimulated alpha-cell proliferation. Mechanistically, the authors describe a novel CBP/p300-Slc7A2 axis, regulating lysine-induced histone acetylation, including that of the glucagon (Gcg), Mafb and Pcsk2 genes. CBP/p300 KO induces autophagic cell death through attenuation of SLC7A2/arginine-stimulated mTORC1 signaling. Collectively, the authors conclude that in alpha-cells, CBP/p300 are central regulators of alpha-cell mass and function by orchestrating SLC7A2-mediated amino acid sensing. In general, the study design is sound, the findings are interesting, and the manuscript is well-written. However, CBP and p300 are ubiquitous nuclear proteins that play a central role in regulating histone acetyl-transferase activity; therefore, it is not surprising that knocking out both genes impair transcription and cellular growth, differentiation and function. The role of this pathway in regulating alpha-cell function in physiology, e.g. under conditions of amino acid deprivation or overload is not clear. It is also not clear whether this pathway is perturbed in diabetes and/or obesity. New experimental data are required to strengthen the core mechanistic components of the proposed model and to demonstrate its relevance to disease pathophysiology.\nMajor Comments\n1. Are there any alterations in CBP, p300 and/or Slc7a2 expression in alpha-cells in animal models of diabetes and/or obesity/metabolic associated liver dysfuncton(MASLD) and even more important in human subjects? Of note, alpha-cell dysfunction was present only under conditions of complete ablation of both CBP and p300, suggesting that the activity of these proteins is partially redundant and dispensable, therefore it is unlikely that partial inhibition of these genes in obesity or diabetes (if exist) is sufficient to induce alpha-cell dysfunction, arguing against the suggestion that this pathway is involved in disease pathophysiology.\nResponse: This is a very good question. To directly address this issue, we first examined whether CBP, p300, or Slc7a2 expression is altered in α cells under diabetic or obese conditions. Single-cell RNA sequencing data and immunofluorescence staining revealed comparable gene and protein expression intensities of CBP, p300, and Slc7a2 in the α cell of db/db mice, high-fat diet (HFD)-fed mice, and type 2 diabetes mellitus (T2DM) human islets compared to corresponding controls (Extended Data Fig.3a, 3e-j). Similarly, no significant alterations in the expression of CBP, p300, or Slc7a2 were observed at either the mRNA or protein level in bulk islets from T2DM patients (Extended Data Fig.3b-d), according to human islet data from HumanIslets.com [8]. These results indicate that the expressions of CBP, p300, and Slc7a2 in α-cell are preserved across metabolic disease states and pathogenic activation of this pathway is unlikely to arise from altered gene abundance alone. Rather, our α cell-specific CBP/p300 knockout model illuminates that this signaling module is nutrient-responsive, pointing toward substrate-driven activation as the more plausible mechanism in metabolic diseases. Supporting this interpretation, our previous work in the 4C cohort, a large-scale human metabolomic study demonstrated a strong correlation of 2-aminoadipic acid (2-AAA), a catabolite of lysine, with the incidence of type 2 diabetes [9]. In our separate study, we further showed that 2-AAA promoted hepatic gluconeogenesis through CBP/p300-mediated transcriptional activation of gluconeogenic genes [10]. Therefore, it is likely that excessive amino acid flux stimulates α cell hyperfunction through intact CBP/p300-SLC7A2 axis, contributing to the subsequent elevation of blood glucose.\nExtended Data Fig. 3. CBP, p300 and Slc7a2 expression levels in alpha cells of several pathological models. (a) Violin plots showing CBP, p300 and Slc7a2 expressions in α cells in 4-week-old db/db and db/dm mice. (b-d) The gene and protein expression levels of CBP, p300, or Slc7a2 in bulk islets from T2DM patients. (e-g) Representative immunofluorescence images of pancreatic islets showing glucagon (Gcg, green), CBP, p300 or SLC7A2 (red), and nuclei (DAPI, blue) in 4-week-old db/db mice (e), 16-week HFD-fed mice (f), and T2DM patients (g). (h-j) Mean fluorescence intensities (MFI) of CBP, p300, and SLC7A2 in α cells in Extended Data Fig.3e-g (n = 3). Scale bar, 10 μm. Data are presented as mean ± SEM.\n2. Glucagon deficiency affected amino acids degradation, but not hepatic glucose production, evident by similar fasting glucose, pyruvate tolerance and expression of gluconeogenic enzymes. Are mouse models of glucagon deficiency relevant for studying the role of glucagon in human physiology? Please explain.\nResponse: Thank you for highlighting this important point. Although glucagon is well known for its rapid glucose-elevating action and clinical use in treating severe hypoglycemia, we did not observe decreased fasting glucose level in CBP/p300αDKO mice despite their hypoglucagonemia. Similar findings have been reported in other models: (i) α-cell ablation using diphtheria toxin in embryos or adult mice minimally affects fasting glucose [11-13], and (ii) Gcg knockout mice also maintain normal fasting glucose levels [14]. These findings suggest that basal hepatic glucose production can be preserved through residual glucagon secretion and compensatory mechanisms that maintain glucose homeostasis. In contrast, amino acid dysregulation is a highly penetrant and conserved hallmark of glucagon deficiency. Elevated circulating amino acid levels have been consistently observed in Gcg knockout, Gcgr knockout, and GCGR antibody blockade mouse models [15]. Li Hong et al. reported a patient with a homozygous GCGR c.958_960del (p.Phe320del) variant who exhibited normal blood glucose level, but persistent hyperaminoacidemia [16]. Patients undergoing pancreatectomy likewise develop severe hyperaminoacidemia that can be alleviated by glucagon administration, whereas individuals with glucagon-producing tumors exhibit profound hypoaminoacidemia [17]. A practical extension of this conserved biology is that plasma amino acid concentrations are now used clinically as pharmacodynamic readouts of GCGR activity in trials of dual (glucagon/GLP-1) or triple (glucagon/GLP-1/GIP) receptor agonists [18].\n3. The authors argue that SLC7A2/arginine, rather than SLC38A5/glutamine regulates mTORC1 and autophagy. Previous studies showed that GCGR antagonism induces alpha-cell proliferation through glutamine transport via SLC38A5, thereby stimulating mTORC1. The authors found that the expression of both Slc7a2 and Slc38a5 decreased in CBP/p300 dKO mice. They should add control experiments of Slc38a5 knockdown and/or over-expression and study the effects on mTORC1 activity and autophagy in alpha-cells to convincingly show that the SLC7A2 transporter is indeed the main regulator of mTORC1-autophagy signaling in alpha-cells. The authors over-expressed Slc7a2 in islets and claim that this enhanced arginine stimulation of mTORC1 (Fig. 7d). This statement is not entirely supported by the data: Slc7a2 over-expression increased mTORC1 activity in absence of arginine, while the response to arginine was in fact abrogated. Fig. 7b-e lack quantifications, and the number of repeated experiments is not mentioned, therefore it is difficult to interpret the findings.\nResponse: We thank you for this excellent and important comment. Our single-cell RNA sequencing analysis revealed that Slc7a2 exhibited high and widespread expression across nearly all WT pancreatic α cells, whereas Slc38a5 was expressed only in a small subset of α cells and at much lower levels (Extended Data Fig.4a-c), consistent with prior studies [2, 13, 19]. Moreover, in α-cell-specific Slc38a5 knockout mice, reduced α-cell proliferation and mass are observed only under GCGR blockade, with no alterations under physiological conditions [2]. Given the robust and ubiquitous expression of Slc7a2 in α cells and the limited prior research, we therefore focused our investigation on this transporter. Although Slc38a5 expression was also reduced in the knockout mice, its low basal expression suggests it is unlikely to be the major contributor under normal conditions; however, a potential auxiliary role cannot be ruled out. We have revised this part in discussion.\nRegarding Fig.7d, a careful review of our experimental records revealed a possibility that the use of DMEM medium led to basal mTORC1 activation in the Slc7a2-overexpressing cells even without additional arginine stimulation. To eliminate this confounding factor, we repeated the experiment using amino acid-free HBSS prior to arginine treatment. Under these corrected conditions, Slc7a2 overexpression clearly enhanced arginine-induced mTORC1 activation (Revised Fig.7d). We sincerely thank you for highlighting this issue. Quantifications for Figure 7b-e (based on n=3 independent experiments) have been incorporated to Supplementary Fig.7a-d.\nExtended Data Fig. 4. Slc7a2 is highly and broadly expressed in wild-type mouse pancreatic α cells. (a-b) UMAP visualization of scRNA-seq data showing the expression of Slc7a2 and Slc38a5 in wild-type (WT) α cells. (c) Scatter plot of Slc7a2 and Slc38a5 expressions in WT α cells. (Supplementary Fig.8)\nExtended Data Fig. 5. Effect of Slc7a2 overexpression on S6 phosphorylation level. Western blot analysis of S6 phosphorylation level in α-TC1-6 cells treated with arginine after Slc7a2 overexpression (Ad Slc7a2). (Revised Fig.7d)\nExtended Data Fig. 6. Quantification of data shown in Figure 7b–e. (a) Quantification of pS6+ Gcg+ /Gcg+ α cells in mouse islets cultured in DMEM, HBSS, or arginine-enriched medium (n = 3 independent experiments). (b-d) Band intensities of pS6ser240/244 normalized to total S6 in α-TC1-6 cells under the indicated conditions (n = 3 independent experiments). Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD test: ***P < 0.001; ###P < 0.001. (Supplementary Fig.7a-d)\n4. The data on autophagy are not convincing. The few autophagy genes shown in Figure 4i are involved in regulating lysosome functon (cathepsins) and Golgi-localized transmembrane localization (Temen59); these are not \"classical\" regulators of autophagy. The same is true for many of the genes shown in Fig. 6g-i. A previous study suggested that in alpha-cells inhibiting mTORC1 induces crinophagy, rather than autophagy. Does aCBP/p300 dKO affect crinophagy? Prompt assessment of autophagic flux requires analysis of LC3 conversion to LC in cells treated with or without a lysosome hydrolases inhibitor, such as chloroquine (as in Fig. 6k) and/or testing the protein level of P62/SQTM1. In Fig. 6j (Slc7a2 knockdown experiment), the authors don't show the effects of Slc7a2 knockdown on autophagic flux. Fig. 6j-k lack quantifications, and the number of repeated experiments is not indicated.\nResponse: We sincerely thank you for these valuable suggestions. According to your suggestions, we expanded our analysis to include classical autophagy-related genes. In the revised Fig.4l (see also Extended Data Fig.7) and revised Fig.6g-i (see also Extended Data Fig.8a-c), we present the expression patterns of core autophagy machinery genes (e.g., Map1lc3a, Map1lc3b, Gabarap, Gabarapl2, Rab7, Vamp8, and Lamp2) under amino acid starvation and CBP/p300 inhibition.\nTo address whether CBP/p300 deletion affects crinophagy, we re-analyzed scRNA-seq results and found that Rab7, a marker of crinophagy due to its essential role in mediating lysosome-secretory granule fusion [20], was upregulated in CBP/p300αDKO α cells (Extended Data Fig.9a). In addition, the expression levels of multiple canonical autophagy genes (e.g., Map1lc3a, Map1lc3b, Atg5, Atg10) were also significantly elevated (Extended Data Fig.9b-e). Given the concurrent induction of Rab7 and autophagy components, it is possible that both crinopghagy and autophagy occur in CBP/p300-deficient α cells.\nAs suggested, we performed additional experiments to provide stronger support for our conclusion in the revised Figure 6j (see also Extended Data Fig.10). The quantifications of Figure 6j-k (n = 3 independent experiments) have been added in Supplementary Fig.6b-c (see also Extended Data Fig.11).\nExtended Data Fig. 7. Autophagy-related gene expression patterns across α-cell subpopulations in CBP/p300αDKO mice. Dot plot showing the expression levels of classical autophagy-related genes in GCG, both, tdTomato, and NONE α cells. Dot size represents the percentage of cells expressing each gene, and dot color represents z-score of gene expression. (Revised Fig.4l)\nExtended Data Fig. 8. Autophagy-related gene expressions in α-TC1-6 cells under the conditions of amino acid starvation, Slc7a2 silencing, and CBP/p300 inhibition. Relative mRNA expression of autophagy-related genes in α-TC1-6 cells treated with HBSS (a), Slc7a2 silencing (b), or 3 μM A-485 (c) (n = 4). Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01. (Revised Fig.6g-i)\nExtended Data Fig. 9. Expression of crinophagy- and autophagy-related genes in WT and CBP/p300αDKO α cells. (a-e) Violin plots and UMAP feature plots showing the expression of Rab7 (a), Map1lc3a (b), Map1lc3b (c), Atg5 (d), and Atg10 (e) in α cells from WT and CBP/p300αDKO mice.\nExtended Data Fig. 10. Western blot analysis of LC3 conversion (LC3-I to LC3-II) in α-TC1-6 cells after Slc7a2 silencing. Chloroquine (CQ) (20 μM), an inhibitor of autophagic flux, was used to block LC3B degradation by inhibiting autolysosomal acidification. (Revised Fig.6j)\nExtended Data Fig. 11. Band intensities of LC3 II normalized to HSP90 in α-TC1-6 cells under the indicated conditions (n = 3 independent experiments) in Figure 6j-k. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD test: ***P < 0.001; ###P < 0.001. (Supplementary Fig.6b-c)\n5. mTORC1 regulates autophagy via phosphorylation of ULK1 and TFEB. Does treatment with arginine, Slc7a2 knockdown and inhibition of CBP/p300 affect ULK1 and TFEB phosphorylation. This may strengthen the conclusion that CBP/p300-SLC7a2 signaling regulates autophagy via mTORC1.\nResponse: Thank you for your valuable and detailed suggestion. Western blots showed that arginine increased the phosphorylation levels of ULK1 and TFEB in α-TC1-6 cells, which was inhibited by Slc7a2 knockdown or CBP/p300 inhibitor A-485 (Extended Data Fig.12a-b).\nExtended Data Fig. 12. Slc7a2 knockdown and CBP/p300 inhibition decrease arginine-stimulated ULK1 and TFEB phosphorylation. (a) Western blot analysis of ULK1 and TFEB phosphorylation levels in α-TC1-6 cells treated with arginine after Slc7a2 knockdown (sh Slc7a2). (b) Western blot analysis of ULK1 and TFEB phosphorylation levels in α-TC1-6 cells treated with arginine in the presence or absence of A-485 (3 μM).\n6. The authors suggest that aCBP/p300 dKO induces autophagic death, however no data are shown throughout the manuscript to support this hypothesis. Autophagy might be an adaptive response to stress and doesn't necessarily induce cell death.\nResponse: We thank you for pointing out this concern. To avoid overstating our findings, we removed all references to “autophagic death” throughout the manuscript and revised the text to ensure that no causal claim between autophagy and cell death is implied.\n7. In the morphometric and lineage trace experiments shown in Figs. 2-3, at the age of 8 weeks, the alpha-cell number in CBP/p300 dKO mice was decreased by ~90%, whereas alpha-cell dedifferentiation (tdT tomato+Gcg+/TdT+ cells) occured in ~66% of the cells. The residual alpha-cell loss may be explained by reduced proliferation, which was observed during the early neonatal period and/or alpha-cell death. I would suggest assessing alpha-cell proliferation and apoptosis at the age of 4 weeks and 8 weeks to quantify the contribution of alpha-cell dedifferentiation, decreased proliferation and cell death to alpha-cell loss in aCBP/p300 dKO mice.\nResponse: We appreciate this constructive criticism. According to your suggestion, we quantified α-cell proliferation (Ki67) and apoptosis (cleaved-Caspase 3) at 4 and 8 weeks in control and CBP/p300αDKO mice. Our results revealed that α-cell proliferation rates in control mice were inherently low at both time points. Although α-cell proliferation in CBP/p300αDKO mice is reduced compared to controls at 4 weeks, the low baseline proliferation rate suggests that decreased proliferation plays a minor role in the observed reduction of α-cell numbers in the knockout mice (Extended Data Fig. 13a). Additionally, we found no increase in α-cell apoptosis in CBP/p300αDKO mice at either 4 or 8 weeks (Extended Data Fig. 13b), indicating that other forms of cell death may contribute to the decline in α-cell numbers. The result of α-cell apoptosis has been added in Supplementary Fig. 2d.\nExtended Data Fig. 13. Effects of CBP/p300 knockout on alpha-cell proliferation and apoptosis. (a-b) The proliferation (Ki67) and apoptosis (cleaved-Caspase 3, CC3) rates at 4 and 8 weeks in control and CBP/p300αDKO mice (n = 3). Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test, compared with WT: ***P< 0.001. (Supplementary Fig.2d)\n8. In Fig. 3f, ~10% of control alpha-cells are stained positive for glucagon and PPY; this seems to be exaggerated. In the aCBP/p300 dKO, the percentage of Ppy+/tdT tomato+ cells was markedly decreased. These findings are puzzling. The colocalization images (Fig. 3e-f) are not convincing; quantification of colocalization on z-stack images would be valuable.\nResponse: We sincerely thank you for this valuable suggestion. This result is comparable with findings from the study by Shiota et al., in which nearly 9 – 16% RFP+ cells are also Ppy-positive in alpha-cells from adult Tamoxifen-treated Gcg CreERT2/w;R26 tdTomato/w mice [1]. Our data analysis revealed that approximately 10% of cells in the control group were tdTomato⁺Ppy⁺. In contrast, tdTomato⁺ cells exhibited significantly elevated cell dedifferentiation in double knockout mice. It is likely that the tdTomato⁺Ppy⁺ subpopulation undergoes even greater cell dedifferentiation, resulting in a relative reduction in its proportion compared to controls. In addition, we are sorry for the confusing of Fig.3e-f. We have performed immunofluorescence staining on 5-μm pancreatic sections using an optimized experimental protocol (Revised Fig.3e). The results are more clearly shown in this version.\nExtended Data Fig. 14. Representative immunofluorescence images showing hormone expression (Ins, Ppy, Sst) in tdTomato+ cells from WT and CBP/p300αDKO mice. Yellow rectangles indicate hormone-positive tdTomato+ cells. Scale bar, 10 μm. (Revised Fig.3e)\n9. The authors suggest that hypoglucagonemia is mainly driven by alpha-cell deficiency. It is possible that CBP/p300 deficiency also affects the machinery of glucagon secretion. Ex vivo static incubations testing the glucagon response of aCBP/P300 dKO islets to secretagogues, such as amino acids would clarify this point.