The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition

Domain: neuroscience
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
The adipokine leptin is a central regulator of energy metabolism. We previously showed that HDAC6 inhibitors enhance central leptin sensitivity. Using integrative analyses of leptin-responsive hypothalamic gene expression signatures, we identified focal adhesion kinases (FAK and PYK2) as essential for the anorectic effect of leptin and the anti-obesity action of HDAC6 inhibitors in male mice. The effect of tubastatin A, an HDAC6 inhibitor, is compromised in Pyk2 knockout mice, and central inhibition of focal adhesion kinases blocks tubastatin-induced weight loss. Focal adhesion kinases phosphorylate and activate the transcription factor STAT3 downstream of leptin receptor, and leptin signaling is attenuated when these kinases are knocked down or inhibited. Finally, hypothalamic knockdown of focal adhesion kinases blunts leptin action, leads to hyperphagic obesity, and attenuates the anti-obesity effect of HDAC6 inhibitors. These findings suggest that FAK and PYK2 are previously uncharacterized members of the leptin receptor signaling and critical mediators of central leptin sensitization.

Introduction
The adipose-derived hormone leptin is secreted in amounts proportional to adiposity. In mammals, leptin exerts an anorectic effect and suppresses food intake upon peripheral or central administration1,2. In humans and rodents, mutations in the gene encoding leptin or its receptor result in profound hyperphagia, severe obesity, hyperglycemia, and impaired endocrine functions, emphasizing the critical contribution of leptin to the maintenance of energy balance3,4.

The effect of leptin on energy homeostasis is predominantly mediated through neurons expressing the long isoform of the leptin receptor (LepRb) within the central nervous system5,6. Binding of leptin to LepRb stimulates several intracellular signal transduction mechanisms including the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Upon LepRb activation, JAK2 is recruited and phosphorylates the intracellular tyrosine residues of LepRb, leading to the recruitment of STAT3. JAK2 phosphorylation of STAT3 tyrosine residue 705 (pSTAT3Y705) promotes dimerization of STAT3 and its subsequent nuclear translocation. STAT3 expression in LepRb neurons is required for the leptin-dependent regulation of energy balance, emphasizing the dominant contribution of STAT3-dependent signal transduction over other pathways activated by leptin7–9. JAK2, however, is not required for the activation of STAT3 in response to leptin invitro10, suggesting that other tyrosine kinases may be involved in mediating this action. Notably, STAT3 is an established target of focal adhesion kinases11–13, however whether focal adhesion kinase (FAK) and protein tyrosine kinase 2b (PYK2) regulate leptin action is unknown.

FAK and PYK2 comprise the focal adhesion kinase family, a subset of non-receptor tyrosine kinases with 46% sequence identity and 65% amino acid similarity14. The focal adhesion kinases play a key role in several cell activities due to their interaction with the cytoskeleton and activation by integrins and G-protein coupled receptors15,16. While FAK is ubiquitously expressed, PYK2 expression is primarily limited to the CNS and cells of hematopoietic lineage, although PYK2 is also expressed in other cell types17,18. Pyk2 knockout (KO) mice are viable and fertile19, however Fak ablation leads to early embryonic lethality because of defects in vasculogenesis20,21, although the knockdown of Fak expression in some tissues (e.g., endothelium) is compensated with elevated PYK2 expression22–25. While both proteins regulate cell motility and migration, polarization, adhesion, and proliferation16,19,26–31, PYK2 is also involved in spatial memory and synaptic plasticity, synaptic remodeling32–35, and signal transduction in response to cytokine and growth factor receptor activation36–39.

The focal adhesion kinases have also been implicated in metabolism. Modest knockdown of Fak in the muscle and liver of lean wild-type mice results in increased food intake and body weight and impaired insulin sensitivity40. Furthermore, adipocyte FAK is necessary for cell survival and insulin sensitivity in the obese state. Consequently, HFD-induced obese mice with an adipose tissue-specific deletion of Fak exhibit greater impairments in obesity-induced insulin sensitivity41. Pyk2 KO mice were reported to be more prone to obesity and exhibit exacerbated obesity-associated impairments in glucose homeostasis42. The metabolic deficits associated with the tissue specific Fak and whole-body Pyk2 KO mouse models suggest a role for the focal adhesion kinases in the regulation of energy homeostasis. The precise pathway(s) and mechanism(s) by which the focal adhesion kinases regulate energy homeostasis, especially in the context of their central action, remains largely unknown. Furthermore, their potential role in hypothalamic leptin receptor signaling has not been studied.

In diet-induced obese (DIO) rodents and humans, circulating leptin concentrations rise in proportion to adiposity. This hyperleptinemic state is causally linked to the development of leptin resistance43. Our group has recently identified that peripheral, but not central, inhibition of HDAC6 activity leads to central leptin sensitization through a systemic factor44. Furthermore, inhibition of endogenous HDAC6 in LepRb-expressing cells does not alter leptin action44. Small molecule HDAC6 inhibitors act as a potent leptin-sensitizers and anti-obesity agents, however the central mediators of these actions are unknown.

Here, we show that focal adhesion kinases are required for the anorectic action of leptin and the anti-obesity effect of HDAC6 inhibitors. FAK and PYK2 physically interact with and phosphorylate STAT3 in response to leptin. Accordingly, pharmacological or genetic inhibition of focal adhesion kinases attenuate leptin signaling. AAV-mediated knockdown of these kinases in the hypothalamus of lean mice results in hyperphagic obesity and resistance to the weight reducing effect of HDAC6 inhibitors. Our results suggest focal adhesion kinases as new regulators of leptin receptor signaling and thus critical components of the homeostatic body weight regulation.

Results
DIO mice treated systemically with HDAC6 inhibitors, including Tubastatin A (TubA), regain their hypothalamic leptin sensitivity and lose weight44. However the central mechanism of action of this leptin sensitization is unclear44,45. To discover genetic and/or pharmacological interventions with effects on gene expression resembling the hypothalamic transcriptomic signatures of leptin sensitization, we utilized Connectivity Map (CMap, clue.io), a database of transcriptomic profiles of human cells subjected to thousands of perturbagens; small molecule compounds or genetic manipulations (overexpression and/or knock-down). To this end, we first used the hypothalamic transcriptome in response to systemic TubA administration in obese mice, as this represents a leptin sensitized state (Fig. 1a and Supplementary Data 1), to query the Connectivity Map (CMap) database (detailed in the “Methods”). Since leptin receptor expressing neurons constitute a small percentage of the total hypothalamic cell pool, we also gathered the leptin regulated and LepR+ neuron-enriched gene lists46 (Fig. 1c), and conducted an independent CMap search with the differentially expressed gene set representing leptin’s signature (differentially expressed genes in LepR+ neurons), Fig. 1d). CMap queries with either input resulted in a set of compounds and genetic perturbations that partially overlapped (Fig. 1b, d and Supplementary Data 2). To narrow down candidate factors that mediate central leptin sensitization conferred by HDAC6 inhibition, we combined the two lists by averaging their connectivity scores (Fig. 1e, f). Based on subsequent analysis of the final candidate list (see “Methods”), we prioritized focal adhesion kinases for further investigation for the following reasons: The compound, PF-562271, which is a dual focal adhesion kinase inhibitor, was the second most significantly and negatively correlated perturbagen when the TubA and leptin signatures were co-analyzed (Fig. 1f). Pyk2, one of the two focal adhesion kinases, was highly upregulated in the hypothalamic of TubA-treated mice (Fig. 1a), and TubA treatment also increased the hypothalamic Fak expression in DIO animals (1.00 ± 0.06 vs. 1.34 ± 0.09 for vehicle vs. TubA, respectively, p=0.018). Furthermore, Pyk2 is significantly associated with body mass index (BMI) and weight in human genome-wide association studies with a Human Genetic Evidence47,48 score of 45 (very strong) (Supplementary Fig. 1a, b). Likewise, Fak, the other focal adhesion kinase, is linked to waist-hip ratio and cardiovascular traits such as blood pressure and elevated cholesterol (Supplementary Fig. 1c, d), collectively suggesting that focal adhesion kinases could be involved in the central regulation of energy balance.