\nResponse: According to your suggestion, isolated islets from control and CBP/p300αDKO mice were incubated with 1mM glucose (1G), 5mM glucose (5G) and 10mM arginine (Arg). CBP/p300αDKO islets showed significant decreases in glucagon secretion under various conditions. The data were added in Supplementary Fig.4 (See also Extended Data Fig.15)\nExtended Data Fig. 15. Glucagon secretion is impaired in the islets from CBP/p300αDKO mice. Glucagon secretion from isolated islets of WT or CBP/p300αDKO mice in response to 1G (1 mM glucose), 5G (5 mM glucose) or 5G + 10 mM Arginine (Arg) (n = 3). Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. Compared with WT group: **P < 0.01, ***P < 0.001. (Supplementary Fig.4)\n10. In Figure 5, the authors show that downregulation of Slc7a2 was associated with loss of alpha-cell identity genes and that pharmacological inhibition of histone acetyl transferase recapitulate these findings, suggesting that CBP/p300-driven H3K27 acetylation at the promoter of key alpha-cell genes mediate alpha-cell dysfunction. The expression of several amino acid transporters, including Slc7a2, Slc38a4 and Slc385 was decreased – are these alterations also mediated via CBP/P300 binding to promoters? Add quantifications to Fig. 5d–f.\nResponse: Thank you for your suggestions. We analyzed H3K27Ac ChIP-seq and chromatin accessibility (ATAC-seq) data of mouse pancreatic islets and related cell lines using the ChIP-Atlas database [21]. The results are summarized as follows:\nFor Slc7a2, a strong H3K27Ac enrichment peak and an open chromatin region were observed in pancreatic islets spanning approximately 300 bp upstream to 800 bp downstream of the transcription start site (TSS) (Extended Data Fig.16a). For Slc38a4, a clear H3K27Ac peak and open chromatin signal were detected in islets within approximately 200 bp upstream to 100 bp downstream of the TSS (Extended Data Fig.16b). In contrast, across the entire Slc38a5 gene locus in islets, open chromatin regions were completely absent (Extended Data Fig.16c). However, we did not find the direct bindings of CBP/p300 to the promoters of Slc7a2, Slc38a4, and Slc38a5 by ChIP-qPCR in α-TC1-6 cells. As suggested, the quantifications of Fig.5d–f were added to Supplementary Fig.5b-d (See also Extended Data Fig.17a-c).\nExtended Data Fig. 16. (a-c) Genome browser tracks showing H3K27Ac ChIP-Seq and ATAC-Seq signals at the Slc7a2 (a), Slc38a4 (b) or Slc38a5 (c) loci in mice islets based on the data from ChIP-Atlas database. Red rectangle indicate region with strong H3K27Ac enrichment peak and an open chromatin region in the promoters of Slc7a2 or Slc38a4 in mice islets.\nExtended Data Fig. 17. (a) Quantification of average SLC7A2 MFI in α cells in WT and CBP/p300αDKO mice (n = 5). (b) Quantification of tdTomato+ MafB+ cells among total tdTomato+ cells in WT and CBP/p300αDKO mice (n = 5). (c) Quantification of tdTomato+ H3K27Ac+ cells among total tdTomato+ cells in WT and CBP/p300αDKO mice (n = 5).（Supplementary Fig.5b-d）\n11. The authors suggest that lysine and its metabolite acetyl CoA mediates SLC7A2 epigenetic regulation, whereas arginine mediates the effects on mTORC1 activity and autophagy. However, it has been shown that mTORC1 is also regulated by acetyl CoA through Raptor acetylation. Does lysine affect mTORC1 activity and autophagy?\nResponse: Thank you for your constructive criticism. Lysine treatment had no effect on the phosphorylation level of S6 in α-TC1-6 cells (Extended Data Fig.18a). Moreover, no significant changes in autophagy-related gene expressions were observed in α-TC1-6 cells treated with lysine (Extended Data Fig.18b).\nExtended Data Fig. 18. Lysine treatment has no effect on mTORC1 signaling in α-TC1-6 cells. (a) Western blot analysis of pS6ser240/244 in α-TC1-6 cells in the presence or absence of 10 mM lysine. (b) Relative mRNA expression of autophagy-related genes (Map1lc3a, Map1lc3b, Gabarap, Gabarapl2, Rab7, Vamp8, Lamp1, Lamp2) in α-TC1-6 cells treated with 10 mM lysine (n =3). Data are presented as mean ± SEM.\nMinor comments\n1. In Fig. 1b-g-Please specify the age of the animals that were studied.\nResponse: As suggested, we added the ages of mice (8 weeks) in figure legend.\n2. In Fig. 4c, Ins1/Ins2 gene expression appears high in delta-cells, which is unexpected. Please explain.\nResponse: We apologize for this oversight, and we thank you for pointing it out. The high Ins1/Ins2 signal in δ-cells was due to an error in the original plot. We have corrected this error and updated the heatmap in the revised Figure 4c (see also Extended Data Fig.19).\nExtended Data Fig. 19. Heatmap of Z-scores for key marker genes across different cell clusters, demonstrating cell-type-specific gene expression patterns. (Revised Fig.4c)\n3. In fig 6e, the yellow arrow annotation may point to an insulin granule, rather than an autophagosome, raising questions regarding the correct identification of alpha-cells vs beta-cells. Please annotate cells and organelles with reference to https://doi.org/10.1369/0022155415583535.\nResponse: Thank you for your constructive criticism. As suggested, we have annotated cells and organelles in Revised Fig.6e (see also Extended Data Fig.20).\nExtended Data Fig. 20. (a) Representative transmission electron microscopy (TEM) images of α cells in islets from WT and CBP/p300αDKO mice, showing autophagosomes and autolysosomes. Yellow arrows indicate autophagosomes. m: mitochondrial, n: nucleus, Scale bar, 500 nm. (b) Representative transmission electron microscopy (TEM) images of α cells and β cells in islets from WT mice. m: mitochondrial, n: nucleus, α: α cells, β: β cells. Scale bar, 500 nm. (Revised Fig.6e and Supplementary Fig.6a)\n4. Line 302: specify mTORC1 reduction is in alpha-cells.\nResponse: As suggested, we added the description in the revised manuscript.\n5. Line 303: “low phosphorylation of S6 by S6K,” not phosphorylation of S6K.\nResponse: We apologize for this mistake and correct it in the revised manuscript.\n6. Fig. 7o–p: text mentions intensity, graph shows % positive cells.\nResponse: Our apologies for this glitch. We corrected this sentence in the new version.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We are sincerely grateful to you for your time, insightful comments, and constructive feedback on our manuscript.\nReviewer #4 (Remarks to the Author):\nWang et al. use Gcg-Cre to delete the histone acetyltransferases p300 and CBP in pancreatic α cells. The KO shows a partial reduction in circulating glucagon and modest increases in several plasma amino acids, which the authors attribute to loss of α cell identity, reduced α cell mass and proliferation, arising from diminished α-cell gene expression. They nominate Slc7a2 as a mediator, and that the reduced Slc7a2 leads to reduced H3K27Ac at the Gcg and Mafb loci and their gene downregulation, and a failure to mount α-cell hyperplasia after anti-Gcgr treatment.\nThe findings on α-cell identity and proliferation overlap with prior Neurog3-Cre genetics showing that p300/CBP are required for islet endocrine cell mass and proliferation. Please cite and discuss this study, since the overlap limits the incremental advance. A significant issue is that most data shown are underpowered, with experiments from Figures 1c, 2, 3, 5, and 6 presented at n = 3 and no justification of sample size.\nFor the core narrative, the authors argue that loss of p300/CBP downregulates Slc7a2, which then maintains α-cell identity through H3K27Ac at Gcg and Mafb, and amino acid-driven proliferation. This interpretation does not fully acknowledge that p300/CBP are fundamental drivers of tissue-specific gene expression that establish endocrine identity. It is therefore equally plausible that Slc7a2 downregulation is a downstream consequence of identity erosion rather than the primary cause. To support causality, one would need in vivo evidence that Slc7a2 loss is sufficient to reduce H3K27Ac at Gcg and Mafb with concordant transcriptional downregulation. A recent Slc7a2-/- report does not show loss of α-cell identity. For proliferation, α cells lacking p300/CBP already show impaired proliferation, so the lack of an amino acid response may reflect a general failure to traverse the cell cycle, in addition to any defect in amino acid sensing.\nResponse: We appreciate your detailed review and valuable suggestions, which have been instrumental in improving the quality of our paper. In the revised manuscript, we cite and discuss the Neurog3-Cre study. Mice lacking all four copies of p300/CBP in pancreatic endocrine progenitors failed to establish ɑ- and β-cell mass postnatally. This innovative study reveals the pivotal role of p300/CBP in pancreatic islet development and provide valuable insights for our research, yet notable differences exist between the studies. Firstly, the referenced study utilized the Ngn3-Cre line, which targets endocrine progenitors during pancreatic development. As Ngn3 expression peaks during embryogenesis (E9.5–E15.5), this model is well-suited for investigating islet cell differentiation and development. Ngn3-Cre-mediated p300/CBP knockout occurs prior to pancreatic α-cell formation, potentially impacting α-cell development. In contrast, our study employed Gcg-Cre to specifically target glucagon-expressing α-cells, focusing on the role of p300/CBP in these cells. Additionally, we used Gcg-CreERT mice to examine the effect of p300/CBP on mature α-cells. Secondly, the Ngn3-Cre model displayed glucose homeostasis dysregulation due to impaired β-cell function and mass, with limited exploration of p300/CBP’s role in α-cells. In the study of Wong et al., Ngn3-Cre-driven pancreatic islet-specific p300 knockout mice displayed reduced proliferation of neonatal α-cells and β-cells. Mice lacking all copies of p300/CBP died postnatally, which prevents further investigation. In our study, the expansion and maintenance of postnatal α cell number were normal in single-gene knockout (CBPαKO, p300αKO) or triple-allele knockout (CBPαKO; p300HE, CBPHE; p300αKO) mice, while α-cell numbers were unchanged at postnatal day 0 (P0) in CBP/p300αDKO mice, with a decline in proliferation observed postnatally. Furthermore, GCGR blockade did not induce α-cell hyperplasia. Using lineage-tracing mice, α-cell specific double knockout of CBP/p300 led to loss of α-cell identity and increased cell death, further highlighting distinct mechanisms in our model compared to the Ngn3-Cre-based approach. Sample sizes were increased to 4-5 per group according to your suggestion.\nOur in vivo and in vitro experiments identified CBP and p300 as the critical regulators of α cell identity, autophagy, and proliferation. Single-cell RNA sequencing revealed a high and widespread expression of Slc7a2 in WT pancreatic α cells and significant downregulation in CBP/p300αDKO α cells. Further in vitro experiments demonstrated the effect of CBP/p300 on Slc7a2 expression and the vital role of Slc7a2 in regulating α cell identity and autophagy-related gene expressions. Moreover, overexpression of Slc7a2 could partially rescue the reduced proliferation caused by CBP/p300 deficiency in α cells. As a result, it is reasonable to suppose that CBP/p300-SLC7A2 axis is critical for the expansion and maintenance of functional pancreatic α cell mass.\nWe agree that p300/CBP loss likely causes broad disruptions in cell cycle regulation, given their role in orchestrating transcriptional programs. However, our study specifically highlights the diminished proliferative response to amino acid stimulation in p300/CBP-deficient α-cells, which correlates with Slc7a2 downregulation. This observation suggests that Slc7a2-mediated amino acid signaling is a critical component of the proliferative defect. Supporting this, our GCGR blockade model failed to induce α-cell hyperplasia, consistent with impaired amino acid-driven proliferation in the absence of p300/CBP. We are grateful for your critical insights, which prompts us to refine our interpretations and design targeted follow-up experiments to strengthen our conclusions.\nSpecific comments\n1. The Gcg-Cre driver is active in pancreatic α cells, intestinal L cells, and preproglucagon neurons. Did p300/CBP loss in gut and brain influence oral glucose tolerance or energy metabolism?\nResponse: We appreciate this constructive criticism. We agree that potential off-target effects in other Gcg-expressing cells, such as intestinal L-cells and GCG+ neurons in brain, should be evaluated to ensure that the phenotype is primarily α-cell specific. To address this issue, we performed additional experiments. Intestinal co-expression of GLP-1 and the tdTomato in Gcg-Cre-driven tdTomato lineage-tracing WT and CBP/p300αDKO mice confirms that Gcg-Cre is active in the intestine (Extended Data Fig.21a). However, the average GLP-1+ cell number per tissue section and plasma levels of active GLP-1 showed no differences between control and CBP/p300αDKO mice (Extended Data Fig.21b-c). Besides, CBP/p300αDKO mice exhibited oral normal glucose tolerance (Extended Data Fig.21d). These above results suggest that intestinal enteroendocrine cells expressing GLP-1 are conserved, perhaps due to the robust regenerative potential of L cells by neogenesis as reported by Shiota et al [1]. Using immunofluorescence staining, we confirmed glucagon-Cre-mediated recombination in neurons of the nucleus tractus solitarius (NTS) (Extended Data Fig.21e). The PPG neurons of NTS have been reported to regulate appetite and energy metabolism [22, 23], but we observed no significant alterations in food intake and energy expenditure in CBP/p300αDKO mice (Extended Data Fig.21f, g), suggesting minimal impact on the central nervous system. These data support that the observed hypoglucagonemia and hyperaminoacidemia are attributed to pancreatic α-cell defects, which have been added to Supplementary Figure 1 and the Result section.\nExtended Data Fig. 21. Effects of Gcg-Cre recombination in the intestinal L-cells and central nervous system neurons. (a) Representative immunofluorescence images of intestinal L-cells from WT and CBP/p300 αDKO mice. Co-staining for tdTomato (red) and GLP-1 (green), with nuclei labeled by DAPI (blue). Yellow arrows indicate double-positive cells. Scale bar, 10 μm. (b) Quantification of GLP-1+ cells per intestinal section in WT and CBP/p300αDKO mice (n = 3 mice per group). (c) Plasma levels of active GLP-1 in 8-week-old WT and CBP/p300αDKO mice (n = 5 mice per group). (d) Oral glucose tolerance test (OGTT) in WT and CBP/p300αDKO mice (n = 5 mice per group), with blood glucose measured at the indicated time points. (e) Representative immunofluorescence images of Glucagon-Cre recombination in neurons of the nucleus tractus solitarius (NTS) using a reporter transgenic mouse CAG-tdTomato. Co-staining for tdTomato (red), CBP or p300 (green), with nuclei labeled by DAPI (blue). Yellow arrows indicate tdTomato-positive cells. 4V: fourth ventricle. Scale bar, 10 μm. (f) Daily food intake in 8-week-old WT and CBP/p300αDKO mice (n = 6 mice per group). (g) The Respiratory Exchange Ratio (RER) was measured in 8-week-old WT and CBP/p300αDKO mice (n = 6 mice per group). (Supplementary Fig.1)\n2. Did the authors measure glucagon secretion from intact islets, and is insulin secretion altered in vivo or ex vivo?\nResponse: Thank you for your constructive criticism. According to your suggestion, we have performed ex vivo secretion assay using intact islets from adult WT and CBP/p300αDKO mice. Glucagon secretion was reduced in islets of CBP/p300αDKO mice (Extended Data Fig.22a) while the plasma insulin level was not altered in vivo (Extended Data Fig.22b).\nExtended Data Fig. 22. Glucagon secretion is impaired in islets isolated from CBP/p300αDKO mice. (a) Glucagon secretion from isolated islets of WT or CBP/p300αDKO mice in response to 1 mM glucose (1G), 5 mM glucose (5G) or 5G + 10 mM Arginine (Arg) (n = 3). (b) Fasting blood insulin levels in 8-week-old WT and CBP/p300αDKO mice (n = 6). Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. Compared with WT group: **P < 0.01, ***P < 0.001. (Supplementary Fig.4, Supplementary Fig.1h)\n3. For Figures 2b and 3c, please report absolute values in addition to fold change.\nResponse: As suggested, we added the absolute values for Fig.2b and 3c in Supplementary Fig.2a and Supplementary Fig.3c (see also Extended Data Fig.23a, b).\nExtended Data Fig 23. (a) Average Gcg+ cell number per pancreatic section in WT and CBP/p300αDKO mice over time (n = 5 mice per group). (b) Average tdTomato+ cell number per pancreatic section in WT and CBP/p300αDKO mice over time (n = 4 - 5 mice per group). Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. Compared with WT group: ***P < 0.001. (Supplementary Fig.2a, Supplementary Fig.3c)\n4. The cell death data regarding tdTomato+ cell loss should be presented as supplementary data. The authors cannot claim cell death without direct experimental evidence.\nResponse: We thank you for your suggestion. We deleted all the description of autophagy-related death in the revised manuscript as suggested. Aa the loss of tdTomato+ cells is a main feature of CBP/p300αDKO mice, we do not remove this result to supplementary data.\n5. Line 195 states that ‘islets with high tdTomato expression in α cells were isolated… and subjected to scRNA-seq,’ yet the dataset is majority Ins+ cells. Please clarify whether single tdTomato+ cells were fluorescence-activated cell sorted after dissociation. If so, please discuss why the single cell data does not show enriched α cells.\nResponse: We appreciate your careful work and apologize for the inaccurate description in the original manuscript. We did not perform fluorescence-activated cell sorting of single tdTomato+ cells. Instead, intact islets were isolated from two groups of mice and directly subjected to scRNA-seq analysis. This approach explains why the dataset predominantly contains Ins+ cells rather than enriched α cells. The description has been corrected in the revised manuscript to avoid any misunderstanding.\n6. Please explain why pseudotime was not computed on a single integrated dataset containing both WT and KO tdTomato+ cells. Figures 4b and 4d appear to analyze only the WT cells, whereas the pseudotime analysis was performed only on the knockout cells. Joint integration followed by trajectory inference is a standard way to place both genotypes on the same manifold so that differences can be attributed to biology.\nResponse: We thank you for this insightful comment. We initially avoided computing pseudotime on a single integrated dataset because tdTomato+ cells reflect different biological states in WT (normal α cells) versus CBP/p300αDKO mice (α cells with Crebbp/Ep300 deletion), which could confound trajectory inference. Nevertheless, as suggested, we performed an integrated Slingshot trajectory analysis combining both WT and CBP/p300αDKO α cells (Extended Data Fig.24a-c). The integrated results showed similar gene expression dynamics and support the conclusions of our original analysis.\nExtended Data Fig. 24. Integrated trajectory analysis of α cells from WT and CBP/p300αDKO mice. (a) UMAP visualization of integrated α cells from WT and CBP/p300αDKO mice, revealing six transcriptionally distinct clusters. (b) UMAP trajectory showing pseudotime progression of α-cell populations from WT and CBP/p300αDKO mice. (c) Module-based hierarchical clustering of differentially expressed genes across α-cell populations, revealing distinct gene modules with characteristic expression dynamics.