FAK and PYK2 mediate the weight reducing effect of TubA
We next set to determine if focal adhesion kinases are required for TubA-induced weight loss. We first tested the effect of dual central inhibition of the focal adhesion kinases on TubA action using PF-431396 (PF), an ATP-mimetic highly selective dual inhibitor of FAK and PYK249. Following daily central infusion of vehicle or PF to DIO wild-type mice, we treated mice with peripheral injection of vehicle or TubA (15 mg/kg) for five consecutive days. Of note, at this dose, TubA reduces obesity in DIO wild-type mice but not in HDAC6 KO animals44. TubA treatment led to a steady weight loss in DIO mice during the treatment period (Fig. 2a, b and Supplementary Fig. 2a). However, central PF infusion conferred significant resistance to TubA-induced weight loss (Fig. 2a, b).

The PF compound is a dual inhibitor of both FAK and PYK2. Since whole body Fak deletion leads to embryonic lethality, but Pyk2 KO mice develop normally, we tested if Pyk2 KO mice exhibited resistance to TubA. We induced obesity by feeding Pyk2 KO mice and their wild-type littermates HFD for 24 weeks. In contrast to a previous study42, Pyk2 KOs displayed similar weight gain and food intake on HFD when compared to wild-type counterparts (Fig. 2c, d), suggesting that embryonic Pyk2 ablation alone does not alter the long term energy metabolism of the animals. Following vehicle acclimation, we treated mice with TubA by daily ip injections. While both groups initially started losing weight, Pyk2 KO mice displayed resistance to TubA treatment and their body weight plateaued after the fifth day of the drug administration (Fig. 2e and Supplementary Fig. 2b). During the approximately 2-week treatment period, the TubA-induced weight loss in DIO mice was almost twice as much as that observed in the Pyk2 KO mice (9.98% ± 0.78 vs. 5.40% ± 1.43, mean ± SEM in wild-type vs. Pyk2 KO mice, respectively, p=0.0018) (Fig. 2f). The food intake of the cohorts was comparable during the acclimation phase (Fig. 2g). However, DIO Pyk2 KO mice displayed resistance to the food intake suppressing effect of TubA, and the wild-type mice consumed less food under TubA treatment (Fig. 2h, i). While the blood glucose of either group decreased following TubA treatments (Fig. 2j), the decrease in the wild-type mice was more pronounced and significantly more than observed in the Pyk2 KO animals (Fig. 2k). Since Pyk2 deletion conferred a partial resistance to TubA-induced weight loss compared to the more robust resistance by dual inhibition of FAK and PYK2, these results collectively suggest that the central action of focal adhesion kinases is required for the anti-obesity effect of TubA.

FAK and PYK2 are required for leptin-induced STAT3 activation
Because TubA action requires leptin signaling, we next tested if focal adhesion kinases act downstream of leptin receptor. FAK and PYK2 are tyrosine kinases. While JAK2 is the canonical kinase that phosphorylates and activates STAT3, JAK2-independent leptin-STAT3 signaling routes have been reported10. To first confirm that JAK2 is not required for the activation of STAT3 by leptin, we assessed STAT3 phosphorylation (pSTAT3Y705) in response to leptin in two independent JAK2-deficient and stably LepRb expressing cell lines, mouse embryonic fibroblasts derived from JAK2 KO mice (MEF JAK2−/−)50 and a human fibroblast cell line genetically deficient of JAK2 (γ2a)51,52. Stimulation of LepRb-expressing JAK2−/− MEFs or γ2a cells resulted in the activation of STAT3 in a dose and time-dependent manner (Supplementary Fig. 3a–d). In both cell lines we observed acute activation of STAT3 within 5 min of leptin stimulation (Supplementary Fig. 3c, d). We also observed a second phase of STAT3 activation after 90 min following the initial leptin stimulation in the γ2a cells (Supplementary Fig. 3d). While such a biphasic activation of STAT3 in response to leptin was previously reported53–55, our results demonstrate that this effect is JAK2 independent. Collectively, these results suggest, consistent with previous reports10, that JAK2-independent, STAT3-dependent leptin signaling mechanisms exist in vitro, implicating the role of other tyrosine kinases in leptin receptor signaling.

Since both FAK and PYK2 have previously been shown to interact with STAT336,56 we tested the focal adhesion kinase family as candidate tyrosine kinases involved in STAT3-dependent leptin action. To determine if either of the focal adhesion kinases activate STAT3, we co-overexpressed STAT3 with either FAK or PYK2 in HEK-293T cells. Both FAK and PYK2 stimulated STAT3 phosphorylation (Fig. 3a, b). Overexpression of the kinase dead (KD) Fak or Pyk2 constructs substantially reduced this effect, suggesting that the catalytic activity of the kinases is necessary for STAT3 activation (Fig. 3a, b). STAT3 co-immunoprecipitated with both PYK2 and FAK in vitro (Fig. 3c) and in the hypothalamic lysates from lean wild-type mice (Fig. 3d). Notably, this interaction was stimulated by leptin in vivo (Fig. 3d), collectively suggesting that both kinases directly interact with STAT3 to promote its activation.

We next evaluated if the focal adhesion kinases were required for leptin-induced activation of STAT3. To this end, we used the immortalized mouse hypothalamic N1-LepRb cells, which endogenously express both focal adhesion kinases44. Knockdown of either Fak or Pyk2 (Fig. 3e–h and Supplementary Fig. 3e–h), or their dual knockdown (Fig. 3i) resulted in a significant attenuation of leptin-induced STAT3 phosphorylation. Knockdown of either kinase was sufficient to attenuate, but not completely impair, the duration and fold induction of STAT3 activation in response to leptin, and a more robust inhibition of leptin signaling was achieved by dual knockdown of Fak and Pyk2.

To see if the results from the knockdown studies can be mimicked by the pharmacological inhibition of FAK and PYK2, we tested if PF also impairs STAT3-dependent transcriptional activation. We used a luciferase construct under the transcriptional control of a STAT3 response element such that expression of luciferase is proportional to the transcriptional activity of STAT344. Stimulation of N1-LepRb cells expressing the STAT3-dependent luciferase construct (N1Stat3Luc-LepRb) with leptin for 24 h dose-dependently increased the luminescence (Fig. 3j, k). Treatment of N1Stat3Luc-LepRb cells with PF, however, dose dependently attenuated leptin induced luciferase expression (Fig. 3j, k). We next utilized the γ2a and MEF JAK2−/− cell lines to examine if the result we observed in N1 cells was restricted to a single cell line, and to control for the potential off-target effects of the PF compound on JAK2. Pre-treatment of stably LepRb-expressing JAK2−/− MEFs or γ2a cells (Supplementary Fig. 3i, j) with PF resulted in reduced phosphorylation of the autophosphorylation site of PYK2 required for its activation (PYK2Y402) (Supplementary Fig. 3i) accompanied by attenuated leptin induced STAT3 phosphorylation (Supplementary Fig. 3i, j) suggesting that the effect of PF is not cell line specific and is independent of JAK2. Results from these studies strongly suggest that FAK and PYK2 are critical regulators of leptin receptor signaling.

FAK and PYK2 mediate leptin action in vivo
Central inhibition of focal adhesion kinases attenuated the weight reducing effect of TubA in DIO mice (Fig. 2a, b), and we showed previously that TubA action is leptin dependent44. Hence, we decided to test if FAK and/or PYK2 regulated leptin action in vivo. To this end, we first analyzed the central expression profile of the focal adhesion kinases, which had not been previously characterized. We conducted RNAscope in situ hybridization to analyze the expression profile of Fak and Pyk2 and their co-localization with LepR in the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), and dorsomedial hypothalamus (DMH), three well-characterized sites of leptin action and TubA-induced leptin sensitivity44. Throughout the hypothalamus, a majority of the LepR+ neurons expressed at least one of the focal adhesion kinases with more LepR+ cells colocalizing with Fak than with Pyk2 (Fig. 4a and Supplementary Figs. 4, 5). Fak has a uniform hypothalamic expression which is higher than Pyk2 expression in the ARC, whereas Pyk2 expression is higher in the DMH compared to the ARC and VMH (Fig. 4b and Supplementary Figs. 4 and 6a–d). We corroborated these findings by reanalyzing a publicly available hypothalamic single cell sequencing dataset57, which corroborated that Fak was ubiquitously expressed and Pyk2 expression was restricted to neurons and microglia (Supplementary Fig. 7).

To assess whether the expression of the focal adhesion kinases responds to nutritional need states or leptin, we conducted a similar study in mice subjected to fasting with or without leptin treatment, and also analyzed the published datasets for the hypothalamic expression profile of the two transcripts. We did not detect a significant effect of fasting on the expression of either gene in the ARC, VMH, or DMH, despite a tendency for increased Pyk2 expression in the ARC (Fig. 4c–k). RNA sequencing of distinct hypothalamic nuclei ARC, VMH, DMH or PVN (paraventricular nucleus of the hypothalamus) did not display a significant regulation of Fak or Pyk2 by fasting (Supplementary Fig. 7a–d). Analyzing two distinct datasets on the transcriptional profile of AgRP neurons, which are critical mediators of leptin action located exclusively in the ARC, revealed that fasting significantly increased the expression of Fak and Pyk2 in the AgRP neurons but not in the POMC neurons58,59 (Supplementary Fig. 7e–g). In agreement with this finding, our LepR-specific expression analysis revealed that fasting increased the expression of Pyk2 specifically in the ARC LepR population (Fig. 4k).

The hypothalamic expression profiling results collectively suggested that a majority (88–94%) of LepR+ neurons expressed at least one of the focal adhesion kinases. We next assessed the physiological relevance of the focal adhesion kinases in mediating leptin dependent feeding and body weight regulation in vivo. To this end, we implanted cannulas into the lateral ventricle of lean wild-type mice for direct intracerebroventricular (icv) infusion of PF. We first confirmed that icv administration of PF did not affect fasting-induced re-feeding (Fig. 5a). Intraperitoneal (ip) administration of leptin prior to the onset of the dark cycle suppressed fasting-induced food intake (Fig. 5a). Central administration of PF, however, was sufficient to suppress leptin-induced anorexia (6.09 g ± 1.14 Veh (icv)/Leptin (ip) vs. 7.22 g ± 1.58 PF (icv)/Leptin (ip) food consumed at 24 h, p=0.0116) (Fig. 5a). Since the hypothalamus is a major site of leptin action, we first analyzed if PF attenuated leptin induced STAT3 phosphorylation in total hypothalamic lysates obtained from lean wild-type mice. Icv PF infusion followed by ip leptin administration impaired the leptin-induced activation of hypothalamic STAT3 (Fig. 5b, c). PF infusion did not block the leptin induced STAT3 phosphorylation completely, presumably due to the involvement of other kinases, including JAK2. Because the extent of leptin action varies across hypothalamic nuclei, we conducted a similar experiment with vehicle or PF infusion into lateral ventricle of lean mice and subsequently analyzed the kinetics of STAT3 phosphorylation in the ARC, VMH and DHM following ip PBS or leptin injections. PF-treated mice had a significantly attenuated leptin response in all three nuclei analyzed (Fig. 5c). Taken together, these results suggest that central inhibition of focal adhesion kinases inhibit leptin signaling.

We next tested the leptin response of the Pyk2 KO mice compared to their wild-type littermates. Pyk2 KO mice exhibited reduced leptin-induced food intake suppression such that they tended to eat more than the wild-type mice even in the presence of peripheral leptin administration (4.38 g ± 1.38 wild-type/Leptin, vs. 5.25 ± 1.21 Pyk2 KO/Leptin food consumed at 24 h) (Fig. 6a, b). While leptin significantly blunted the body weight gain in wild-type mice after 6 and 18 h of re-feeding, this effect was not observed in the Pyk2 KO mice (Fig. 6c). Finally, leptin-induced STAT3 phosphorylation was diminished in the KO cohort (Fig. 6d, e), in line with our previous findings. Altogether, these results suggest that focal adhesion kinases are required for the full anorectic effect of leptin.