\n7. In Figure 4g, the inferred trajectory appears to terminate in the cluster that shows high Gcg expression in Figure 4i, yet in Figure 4h the Gcg signal does not peak at the end of the trajectory. In addition, Figure 4i appears to combine wild type and knockout cells, whereas Figures 4g and 4h do not. Please clarify.\nResponse: We thank you for this thoughtful comment. The apparent discrepancy arises from differences in data display between panels. Figure 4h shows normalized relative expression, which emphasizes inter-gene differences, whereas Figure 4i presents absolute expression levels, where any detectable signal is rendered in red. Thus, Gcg expression remains detectable at the trajectory endpoint in Figure 4i, albeit at low levels and intermixed with a subset of Gcg-negative cells. We have optimized the color scale in Revised Fig.4i to improve clarity (Extended Data Fig.25). Regarding the inclusion of WT cells, our intention was to overlay WT α cells in gray as a reference map to illustrate the altered clustering structure of CBP/p300αDKO α cells. Both Gcg+ and Gcg– subpopulations exist within the KO cells. We have clarified this annotation in the revised figure legend.\nExtended Data Fig. 25. UMAP feature plots illustrating Gcg and tdTomato expression in CBP/p300αDKO α cells. UMAP feature plots showing the expression patterns of Gcg, tdTomato, and their co-expression in CBP/p300αDKO α cells. WT α cells were overlaid in gray to serve as a reference map for the normal α-cell distribution. The color scale represents normalized expression intensity for each gene. (Revised Fig.4i)\n8. Please define the thresholds used to call ‘Gcg+’ and ‘Gcg-’ in the single-cell data (for example unique molecular identifier cutoffs, scaled expression, or module scores), and state where this is documented in the Results.\nResponse: We thank you for this valuable comment. Gcg+ and Gcg– cells were defined following the strategy of Perez-Frances et al. [24], using normalized expression values and Pareto density-defined cutoffs to distinguish hormone-positive from hormone-negative cells. Cells with normalized Gcg expression above the Pareto-derived threshold were classified as Gcg+, and those below as Gcg-. The same approach was applied to define tdTomato+ and tdTomato- cells. The cutoff values are shown in Extended Data Fig.26a-b (Gcg, 4.384858; tdTomato, 2.281258), and the processed matrices and thresholds are provided in Extended Table 1. This criterion is now explicitly described in the Methods section of the revised manuscript.\nExtended Data Fig. 26. Determination of expression thresholds for Gcg and tdTomato in scRNA-seq. (a, b) Pareto density plots illustrating the distribution of normalized expression values for Gcg (a) and tdTomato (b) in α cells from CBP/p300αDKO mice. Vertical green lines indicate the cutoff values (4.384858 for Gcg; 2.281258 for tdTomato) used to classify cells as positive (Gcg⁺, tdTomato⁺) or negative (Gcg⁻, tdTomato⁻).\n9. In Figure 4i, z-score dot plots can exaggerate differences across genes with narrow dynamic ranges. Please use violin plots or dot plots of normalized expression so readers can see absolute distributions. This information should be presented as early as possible, since as of now it is obscure to interpret the effect size of the α cell gene downregulation.\nResponse: We appreciate this constructive suggestion. We agree that z-score dot plots can exaggerate differences for genes with narrow dynamic ranges. To address this, we added violin plots showing normalized absolute expression values of key α-cell genes (Extended Data Fig.27a), which allow direct visualization of their distributions across subpopulations. We also provide the corresponding z-score dot plots (Extended Data Fig.27b). Importantly, both representations reveal consistent expression trends. We believe this addition substantially improves the clarity of data presentation.\nExtended Data Fig. 27. Absolute and normalized expression patterns of key genes across α-cell subpopulations. (a) Violin plot showing absolute expression levels of key genes grouped by functional categories (e.g., Known markers, α cell identity, autophagy, transdifferentiation, and amino acid transporters) in GCG, both, tdTomato, and NONE groups. These plots reveal distinct expression patterns among the subgroups, with notably increased expression of autophagy-related genes in the tdTomato subset. (b) Corresponding z-score dot plots for the same gene sets, illustrating relative expression differences across subpopulations. (Revised Fig.4l)\n10. Is Arx differentially regulated in the KO α cells?\nResponse: Thank you for raising this important point. Our single-cell RNA-seq analysis showed that the expression of the Arx gene was not decreased but instead upregulated in CBP/p300αDKO α cells, as shown in Extended Data Fig.28.\nExtended Data Fig. 28. Upregulation of Arx expression in CBP/p300αDKO α cells. (a-b) Violin plots (a) and UMAP feature plots (b) showing Arx expression levels in WT and CBP/p300αDKO pancreatic α cells.\n11. Figures 5k and 5l: the conclusion that Slc7a2-mediated lysine uptake drives H3K27Ac would be stronger with lysine-rescue experiments in shRNA-Slc7a2 cells.\nResponse: Thank you for your constructive criticism. According to your suggestion, we performed lysine-rescue experiments in shRNA-Slc7a2 cells and a modest increase of indicated gene expressions was detected in the lysine-rescue group (Extended Data Fig.29). The new data were added in the revised Fig.5l.\nExtended Data Fig. 29. Lysine partially rescues Slc7a2 silencing-repressed α-cell function gene expression. Relative mRNA expression of Gcg, Mafb, and Pcsk2 in α-TC1-6 cells treated with 5 mM Lysine, after Slc7a2 knockdown (sh Slc7a2) (n = 3 independent experiments). Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD test, compared with CON: *P < 0.05, **P < 0.01, ***P < 0.001; compared with shSlc7a2: #P < 0.05, ##P < 0.01. (Revised Fig.5l)\n12. Figure 7d: can the authors comment on why overexpression of Slc7a2 increases basal pS6?\nResponse: We thank you for your insight. Regarding Fig. 7d, upon thorough review of our experimental records, it is possible that the use of DMEM medium results in basal mTORC1 signaling activation in the Slc7a2 overexpression group. To address this, we modified the experimental conditions by employing amino acid starvation with HBSS and repeated the experiment (Extended Data Fig.30). The new result was added in the revised Fig.7d.\nExtended Data Fig. 30. Western blot analysis of S6 phosphorylation level in α-TC1-6 cells treated with arginine after Slc7a2 overexpression (Ad Slc7a2). (Revised Fig.7d)\n13. Figure 7e: the p300/CBP inhibitor reduces pS6 irrespective of arginine, so it is impossible to conclude that blocking p300/CBP specifically suppresses arginine-mediated pS6 induction.\nResponse: As aforementioned in the previous response, we modified the experimental conditions by employing amino acid starvation with HBSS. When the basal mTORC1 activity in α-cells was extremely low at the amino acid-free status, A-485 could not further inhibit it. While arginine-stimulated pS6 phosphorylation was significantly suppressed by A-485 (Extended Data Fig.31). The new data were added in the revised Fig.7e. We sincerely thank you for pointing this out.\nExtended Data Fig. 31.Western blot analysis of pS6 ser240/244 in α-TC1-6 cells treated with arginine in amino acid-free medium (HBSS) in the presence or absence of CBP/p300 inhibitor A-485 (3 μM). (Revised Fig.7e)\n14. Line 445: “These results indicate that chronic amino acid exposure could stimulate α cell function through epigenetic modification-mediated gene expression” is not supported by the data. The reported effects are chiefly on alpha cell proliferation rather than function, and while they may involve p300/CBP, they do not establish a requirement for epigenetic modification, since these proteins have numerous non-epigenetic functions.\nResponse: Thank you for your constructive criticism. Based on your comment, we performed ex-vivo glucagon secretion assays in islets isolated from WT and CBP/p300αDKO mice. Glucagon secretion was reduced in islets of CBP/p300αDKO mice in response to arginine (Extended Data Fig.22a), indicating that CBP/p300 deficiency compromises α-cell function. In addition, chronic treatment of lysine enhanced glucagon secretion from wild-type mouse islets (Extended Data Fig.32b). Moreover, lysine treatment increased H3K27Ac levels at the promoters of Gcg and MafB (Fig.5m, n). Its catabolite 2-aminoadipic acid (2-AAA) robustly elicited the expression of Gcg, Mafb, and Pcsk2 (Fig. 5o), with elevated H3K27Ac levels at their promoters (Fig.5p, q). As well known, p300/CBP indeed have numerous non-epigenetic functions, such as the important functional domain: bromodomain. We treated α-TC1-6 cells with another well-known CBP/p300-HAT inhibitor C646 and the specific CBP/p300-bromodomain inhibitor, I-CBP112. Like A-485, C646 significantly suppressed the expression of functional genes in α cells. However, I-CBP112 showed no such effect (Extended Data Fig.32b), indicating that the HAT domain of CBP/p300 play the vital role in regulating α cell function. The new added data further support our conclusion.\nExtended Data Fig. 32. Effects of CBP/p300 inhibitors and lysine on the expression of α-cell identity genes and glucagon secretion. (a) Glucagon secretion from WT mouse islets treated with 5 mM glucose (5G) or 5G + 10 mM Arginine (Arg) in the presence or absence of lysine pretreatment for 16 h (n = 3). (b) Relative mRNA expression of Gcg, Mafb, and Pcsk2 in α-TC1-6 cells treated with 30 μM C646 or 5 μM I-CBP112 (n = 3). Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test, compared with CON: *P < 0.05, **P < 0.01, ***P < 0.001. (Supplementary Fig.5f)\nReviewer #5 (Remarks to the Author):\nIn this paper, the authors demonstrated elegantly that CBP/p300 plays an essential role in pancreatic α cell function and expansion in the postnatal period. They also found cationic amino acid transporter Slc7a2 as a key mediator of the maintenance of αcells.\n1) Slc7A2 has been well studied in a lot of cell types including immune cells and cancer cells and its role in cell proliferation already established. The evidence of the essential role of Slc7A2 in this study should be proved by transgenic overexpression of Slc7a2 in the α cells of DKO mice.\nResponse: It is very difficult to complete the in vivo mouse experiment within such a short time. To nevertheless address this important point, we performed the following targeted experiments in cell lines. As shown below (Extended Data Fig.33), the knockdown of CBP/p300 significantly inhibited the proliferation rate of α-TC1-6 cells, which was partially rescued by Slc7a2 overexpression.\nExtended Data Fig. 33. Slc7a2 overexpression partially rescues the proliferative defect caused by CBP/p300 knockdown in α-TC1-6 cells. Proliferation rates (percentage of Ki67⁺ cells) in α-TC1-6 cells subjected to CBP/p300 silencing (si CBP/p300) and Slc7a2 overexpression (Ad Slc7a2) (n = 4). Scale bar, 10 μm. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD test: ***P< 0.001; ###P< 0.001.\n2) One of the previous studies of glucagon signal blockade by Coate et al. (Mol Metab2024) demonstrated the increase of non-alpha cells (mainly delta or beta cells) in a Slc7a2-mTORC1 dependent manner, while in this study, Ppy-positive glucagon cells were increased. Please discuss the apparent discrepancy.\nResponse: We appreciate this constructive criticism. In the referenced study, alpha cell hyperplasia and increased proliferation rates of beta and delta cells were both observed in the different models of interrupted glucagon signaling. However, the study centered on the proliferation mechanisms of non-α cells. Our study focuses on exploring the role of CBP/p300 in α cell identity and mass. And we did not report an increase in Ppy-positive glucagon cells in our study.\n3) The scRNA-seq revealed four subgroups of alpha cells (Fig4K), which I think are important findings. It should be noted that tdTomato+ Gcg- (tdTomato) cells, consisted of 39%. They were considered as denoting identity-compromised α cells. However, they may have been caused by either impaired differentiation of progenitor cells or dedifferentiation of mature α cells. Please discuss this point by analyzing the expression profiles. As for pancreatic beta cells, the impairment of healthy differentiation resulted in the expression of “disallowed genes” such as alpha cell genes. In this sense, the expression of Mafa, Ngn3, or Pdx1 should be examined.\nResponse: Good suggestion! In our dataset, the tdTomato+ Gcg– population indeed represented a substantial fraction of α-lineage-traced cells. To clarify whether these cells reflect impaired progenitor differentiation or dedifferentiation of mature α cells, we analyzed three levels of markers. First, mature α-cell identity genes were uniformly reduced in this subset (Extended Data Fig. 34), consistent with an identity-compromised state. Second, Ngn3 was undetectable and other progenitor-related genes showed no significant changes (Extended Data Fig.34), arguing against persistence of progenitor-like cells. Third, Mafa and Ucn3 remained unchanged, while Pdx1 showed only a mild increase (Extended Data Fig.34), suggesting that a β-cell lineage shift is unlikely. Collectively, these findings support that the tdTomato+ Gcg– subset most likely represents dedifferentiated mature α cells rather than impaired progenitor differentiation or β-cell transdifferentiation.\nExtended Data Fig. 34. Expression of α-cell identity, progenitor, and β-cell identity genes across α-cell subpopulations. Violin plots displaying absolute expression levels of mature α-cell identity genes, progenitor markers, and β-cell identity genes across GCG, Both, tdTomato, and NONE groups.\n4) Related to the point above, tdTomato+Gcg+(Both) cells were considered as escaping the recombination, but the evidence (expression of either CBP or p300) were not provided. If these are the intact alpha cells, then high levels of amino acids should have induced the expansion of these ‘Both cells’. To examine whether the ‘Both cells ‘ were ‘healthy’, nutrient-induced glucagon secretion, either from isolated islets or alpha cells, should be tested.\nResponse: We thank you for the comment and would like to clarify the “Both” (Gcg+tdTomato+) cells were not defined as recombination-escaping cells in our analysis. Instead, the “GCG” (Gcg+tdTomato-) subset represents true escapees, whereas the “Both” population reflects α cells that undergo Cre recombination but retain partial Gcg expression. Consistent with this, “Both” cells exhibit reduced expression of α-cell identity and amino-acid transporter genes compared to the “GCG” subset, yet remain higher than the tdTomato (Gcg- tdTomato+) population (Extended Data Fig.35a), suggesting that they represent an intermediate, partially dedifferentiated state rather than fully intact α cells. Glucagon secretion was reduced in islets isolated from CBP/p300αDKO mice under the conditions of 1 mM glucose, 5 mM glucose and 10 mM Arginine (Extended Data Fig.35b).\nExtended Data Fig. 35. Pathway enrichment profiles of α-cell subpopulations based on GSVA analysis. (a) GSVA heatmap showing enrichment scores of selected GO biological processes across the four α-cell subpopulations. (b) Glucagon secretion from isolated islets of WT or CBP/p300αDKO mice in response to 1 mM glucose (1G), 5 mM glucose (5G) or 5G + 10 mM Arginine (Arg) (n = 3). Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. Compared with WT group: **P < 0.01, ***P < 0.001. (Supplementary Fig.4)\n5) The transcriptional regulation of Slc7a2 is of great significance. However, it is not clear whether CBP/p300 directly regulate transcription of Slc7a2. Please demonstrate the CBP/p300 location on the promoter/enhancer sites of Slc7a2 genes, and their absence in the DKO mice.\nResponse: We appreciate this constructive criticism. We analyzed H3K27Ac ChIP-seq and chromatin accessibility (ATAC-seq) data of mouse pancreatic islets and related cell lines using the ChIP-Atlas database [21]. For Slc7a2, a strong H3K27ac enrichment peak and an open chromatin region were observed in pancreatic islets spanning approximately 300 bp upstream to 800 bp downstream of the transcription start site (TSS) (Extended Data Fig.36). However, we did not detect the binding of CBP/p300 to the promoter regions of Slc7a2 by ChIP-qPCR in α-TC1-6 cells.\nExtended Data Fig. 36. Genome browser tracks showing H3K27Ac ChIP-Seq and ATAC-Seq signals at the Slc7a2 loci in mice islets based on the data from ChIP-Atlas database. Red rectangle indicate region with strong H3K27Ac enrichment peak and an open chromatin region in pancreatic islets spanning approximately 300 bp upstream to 800 bp downstream of the transcription start site (TSS).\nReference:\n1. Shiota, C. et al. Gcg CreERT2 knockin mice as a tool for genetic manipulation in pancreatic alpha cells. Diabetologia 60, 2399–2408 (2017).\n2. Kim, J. et al. Amino Acid Transporter Slc38a5 Controls Glucagon Receptor Inhibition-Induced Pancreatic α Cell Hyperplasia in Mice. Cell Metab 25, 1348-1361.e1348 (2017).\n3. Gong, Y. et al. Hyperaminoacidemia induces pancreatic α cell proliferation via synergism between the mTORC1 and CaSR-Gq signaling pathways. Nat Commun 14, 235 (2023).\n4. Bao, K. et al. Hepatic PKA Mediates Liver and Pancreatic α-Cell Cross Talk. Diabetes 74, 885- 587897 (2025).\n5. Solloway, M. J. et al. Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass. Cell Rep 12, 495-510 (2015).\n6. Bozadjieva, N. et al. Loss of mTORC1 signaling alters pancreatic α cell mass and impairs glucagon secretion. J Clin Invest 127(12), 4379–4393 (2017).\n7. Weinert, Brian T. et al. Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome. Cell 174(1), 231 - 244.e12 (2018).\n8. https://www.humanislets.com\n9. Wang, S. et al. Amino acids, microbiota-related metabolites, and the risk of incident diabetes among normoglycemic Chinese adults: Findings from the 4C study. Cell Rep Med 3(9), 100727 (2022).\n10. Sheng, C. et al. Hepatic CBP/p300 Orchestrate Amino Acid-Driven Gluconeogenesis through Histone Crotonylation. Adv Sci 12, no. 41: e07635 (2025).\n11. Thorel, F. et al. Normal glucagon signaling and β-cell function after near-total α-cell ablation in adult mice. Diabetes 60, 2872-2882 (2011).\n12. Pedersen, J. et al. Glucose metabolism is altered after loss of L cells and α-cells but not influenced by loss of K cells. Am J Physiol Endocrinol Metab 304, E60-73 (2013).\n13. Shiota, C. et al. α-Cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets. Am J Physiol Endocrinol Metab 305, E1030-1040 (2013).\n14. Watanabe, C. et al. Remodeling of hepatic metabolism and hyperaminoacidemia in mice deficient in proglucagon-derived peptides. Diabetes 61, 74-84 (2012).\n15. Dean, ED. et al. A Primary Role for α-Cells as Amino Acid Sensors. Diabetes 1, 69(4), 542–549 (2020).\n16. Li, H. et al. The first pediatric case of glucagon receptor defect due to biallelic mutations in GCGR is identified by newborn screening of elevated arginine. Mol Genet Metab Rep 17,46-52, (2018).