To directly determine the role of hypothalamic focal adhesion kinases in the regulation of energy metabolism, we first designed AAV particles encoding a scrambled shRNA sequence or two tandem shRNA sequences targeting Pyk2 and Fak (shPyk2/Fak). We aimed for dual knockdown due to the redundant function of the two kinases and to avoid potential compensatory expression in response to a single focal adhesion kinase gene knockdown22–25 (Fig. 3e). We injected these AAV particles into the mediobasal hypothalamus (covering ARC and VMH) and DMH of adult lean mice (Supplementary Fig. 8a). Two weeks after viral injection, we placed half of the mice in either AAV group (shScr versus shPyk2/Fak) on HFD. Mice in the shPyk2/Fak groups developed obesity on both regular chow and HFD (Fig. 7a–c). On either diet, shPyk2/Fak cohorts consumed more food (Fig. 7d), accounting for their rapid weight gains, which was reflected in significant accumulation of fat mass (Fig. 7e–h) and elevated fasting blood glucose (Fig. 7i).

Before the development of any significant weight difference, we also tested the leptin response of the mice on chow diet. While leptin suppressed the food intake and weight gain of the shScr group, dual knockdown of the kinases in the hypothalamus (shPyk2/Fak group) impaired the anorectic action of leptin (Fig. 7j, k), confirming our previous findings that focal adhesion kinases are indispensable for leptin action in vivo. Taken together, these findings strongly suggest that hypothalamic action of focal adhesion kinases is required for leptin receptor signaling and the maintenance of energy homeostasis. HDAC6 inhibitors induce weight loss in a leptin dependent manner44,45,60. Therefore, we finally tested the weight reducing effect of HDAC6 inhibitors on DIO shPyk2/Fak cohort and their control group. Following an acclimation period, where mice maintained stable body weights, we treated the DIO cohorts with TubA. Animals in the shScr group displayed decreased food intake and weight loss following TubA administration (Fig. 7l–n). However, dual knockdown of the kinases led to resistance to the anti-obesity effect of TubA (Fig. 7l–n). Loss of response to TubA was not due to excess obesity of the shPyk2/Fak cohort as we observed a similar effect in a sub-cohort of mice selected for matched pre-treatment body weights (Fig. 7o). Notably, these results were not restricted to TubA. Mice in the DIO shPyk2/Fak group maintained their body weight during treatment with the brain penetrant HDAC6 inhibitor ACY775, which induced weight loss in DIO shScr mice (Supplementary Fig. 8b–f), collectively suggesting that central action of focal adhesion kinases is required for the anti-obesity effect of HDAC6 inhibitors.

Methods
Mice
All animal studies were performed following procedures approved by the Institutional Animal Care and Use Committees (IACUC) at the University of Michigan and University of Pittsburgh, and by the Committees on Ethics of Animal Experiments of the University of Pavia. Mice were fed either regular chow (5LOD from LabDiet, Purina, St. Louis, MO) or high-fat diet (HFD, 60 kcal% fat, Research Diets, cat. no. D12492) and had free access to food and water unless noted otherwise and kept on a 12h light/dark cycle in a temperature and humidity-controlled environment. Mice had ad libitum access to food and water unless otherwise indicated. Wild-type male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were used. Pyk2 KO mice generation was previously reported19 and colonies were maintained as described26. Only male mice were included in this study.

Cannulation surgery
Mice were anesthetized with isoflurane, and a stainless-steel guide cannula (Plastics One, Roanoke, VA) was implanted into the lateral ventricle using the following stereotaxic coordinates: AP: −0.46 mm, ML: −1.00 mm, DV: −2.20 mm. All intracerebroventricular (icv) injections were performed using a 2-µL Hamilton syringe (Plastics One, Roanoke, VA). Following recovery from the surgery, accurate placement of the cannula was confirmed by central angiotensin II administration. Animals that did not exhibit an elevation in water intake in response to central angiotensin II were excluded from the study. PF was dissolved in DMSO and infused at 500 nL per mouse into the right lateral ventricle at the indicated doses.

Stereotaxic viral injections
Mouse Pyk2- and Fak-targeting shRNA sequences described above were cloned into an AAV vector in tandem reading frames (VectorBuilder). AAV2-CMV-Pyk2/Fak shRNA and AAV2-CMV-scramble shRNA viruses were generated by the University of Michigan Vector Core. For stereotaxic surgical procedures, 8 to 10-week-old wild-type mice were anesthetized with isoflurane and positioned in a stereotaxic frame (Kopf). A micro-precision drill was used to create a small burr hole directly above the site of viral injection, followed by the careful removal of the dura. A total of 500 nL of AAV was injected bilaterally into the arcuate nucleus and dorsomedial hypothalamus using a micromanipulator (Narishige) attached to a pulled glass pipette. The coordinates from bregma for the arcuate nucleus: anterior/posterior: −1.40 mm, medial/lateral: ±0.25 mm, dorsal/ventral: 5.95 mm; for DMH: anterior/posterior: −1.70 mm, lateral: ±0.30 mm, dorsal/ventral: −5.60 mm. The virus was administered at a rate of 50 nL/min. After the injection, the glass pipette was left in place for an additional 10 min to prevent the viral solution from leaking outside the targeted brain region. A recovery period of 3 weeks was allowed post-surgery to ensure viral expression and healing from the surgical procedures. Viral expression was verified post hoc in all animals. Data from animals in which the viral expression was located outside the targeted area were excluded from analysis.

Fasting and re-feeding studies
For experiments involving wild-type lean mice, food was removed from the cages of cannulated mice prior to the beginning of the dark cycle (6:00 p.m.). After 23 h of fasting, mice were infused with PF (2.5 µg as indicated in Fig. 5 legend) or DMSO vehicle of equal volume (500 nL per mouse) icv 30 min later, mice were injected with 5 or 10 mg/kg leptin or PBS as vehicle intraperitoneally (ip). Food intake was monitored 3, 6, 16, and 24 h. Body weight was measured 6, 16, and 24 h post re-feeding. This experiment was conducted on two separate cohorts of mice with both cohorts exhibiting similar results. For each cohort of mice, the fasting-induced re-feeding experiment was performed 2–3 times, each time rotating the mice in the experimental groups. Each trial was separated by a minimum of 5 days. Following the final trial, mice were fasted overnight for 16 h (6:00 p.m. to 10:00 a.m.). After the fast, PF or DMSO was injected icv, followed by leptin or PBS ip injection 15 min later. Thirty minutes after administration of leptin or PBS, hypothalamic tissue punches were collected and snap frozen in liquid nitrogen, and stored at −80°C until further analysis. To test the effect of PF on TubA-induced weight loss (Fig. 2a, b), wild-type DIO mice on HFD were cannulated into the lateral ventricle, and treated with PF or DMSO icv and TubA or Vehicle ip once daily. For experiments involving Pyk2 KO mice, food was removed from the cages for the last 6 h prior to the onset of dark cycle. Fifteen minutes prior to the onset of the dark cycle, Pyk2 KO or littermate control mice were injected with PBS or leptin (5 mg/kg) followed by returning food to the mice. Body weight and food intake were measured 3, 6, 18, and 24 h following re-administration of food.

Antibodies and plasmids
Anti-phosphoSTAT3Y705 (#9145 and #9131, 1:1000), anti-STAT3 (#9139, 1:1000), anti-phosphoPYK2Y402 (#3291, 1:1000), anti-PYK2 (#3480, 1:1000), anti- GAPDH HRP conjugate (#8884, 1:10,000), anti-phosphoFAKY397 (#8556, 1:1000), and anti-FAK (#13009, 1:1000), anti-alpha Tubulin (#3873, 1:5000), anti-acetyl alpha tubulin (#5335, 1:2000), anti-FLAG tag (#14793, 1:5000), anti-myc tag (#2278, 1:2000) HRP-conjugated anti-rabbit IgG (#7074, 1:2000), HRP-conjugated anti-mouse IgG (#7076, 1:2000), DyLight 800-conjugated anti-rabbit IgG (#5151, 1:10,000), DyLight 680-conjugated anti-mouse IgG (#5470, 1:10,000) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA) and were diluted as indicated. The anti-α-FLAG (#SAB4301135, 1:10,000) antibody was purchased from Sigma-Aldrich (St. Louis, MO).

The FLAG-tagged human leptin receptor ORF was purchased from Sino Biologicals (Beijing, China). The wild-type human Pyk2 ORF was purchased from OriGene (Rockville, MD). The GFP-tagged kinase-dead PYK2 mutant (PYK2 KD) was kindly provided by Dr. Kristiina Vuori (Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA). The wild-type Fak (#50515), autophosphorylation-site mutant FAKY397F (#50516), FLAG-tagged Stat3, and LacZ constructs were obtained from Addgene (Cambridge, MA).

In silico connectivity map (CMap) analysis
Bulk RNA sequencing was conducted using hypothalamic RNA obtained from male wild-type DIO mice treated with vehicle (dimethyl sulfoxide, DMSO) or TubA for four consecutive days. The leptin-induced gene expression changes in LepR neurons was previously published46, and in our analysis we used the results obtained 10 h after leptin treatment. Upregulated (fold change ≥ 1.50) and downregulated (fold change ≤ 0.70) transcripts were entered into the 1.0 query version of CMap (clue.io). TubA and leptin gene expression signatures were analyzed as individual queries. The arithmetic mean of CMap derived connectivity scores for these two queries is used as the TubA/Leptin combined score. The top four perturbagens that best correlated with the combined scores were Serpin1d knockdown and kinetin-riboside (apoptosis/mitophagy inducer) as positively associated factors (changing the gene expression in the same direction as input), and ZG-10 (JNK inhibitor) and PF-562271 (PF, focal adhesion kinase inhibitor) as negatively associated factors, i.e., changing the gene expression in the opposite direction compared to the input query (Fig. 1e, f). This final list was manually sorted and some of the factors were eliminated as non-likely candidates. For example, Serpind1 is a serine protease inhibitor that is a predominantly liver-specific gene with a restricted expression to the vascular leptomeningeal cells in the brain (http://mousebrain.org/). We further ruled out Serpind1 because its overexpression had a very low similarity score (−3.125) in contrast to the relatively high score of its knockdown (85.08, with CMap similarity scores ranging between −100 to +100). Some factors in either list appeared as both negative and positive correlators, e.g., JNK inhibitors and topoisomerase inhibitors appeared with combined connectivity scores ranging from −97.49 to +35.63. We reasoned those factors were random associations due to lack of a uniform direction in their scores.

Generation of N1Stat3Luc stable cell line and experimentation
Lentivirus encoding a luciferase reporter construct under a minimal CMV promoter and a STAT3 transcriptional response element was purchased from Cellomics Technology (Halethorpe, MD). For generating the N1Stat3Luc cell line, 10 µg/mL polybrene (Santa Cruz Biotechnology, Dallas, TX) was added to the viral particles prior to addition of lentiviral medium to N1 cells. Cells were supplemented with equal volume of 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) approximately 4 h after lentiviral medium addition. Two days after infection, the medium was removed and replaced with DMEM supplemented with 10% FBS and 10 µg/mL puromycin antibiotic selection (Thermo Fisher Scientific, Waltham, MA). Surviving cells which had stably integrated the construct (denoted as N1Stat3Luc) were used for experiments. For experiments using the N1Stat3Luc cells, cells were plated in 384 well plates. On the following day, cells were transfected with 80 ng LepRb and 0.12 µL Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in reduced serum medium (Opti-MEM, Invitrogen, Carlsbad, CA). The following day, the indicated concentration of PF and leptin was added. The following day, 1x D-luciferin substrate was added to each well. Within 10 min of D-luciferin addition, fluorescence was measured using an EnSpier plate reader (PerkinElmer, Waltham, MA).

Cell culture and transfection
All cell lines were maintained at 37°C in 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS and 100 units/mL penicillin, 100 µg/mL streptomycin or selection antibiotic. The embryonic mouse hypothalamus cell line (N1) was purchased from Cedarlane Labs (Burlington, Canada). The JAK2−/− mouse embryonic fibroblasts and γ2a human fibroblasts, which were both kindly provided by Dr. Liangyou Rui (University of Michigan, Ann Arbor, MI), stably express LepRb as previously described10 and were maintained in 100 µg/mL hygromycin B (Invitrogen, Carlsbad, CA) selection. Upon transfection, medium containing antibiotics was removed and replaced with DMEM with 10% FBS. Plasmids were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s protocol.

Generation of Fak and Pyk2 stable knockdown cell lines
Fak (CCGGGCCTTAACAATGCGTCAGTTTCTCGAGAAACTGACGCATTGTTAAGGCTTTTTG) and Pyk2 (CCGGATTGAGGACGAAGACTATTACCTCGAGGTAATAGTCTTCGTCCTCAATTTTTTG) shRNA constructs were purchased from Sigma (St. Louis, MO). Scramble shRNA (plasmid #1864) was purchased from Addgene. Scramble shRNA construct in pLKO.1 vectors was subcloned into pLL3.7 (#11795, Addgene), which also encodes GFP. Lentivirus was generated by transfecting 293T cells plated in 15 cm dishes with the following plasmids: 6.3 µg each of pRSV-Rev, pVSVG, and pMDLp/g (gift of Dr. Roger Colbran, Vanderbilt University) and 12.6 µg of the shRNA construct. Ninety-six hours following transfection, the medium was collected, briefly centrifuged, and filtered through a 0.45 µm filter. Lentiviral medium with 10 µg/mL polybrene (Santa Cruz Biotechnology, Dallas, TX) was added to N1 cells which were supplemented with equal volume of 10% FBS DMEM approximately 4 h after lentiviral medium addition. Two days after infection, the medium was removed and replaced with DMEM supplemented with 10% FBS and 10 µg/mL puromycin antibiotic selection (Thermo Fisher Scientific, Waltham, MA). Surviving cells which had stably integrated the construct were used for experiments as indicated.