\n17. Almdal, TP. et al. Increased amino acid clearance and urea synthesis in a patient with glucagonoma. Gut 31, 946–948 (1990).\n18. Bossart, M. et al. Effects on weight loss and glycemic control with SAR441255, a potent unimolecular peptide GLP-1/GIP/GCG receptor triagonist. Cell Metab 34, 59–74.e10 (2022).\n19. Diana E. et al. Single cell transcriptomic profiling of mouse pancreatic progenitors. Physiol Genomics 49(2), 105-114 (2017).\n20. Rajak, S. et al. MTORC1 inhibition drives crinophagic degradation of glucagon. Mol Metab 53, 101286 (2021).\n21. https://chip-atlas.org/\n22. Stefan, W. et al. Obesity medication lorcaserin activates brainstem GLP-1 neurons to reduce food intake and augments GLP-1 receptor agonist induced appetite suppression. Mol Metab 68 101665 (2023).\n23. Kabahizi, A. et al. Glucagon-like peptide-1 (GLP-1) signalling in the brain: From neural circuits and metabolism to therapeutics. Brit J Pharmacol 179(4), 600–624 (2022).\n24. Perez-Frances, M. et al. Pancreatic Ppy-expressing gamma-cells display mixed phenotypic traits and the adaptive plasticity to engage insulin production. Nat Commun 12, 4458 (2021).\nResponse to Reviewers\nWe sincerely thank the editor and all reviewers for reviewing our revised manuscript and providing detailed feedback. Below, we provide a detailed point-by-point response to all comments. Newly added data are included in the revised figures and text as indicated.\nReviewer #4 (Remarks to the Author):\n1. A major missing link in the proposed model is the claim that loss of p300/CBP downregulates Slc7a2 as a key mechanistic step, as implied by the title (“CBP/p300-Slc7a2 axis is critical for the expansion and maintenance of functional pancreatic α cell mass”). This is crucial to demonstrate because p300/CBP broadly regulates cell type-specific transcription; therefore, the loss of α-cell mass/identity cannot be attributed specifically to Slc7a2 downregulation. Moreover, Slc7a2 downregulation in the p300/CBP knockout is unlikely to be a major driver of the phenotype, given that p300/CBP HAT inhibitors have a much stronger effect on α-cell gene transcription than Slc7a2 silencing (Figure 5). The authors should explicitly discuss that Slc7a2 is unlikely to be the primary mechanism linking p300/CBP loss to the α-cell phenotypes and revise the “axis/through” framing accordingly.\nResponse: We sincerely thank you for your thoughtful comments. Indeed, CBP/p300 deficiency induces profound alterations in the epigenome and transcriptome in pancreatic α-cells of CBP/p300αDKO mice. Slc7a2 is unlikely to be a dominator of the process. However, Slc7a2 downregulation is, at least in part, involved in this phenotype. Following your suggestions, we added this discussion and revised corresponding descriptions to avoid overstatement.\n2. If the authors wish to retain some claims of a direct p300/CBP-Slc7a2 axis, the current revision does not provide locus-level evidence to support this. Because p300/CBP recruitment is typically indirect, formaldehyde-only ChIP may be insufficient to assess occupancy. Supportive experiments would include (i) p300/CBP ChIP using a longer-range crosslinker (e.g., EGS) at the Slc7a2 locus, or (ii) ChIP-qPCR for H3K27ac at the Slc7a2 locus after manipulating p300/CBP activity. These would strengthen the locus-level association, though they would still not fully distinguish direct effects from indirect consequences of altered α-cell identity programs from loss of p300/CBP.\nResponse: Thank you for this constructive feedback. To address your concerns, we treated α-TC1-6 cells with the selective p300/CBP HAT inhibitor A-485 and performed ChIP-qPCR for H3K27ac at the Slc7a2 promoter. As shown below, A-485 treatment significantly reduced H3K27ac enrichment at the Slc7a2 promoter region (Extended Data Fig.1a). Moreover, our previous H3K27Ac ChIP-seq data from A-485-treated rat islets showed similar changes (Extended Data Fig.1b). While indirect recruitment via α-cell lineage TFs likely mediates initial p300/CBP localization, our results highlight the essential role of their acetyltransferase function in locus activation and α-cell maturation. To avoid overstatement, we deleted the description of p300/CBP-Slc7a2 axis according to your suggestion.\nExtended Data Fig. 1. Diminished H3K27ac occupation at the Slc7a2 promoter upon CBP/p300-HAT inhibition. (a) ChIP-qPCR analysis showing enrichment of H3K27Ac at the Slc7a2 promoters in α-TC1-6 cells treated with A-485 or control (CON) (n = 3). (b) Genome browser tracks showing H3K27Ac ChIP-seq signals at the Slc7a2 loci in rat islets treated with vehicle (CON) or A-485 (3 μM). Green rectangles indicate regions with significantly reduced H3K27Ac enrichment upon A-485 treatment. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. **P < 0.01. (Fig.5k and Supplementary Fig. 5e)\n3. Line 999 (“Fig. 6 Knockout of CBP/p300 triggers α cell autophagy through SLC7A2/arginine-regulated mTORC1 signaling”). The data support that CBP/p300 knockout and Slc7a2 silencing can each trigger α-cell autophagy, but do not establish that CBP/p300 loss induces autophagy through Slc7a2. Please revise the figure title and related text to avoid implying causality between the two.\nResponse: We appreciate your valuable and insightful critique. As suggested, we revised the title of Figure 6 to avoid a causal relationship.\n4. The authors should discuss non-histone acetylation as a plausible contributor to α-cell regulation, given the broad effects of CBP/p300 perturbation.\nResponse: Good suggestion! We added this relevant discussion in our revised manuscript.\n5. Line 101, revise to “oral glucose tolerance test” for clarity.\nResponse: As suggested, we revised the description in the new version.\n6. The claim that loss of p300/CBP leads to cell death remains unaddressed (e.g., lines 440-441, 897 etc). The authors should revisit these statements and rephrase them precisely as cell loss/reduced α-cell mass.\nResponse: We appreciate your valuable suggestion. We have carefully revised all relevant statements throughout the manuscript to replace \"cell death\" with precise terminology.\nReviewer #5 (Remarks to the Author):\nThe revised manuscript was modified according to the reviewers’ comments including mine, and has been improved significantly. My major concerns about the origin and the “healthiness” of the alpha cells of each subgroup have been well taken and discussed. I have only minor comment related to the previous points.\nI think the discussion on the identity of glucagon cells in the DKO was quite essential, and the information on the expression of disallowed genes in the authors’ responses was very important. It should be noted that the highly differentiated alpha cells exhibited not only amino acid-induced glucagon secretion but also glucose-suppressed glucagon secretion, which is a marked difference from beta cells. Now that the authors included the data on glucagon secretion in response to glucose in the revised manuscript, I wonder if the authors can add information on the expression of the genes related to glucose responsiveness (Glucose transporters, Gck, Abcc8, Kir6.2 and others). I also suggest that these data should be combined with Extended data Fig34 and be briefly mentioned in the text with Supplemental data.\nResponse: We sincerely thank you for the time and effort dedicated to reviewing our manuscript with the thoughtful and constructive comments, which have been instrumental in improving the quality of our work. As suggested, we added the new data on glucose-responsive gene expressions to Supplementary Fig. 4b and updated the text accordingly.\nExtended Data Fig. 2. Expression of islets progenitor, β-cell identity and α-cell glucose-responsive genes across α-cell subpopulations. Violin plots displaying absolute expression levels of islets progenitor, β-cell identity and α-cell glucose-responsive genes across GCG, Both, tdTomato, and NONE groups. (Supplementary Fig. 4b)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the authors’ careful responses and revisions, which adequately address my concerns.", "reviewer_2": "The authors have done an excellent job revising the manuscript. They added new experimental data that strengthen the study and adequately addressed all of my previous critiques. I have no further comments. Well done.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors made a substantial effort to address many of the comments and have significantly improved the manuscript. However, several claims remain overstated and should be revised to more accurately reflect what the data support.\n1. A major missing link in the proposed model is the claim that loss of p300/CBP downregulates Slc7a2 as a key mechanistic step, as implied by the title (“CBP/p300-Slc7a2 axis is critical for the expansion and maintenance of functional pancreatic α cell mass”). This is crucial to demonstrate because p300/CBP broadly regulates cell type-specific transcription; therefore, the loss of α-cell mass/identity cannot be attributed specifically to Slc7a2 downregulation. Moreover, Slc7a2 downregulation in the p300/CBP knockout is unlikely to be a major driver of the phenotype, given that p300/CBP HAT inhibitors have a much stronger effect on α-cell gene transcription than Slc7a2 silencing (Figure 5). The authors should explicitly discuss that Slc7a2 is unlikely to be the primary mechanism linking p300/CBP loss to the α-cell phenotypes and revise the “axis/through” framing accordingly.