RT-qPCR
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). Isolated RNA was converted to cDNA using a cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA). Using the generated cDNA, gene expression was analyzed by qPCR using forward and reverse primers (listed below) for the indicated genes and SYBR Green qPCR Master Mix (Invitrogen, Carlsbad, CA). All primers used for qPCR were purchased from Sigma-Aldrich (St. Louis, MO). An ABI Prism 7500 FAST sequence detector (Applied Biosystems) was used to run the qPCR reaction. The ΔΔCT method was used to determine the expression level of a given transcript which was normalized to a housekeeping gene (TATA-binding protein, TBP). The primers used are as follows: Fak (Fwd 5′-CCATGCCCTCGAAAAGCTATG; Rev 5′-TCCAATACAGCGTCCAAGTTCTA), Pyk2 (Fwd 5′-CAGCAGAAGCAGATGGTGGA; Rev 5′-CCCTGTGAACTCCGTGTAGC), and TBP (Fwd 5′-GAAGCTGCGGTACAA TTCCAG; Rev 5′-CCCCTTGTACCCTTCACCAAT).

Drugs and peptides
The dual FAK/PYK2 inhibitor, PF-431396 (PF, Tocris Bioscience, Bristol, UK), was dissolved in sterile dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO). Recombinant mouse leptin was from A. F. Parlow (National Hormone and Peptide Program, Torrance, CA) and Sigma-Aldrich (#L3772, St. Louis, MO) and was dissolved in sterile phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA) or sterile 20 mM Tris/HCl, pH 8.0. The angiotensin II (Sigma-Aldrich, St. Louis, MO) was dissolved in PBS. Tubastatin A Hydrochloride (MCE, MedchemExpress) was dissolved in 20% DMSO/30% PBS/50% polyethylene glycol (PEG)-400 (Sigma-Aldrich).

Immunoprecipitation and immunoblotting
Cell plates for immunoblot analysis were removed from −80°C and briefly thawed on ice prior to addition of RIPA buffer (50 mM TRIS pH: 7.50, 25 mM NaF, 100 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100) supplemented with protease (complete EDTA-free protease inhibitor tablets, Roche, Basel, Switzerland) and phosphatase inhibitors (PhosSTOP phosphatase inhibitor tablets, Roche, Basel, Switzerland). For immunoprecipitation experiments, cells were washed one time with ice-cold PBS prior to addition of RIPA buffer without SDS (50 mM TRIS pH: 7.50, 25 mM NaF, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors. Cells lysates were gently agitated for 15 min at 4°C prior to centrifugation at 17,000 × g for 10 min at 4°C. Total protein concentration of the cleared lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Immunoprecipitation was carried out overnight at 4°C using 1 µg antibody per mg protein in the lysate. The immunoprecipitation experiments using hypothalamic lysates were carried out after crosslinking freshly dissected tissues in 10% formalin for 30 min at 4°C. Tissues were next treated with glycine for 30 min, and then homogenized in RIPA buffer as described above. Cleared lysates were incubated with STAT3 or IgG antibodies for immunoprecipitation. Protein A/G beads were added for another 2 h, and the beads were washed 3 times with RIPA buffer and bound proteins were eluted in 2x laemmli buffer. Lysates were briefly boiled for 3–4 min, except for when blotting for LepRb, prior to being loaded on an SDS-PAGE gel (BioRad, Hercules, CA). Electrophoresed proteins were transferred to a PVDF membrane (Millipore, Burlington, MA). After transfer, membranes were blocked in a solution of bovine serum albumin (Research Products International, Mount Prospect, IL) in PBS prior to incubation with indicated primary antibodies for 1 h at room temperature or overnight at 4°C. Blots were washed with PBST (PBS with 0.1% Tween-20) for 30 min. Blots were then incubated with an HRP- or a fluorophore-conjugated secondary antibody for 1 h at room temperature followed by washing for 30 min with PBST. Blots were developed immediately following the first 30 min wash. Blots incubated with an HRP-conjugated secondary antibody were developed using West Femto ECL Substrate (Thermo Fisher Scientific, Waltham, MA) and visualized using a Bio-Rad ChemiDoc Touch Imaging System (BioRad, Hercules, CA). Blots incubated with a fluorophore-conjugated secondary antibody were visualized using an Odyssey CLx imager (LI-COR Biosciences, Lincoln, NE). Some membranes were incubated at 37°C in stripping buffer (Thermo Fisher Scientific, Waltham, MA) for 1 h prior to re-blocking and re-probing the membrane with another antibody. A relevant molecular weight marker is labeled in each immunoblot.

Immunoblot quantification
ImageJ (https://imagej.nih.gov/) was used for all immunoblot quantifications. To calculate the relative leptin induced pSTAT3/STAT3 values, each quantified pSTAT3 band was normalized to the quantified total STAT3 band. Then, each sample was normalized to the average of the unstimulated control sample(s).

RNAScope and microscopy
RNAScope fluorescent in situ hybridization. To quantify Ptk2, Ptk2b and Lepr mRNA abundance in the hypothalamus, RNAScope fluorescent multiplex v2 assay (Advanced Cell Diagnostics) was used. Fresh brain samples were collected from ad lib fed, overnight fasted, and leptin-treated fasted mice. Following dissection, brain samples were embedded in OCT and immediately frozen on dry ice and stored at −80°C until further processing. A total of 16 µm-thick frozen sections covering the hypothalamus were cryostat sectioned and mounted onto Super Frost Plus slides (Fisher Scientific). In situ hybridization was performed according to the RNAscope fluorescent v2 multiplex kit user manual for fresh frozen tissue (Advanced Cell Diagnostics), using RNAscope Probe-Mm-Ptk2-C1 (#511721), RNAscope Probe-Mm-Ob-r-C2 (#402731-C2), and RNAscope Probe-Mm-Ptk2b-C3 (#506861-C3).

Image capture. Confocal Z-stacks comprised of 1.0-μm optical sections through triple-labeled sections were captured at ×20 magnification using a Leica SP8 confocal laser scanning system attached to a motorized inverted microscope (Leica Microsystems). Fluorophores were detected by four lasers at wavelengths of 405, 488, 561, and 640 nm.

Analysis of mRNA transcripts. Cells in each area (DMH, VMH, and ARH) were analyzed using Leica LAS X Life Science Microscope Software (Leica Microsystems). A fixed threshold and uniform roi was applied to all images, but separately for each channel and for each nucleus. Pixels above background were measured in optical sections through the entirety of each cell (indicated by a defined nucleus) and averaged per cell, to calculate animal averages and group means.

Co-localization analysis of transcripts. To examine whether there was a difference in the percentage of co-expression of LepR/Pyk2/Fak transcripts within a nucleus, images were taken on a Leica SP8 confocal laser scanning system at ×20 magnification. The total number of cells expressing either LepR, Pyk2 and/or Fak was counted in sections of the DMH, VMH and ARH for each group, and the mean percentage of triple-labeled (LepR+Pyk2+Fak), double-labeled (LepR+Pyk2, LepR+Fak, or Pyk2+Fak), or single-labeled (LepR, Pyk2, or Fak) cells was calculated.