\n2. If the authors wish to retain some claims of a direct p300/CBP-Slc7a2 axis, the current revision does not provide locus-level evidence to support this. Because p300/CBP recruitment is typically indirect, formaldehyde-only ChIP may be insufficient to assess occupancy. Supportive experiments would include (i) p300/CBP ChIP using a longer-range crosslinker (e.g., EGS) at the Slc7a2 locus, or (ii) ChIP-qPCR for H3K27ac at the Slc7a2 locus after manipulating p300/CBP activity. These would strengthen the locus-level association, though they would still not fully distinguish direct effects from indirect consequences of altered α-cell identity programs from loss of p300/CBP.\n3. Line 999 (“Fig. 6 Knockout of CBP/p300 triggers α cell autophagy through SLC7A2/arginine-regulated mTORC1 signaling”). The data support that CBP/p300 knockout and Slc7a2 silencing can each trigger α-cell autophagy, but do not establish that CBP/p300 loss induces autophagy through Slc7a2. Please revise the figure title and related text to avoid implying causality between the two.\n4. The authors should discuss non-histone acetylation as a plausible contributor to α-cell regulation, given the broad effects of CBP/p300 perturbation.\n5. Line 101, revise to “oral glucose tolerance test” for clarity.\n6. The claim that loss of p300/CBP leads to cell death remains unaddressed (e.g., lines 440-441, 897 etc). The authors should revisit these statements and rephrase them precisely as cell loss/reduced α-cell mass.", "reviewer_5": "The revised manuscript was modified according to the reviewers’ comments including mine, and has been improved significantly. My major concerns about the origin and the “healthiness” of the alpha cells of each subgroup have been well taken and discussed. I have only minor comment related to the previous points.\nI think the discussion on the identity of glucagon cells in the DKO was quite essential, and the information on the expression of disallowed genes in the authors’ responses was very important. It should be noted that the highly differentiated alpha cells exhibited not only amino acid-induced glucagon secretion but also glucose-suppressed glucagon secretion, which is a marked difference from beta cells. Now that the authors included the data on glucagon secretion in response to glucose in the revised manuscript, I wonder if the authors can add information on the expression of the genes related to glucose responsiveness (Glucose transporters, Gck, Abcc8, Kir6.2 and others) . I also suggest that these data should be combined with Extended data Fig34 and be briefly mentioned in the text with Supplemental data."}, "author_response": "Response to Reviewers\nWe sincerely thank the editor and all reviewers for reviewing our revised manuscript and providing detailed feedback. Below, we provide a detailed point-by-point response to all comments. Newly added data are included in the revised figures and text as indicated.\nReviewer #4 (Remarks to the Author):\n1. A major missing link in the proposed model is the claim that loss of p300/CBP downregulates Slc7a2 as a key mechanistic step, as implied by the title (“CBP/p300-Slc7a2 axis is critical for the expansion and maintenance of functional pancreatic α cell mass”). This is crucial to demonstrate because p300/CBP broadly regulates cell type-specific transcription; therefore, the loss of α-cell mass/identity cannot be attributed specifically to Slc7a2 downregulation. Moreover, Slc7a2 downregulation in the p300/CBP knockout is unlikely to be a major driver of the phenotype, given that p300/CBP HAT inhibitors have a much stronger effect on α-cell gene transcription than Slc7a2 silencing (Figure 5). The authors should explicitly discuss that Slc7a2 is unlikely to be the primary mechanism linking p300/CBP loss to the α-cell phenotypes and revise the “axis/through” framing accordingly.\nResponse: We sincerely thank you for your thoughtful comments. Indeed, CBP/p300 deficiency induces profound alterations in the epigenome and transcriptome in pancreatic α-cells of CBP/p300αDKO mice. Slc7a2 is unlikely to be a dominator of the process. However, Slc7a2 downregulation is, at least in part, involved in this phenotype. Following your suggestions, we added this discussion and revised corresponding descriptions to avoid overstatement.\n2. If the authors wish to retain some claims of a direct p300/CBP-Slc7a2 axis, the current revision does not provide locus-level evidence to support this. Because p300/CBP recruitment is typically indirect, formaldehyde-only ChIP may be insufficient to assess occupancy. Supportive experiments would include (i) p300/CBP ChIP using a longer-range crosslinker (e.g., EGS) at the Slc7a2 locus, or (ii) ChIP-qPCR for H3K27ac at the Slc7a2 locus after manipulating p300/CBP activity. These would strengthen the locus-level association, though they would still not fully distinguish direct effects from indirect consequences of altered α-cell identity programs from loss of p300/CBP.\nResponse: Thank you for this constructive feedback. To address your concerns, we treated α-TC1-6 cells with the selective p300/CBP HAT inhibitor A-485 and performed ChIP-qPCR for H3K27ac at the Slc7a2 promoter. As shown below, A-485 treatment significantly reduced H3K27ac enrichment at the Slc7a2 promoter region (Extended Data Fig.1a). Moreover, our previous H3K27Ac ChIP-seq data from A-485-treated rat islets showed similar changes (Extended Data Fig.1b). While indirect recruitment via α-cell lineage TFs likely mediates initial p300/CBP localization, our results highlight the essential role of their acetyltransferase function in locus activation and α-cell maturation. To avoid overstatement, we deleted the description of p300/CBP-Slc7a2 axis according to your suggestion.\nExtended Data Fig. 1. Diminished H3K27ac occupation at the Slc7a2 promoter upon CBP/p300-HAT inhibition. (a) ChIP-qPCR analysis showing enrichment of H3K27Ac at the Slc7a2 promoters in α-TC1-6 cells treated with A-485 or control (CON) (n = 3). (b) Genome browser tracks showing H3K27Ac ChIP-seq signals at the Slc7a2 loci in rat islets treated with vehicle (CON) or A-485 (3 μM). Green rectangles indicate regions with significantly reduced H3K27Ac enrichment upon A-485 treatment. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t-test. **P < 0.01. (Fig.5k and Supplementary Fig. 5e)\n3. Line 999 (“Fig. 6 Knockout of CBP/p300 triggers α cell autophagy through SLC7A2/arginine-regulated mTORC1 signaling”). The data support that CBP/p300 knockout and Slc7a2 silencing can each trigger α-cell autophagy, but do not establish that CBP/p300 loss induces autophagy through Slc7a2. Please revise the figure title and related text to avoid implying causality between the two.\nResponse: We appreciate your valuable and insightful critique. As suggested, we revised the title of Figure 6 to avoid a causal relationship.\n4. The authors should discuss non-histone acetylation as a plausible contributor to α-cell regulation, given the broad effects of CBP/p300 perturbation.\nResponse: Good suggestion! We added this relevant discussion in our revised manuscript.\n5. Line 101, revise to “oral glucose tolerance test” for clarity.\nResponse: As suggested, we revised the description in the new version.\n6. The claim that loss of p300/CBP leads to cell death remains unaddressed (e.g., lines 440-441, 897 etc). The authors should revisit these statements and rephrase them precisely as cell loss/reduced α-cell mass.\nResponse: We appreciate your valuable suggestion. We have carefully revised all relevant statements throughout the manuscript to replace \"cell death\" with precise terminology.\nReviewer #5 (Remarks to the Author):\nThe revised manuscript was modified according to the reviewers’ comments including mine, and has been improved significantly. My major concerns about the origin and the “healthiness” of the alpha cells of each subgroup have been well taken and discussed. I have only minor comment related to the previous points.\nI think the discussion on the identity of glucagon cells in the DKO was quite essential, and the information on the expression of disallowed genes in the authors’ responses was very important. It should be noted that the highly differentiated alpha cells exhibited not only amino acid-induced glucagon secretion but also glucose-suppressed glucagon secretion, which is a marked difference from beta cells. Now that the authors included the data on glucagon secretion in response to glucose in the revised manuscript, I wonder if the authors can add information on the expression of the genes related to glucose responsiveness (Glucose transporters, Gck, Abcc8, Kir6.2 and others). I also suggest that these data should be combined with Extended data Fig34 and be briefly mentioned in the text with Supplemental data.\nResponse: We sincerely thank you for the time and effort dedicated to reviewing our manuscript with the thoughtful and constructive comments, which have been instrumental in improving the quality of our work. As suggested, we added the new data on glucose-responsive gene expressions to Supplementary Fig. 4b and updated the text accordingly.\nExtended Data Fig. 2. Expression of islets progenitor, β-cell identity and α-cell glucose-responsive genes across α-cell subpopulations. Violin plots displaying absolute expression levels of islets progenitor, β-cell identity and α-cell glucose-responsive genes across GCG, Both, tdTomato, and NONE groups. (Supplementary Fig. 4b)"}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have addressed all concerns raised satisfactorily."}, "author_response": ""}]

Decision letter