Single-cell RNA sequencing analysis
Processed single-cell RNA sequencing data of the hypothalamus57 was downloaded from GEO (GSE87544). All analyses were conducted using the R package Seurat (v. 4.3.0)74,75. The raw reads from 14,437 hypothalamic single cells were normalized and scaled. Top 2000 highly variable genes were identified and used as input for principal component analysis. Top 15 principal components (PCs) were determined to capture the greatest variance using the ElbowPlot function, and were used as input for the clustering analysis (resolution of 0.1) which was visualized with UMAP. Robustness of the clustering was evaluated by varying the PC and resolution parameters. The expression pattern of features of interest was evaluated using the FeaturePlot function. The scripts used to perform the scRNA sequencing analysis are available in Supplementary Note 1.

Statistics and reproducibility
All data are presented as the mean ± standard error of the mean (SEM) with a p value < 0.05 considered statistically significant. Unpaired and two-tailed t-test and one- and two-way ANOVA followed by Tukey’s or Sidak multiple comparison test were applied. For N1Stat3Luc analysis (Fig. 3j, k), following transformation of the data, a nonlinear regression was used. All statistical analyses were conducted using GraphPad Prism (GraphPad, La Jolla, CA). p values are indicated in the figures. Sample sizes are indicated in figure legends. No statistical method was used to predetermine sample size. For the studies that involve stereotaxic surgeries into the lateral ventricle, the animals that tested negative during the water intake test (Angiotensin II test) were excluded from the study. Mice that were transduced with AAV particles were analyzed after the completion of the experiments and negatively targeted animals were excluded from the post hoc analysis. Allocation of samples or animals were random. The Investigators were not blinded to allocation during experiments and outcome assessment except for the experiments presented in Figs. 4, 5c and Supplementary Figs. 4, 5.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript entitled “The focal adhesion kinases regulate leptin action and the weight reducing effect of HDAC6 inhibition”, Hadley and colleagues follow up on their recent published work demonstrating that pharmacological HDAC6 inhibition by TubA leads to peripheral production of a yet unidentified factor that increases hypothalamic leptin sensitivity and reduces body weight in Diet induced obese mice. In the present study they find that the increase in hypothalamic leptin sensitivity in this model requires the focal adhesion kinases FAK and Pyk2 and that these kinases sensitize leptin signaling by interacting and phosphorylating Stat3, the main downstream regulator of leptin action.\nSpecifically:\n- Using comparative in silico analysis of hypothalamic transcriptome profiles obtained experimentally to those of the\nConnectivity Map database, the authors identify FAK and Pyk2 as candidates for mediating the leptin sensitizing effect of\nTub-A treatment.\n- Using pharmacological inhibition of focal kinases and genetic deletion of Pyk2 the authors demonstrate that the central\naction of these focal adhesion kinases is required for the anti-obesity effect of TubA.\n- In cell culture assays, the authors confirm that leptin can activate Stat3 in a Jak2 independent manner and that Stat3\nactivation (ie phosphorylation) can be mediated by FAK or Pyk2.\n- Finally, in further in vivo studies the authors determine that FAK or Pyk2 are expressed in LepR expressing neurons, that\nPyk2 expression is increased by fasting in an ARC Lepr neuron population, that pharmacological inhibition of focal kinases\nor genetic ablation of Pyk2 attenuates pharmacological leptin induced activation of stat3 and its short-term anorectic effects\nin a refeeding paradigm.\nIn general, the study is well constructed and logically presented and within the confine of my expertise I find no major issues\nwith both the in vivo and in vitro experimental approaches and data.\nSome clarifications and modifications in the presentation would benefit the manuscript:\n1) The initial model should be better described in the abstract and introduction. As currently written the reader is led to\nbelieve that inhibitors of the cytosolic deacetylase HDAC6 increase central leptin sensitivity directly, which is not what the\nauthors have demonstrated in their previous publication nor what they are actually proposing here. The authors should make\nit much clearer that the manuscript is about the hypothalamic effects of an unknown peripheral factor induced by HDAC6\ninhibition by TubA and NOT by HDAC6 inhibition by TubA directly on the hypothalamus. Maybe adding a schematic\nrepresentation of the model as a first panel in figure 1 or as a supplemental figure could be useful.\nThe discussion should also be clearer in that respect and address this upfront rather than in a convoluted way in the last\nparagraph. As written, the discussion seems to imply that the study was motivated by the search of a non-canonical JAK2-\nindependent but STAT3-dependent leptin signaling mechanisms when instead it was initially motivated by understanding\nthe leptin sensitizing effect of a pharmacologically induced peripheral factor, leading to the identification of a JAK2-\nindependent but STAT3-dependent leptin signaling mechanisms of potential physiological relevance.\n2) There is no method section for the in silico analysis. The first paragraph of the result describing this analysis should be\nmore concise and most of the methodological description should be moved to the method sections were some of its aspects\nshould be expanded and more detailed.", "reviewer_2": "The authors examined potential roles of Fak and Pyk2 in central leptin actions, independent of JAK2 pathway. Both in vitro and in vivo evidence was provided to try to support the possibility that Fak/Pyk2 is a novel downstream signal of leptin, contributes to stat3 phosphorylation in leptin-responsive neurons, and mediates leptin-induced anorexia. While the\nhypothesis or model, if proven true, is quite significant and novel, the evidence as presented in the current manuscript falls\nshort and is not as convincing. A number of issues were noted as detailed below.\n1. A major concern is the study did not use a cell type-specific (e.g. LepR+ neurons) Fak or Pyk2 deletion model to really\nexamine the functional involvement of these factors in central leptin actions. Data obtained from the global Pyk2 KO mice\ncould not exclude the possibility that Pyk2 in peripheral tissues are somehow involved.\n2. Fig. 2a-b is missing important control groups: vehicle+vehicle, and vehicle+PF. Without such groups, data as presented\nare very hard to interpret.\n3. For all body weight data, absolute body weight values should be presented, instead of the % change. The different\n%change could be due to different baseline body weight when the treatment started.\n4. Pyk2 KO mice showed similar body weight as WT mice after a 24-week HFD feeding, while a previous study (ref#28)\nreported a dramatic weight increase in Pyk2 KO mice after a 8-week HFD feeding compared to WT controls. The authors\nshould show the body weight curves for the entire 24-week HFD study. The lack of body weight phenotype in HFD-fed mice\nsuggests a minor role of Pyk2 in leptin signaling, which actually does not support the hypothesis.\n5. The protein interactions observed in vitro should be also examined in vivo using mouse hypothalamic tissues. In addition,\nis this interaction regulated by leptin, fasting, HFD feeding, etc?\n6. Fig. 4c, f, and i are not convincing at all to support the statement that the majority of LepR+ cells at least express either\nFak or Pyk2.\n7. In Fig. 5c, PF pretreatment did not blunt leptin-induced stat3 phosphorylation. It is probably due to the short (30 min) leptin\naction. In Fig. 5h, Pyk2 KO mice only blunted leptin-induced stat3 phosphorylation at 120 min time point. The same time\npoints should have been checked in Fig. 5c. This also brings up another possibility that is worth examining: whether JAK2\nand FAK/Pyk2 pathways mediate leptin signaling at different time points. Given the focus of this manuscript, such a question\nshould be addressed to further enhance the significance of the study.\n8. The study seems to be exclusively done in male animals without examining similar phenotypes in female mice. At the\nvery least, this limitation should be acknowledged.", "reviewer_3": "Hadley et al. used a combination of mouse genetics and in vitro cell culture systems to examine whether focal adhesion\nkinases (FAK and Pyk2) mediate leptin action in body weight regulation. This study appears to be a continuation of the\nauthors' previous study that revealed improved leptin action as a mechanism underlying the body weight reducing effect of\nHDAC6 inhibition. Physiologically, the current studies presented data supporting that body weight reducing effect of HDAC6\ninhibition and food intake reducing effect were both blunted by pharmacological inhibition of FAK and Pyk2, and by genetic\nknock out of Pyk2. Additional extensive cell culture assays showed that FAK and Pyk2 interacted with STAT3 and were\nrequired for leptin action on activating pSTAT3. Although the study on potential mediators of leptin signaling is important and\nof the effect of HDAC6 inhibition is significant, there appear to be some major concerns on the consistency issues regarding\nthe proposal mechanism and the observed results.\n1) If FAK and Pyk2 represent real important mediators of leptin induced pSTAT3 action, then Pyk2 KO or pharmacological\ninhibition of FAK and Pyk2 should produce increased feeding and body weight. Loss of function of leptin signaling is known\nto produce robust effects on hyperphagia and obesity as previously shown in hypothalamic STAT3 KO, leptin receptor point\nmutation causing specific defective STAT3 signaling etc. However, the data presented in this study with Pyk2 KO and\ninhibition of FAK and Pyk2 both failed to produce any effect on feeding or body weight, which argue against the key\nhypothesis the authors proposed that FAK and Pyk2 mediate leptin action on pSTAT3 signaling.\n2) The colocalization data on FAK and PyK2 with leptin receptors are also not convincing. To bolster the authors' claim, it is\nnecessary to produce additional in vivo data on hypothalamic or leptin receptor specific KO of FAK and Pyk2 (baseline body\nweight as well as the effect of leptin action) that can be used to support the claim.\n3) It is not clear what is the functional relationship between FAK and Pyk2. The role of FAK is purely estimated from relative\ndifference in the degree of body weight reduction of HDAC6 inhibition between Pyk2 and TubA pharmacology, which may\nnot be reliable at all given the potential differences between mouse genetics and pharmacology. Pharmacological effects of\nPF between control and Pyk2 KO mice related to feeding and body weight may be able to tease out the effect of PF\ninhibition of FAK.\n4) Supplementing the data presented in Fig 5, alternative data on pSTAT3 immunostaining of the hypothalamic section will\nreveal details of changes in pSTAT3 responses, which will add more confidence to the observed findings.\n5) It will be more helpful if the authors could provide more information on the background and function of FAK and Pyk2 as\nwell as speculation on how these kinases can be involved in leptin signaling.", "reviewer_4": "This is a very interesting manuscript, original and well achieved. However, there several key questions and some\nimprovement to deal with before accepting the manuscript\n1) PYK2 and FAK came out from the transcriptomic in DIO mice treated with TubA, however, it is unclear whether the level of\nthe two proteins increased with respect of Stat3 activation. Confirmation that the Pyk2 and Fak proteins and activity\nincreased with Phospho-Stat3 in hypothalamic neurons of DIO-induced mice treated with TubA is required. This can be\ndone by Immunohistochemistry for example.\n2) How does cytoplasmic HDAC6 inhibition links the increase of the Pyk2 and Fak mRNA level? What is the working\nhypothesis? The authors focused on the roles of the kinases on Stat3 activation, but the main finding that HDAC6 inhibition\nresults in the change of the mRNA levels remains unmet. This link can be confirmed in in vitro experiments using Pyk2 and\nFak cell lines expressing that are exposed to TubA or Leptin. Are Pyk2 and Fak mRNA directly or indirectly regulated by\nHDAC6? What are the HDAC6 targets modified by TubA that modulate mRNA expression. Does it happen at transcription\nlevel or at posttranscription level? Effect of TubA in the presence and absence of Actinomycin D in any adequate cell\nmodel as suggested above would help in unraveling this hypothesis\n3) In the Fig. 2a, mice were under HFD for the treatment with TubA. However; it is unclear whether the diet started with the\nTubA treatment at the same time or whether mice were previously under HFD for several weeks or days before starting the\nTubA (Veh or PF) treatment. The procedure is not well described and should be indicated in the material and.\nmethods section and in the legend.\n4) TubA restores leptin sensitivity via FAK and PYK2. However, it is unclear if LepR is required for Leptin to induce FAK and\nPYK2. The authors must treat db/db mice with TubA (with or without PF) and monitor the weight changes overtime as\nachieved in the Fig. 2a. In vitro experiments, should confirm the role of Leptin in the effect of LepR\n5) Why did PYK2 KO have normal food intake?\n6) In Fig. 3, experiments using ShRNA against HDAC6 are required to confirm the effect of Leptin on the upregulation of\nFAK and PYK2.\n7) In the literature, some articles reveal that HDAC6 directly interacts with Stat3. If so, this confirms the link between HDAC6\nand Leptin receptor signaling, and that, in anorexigen neurons, leptin may regulate Stat3 via association/dissociation of\nHDAC6 with Stat3. This mechanism can synergize with the upregulation of FAK and PYK2. This may also explain the\nsimilarities between TubA and Leptin\n8) A figure showing a schema summarizing the mechanism unraveled is useful.\n9) Were Genuine Western Blotting (uncropped and with molecular weights) deposited in repository? Can the authors\nprovide the original raw data?"}, "author_response": "Introduction to the revised manuscript and reviewers’ points\nWe thank the reviewers for their constructive criticism and appreciate their unanimous tone expressing the\nsignificance of our work. To address the questions raised by the reviewers, we conducted new experiments\nand revised the text to make it both more concise and clearer. The main novelty in the revised manuscript is\nnow listed in the last figure (Fig. 6) where we show that hypothalamus specific knockdown of focal adhesion\nkinases blunts leptin action, leads to hyperphagic obesity, and attenuates the anti-obesity effect of HDAC6\ninhibitors. The revised manuscript has 6 main figures and 10 supplementary figures and 2 supplementary\ntables. We also included an illustration as requested by the reviewers, summarizing the main theme of the\ncurrent study and considering the previous findings. Below, we present a point-by-point response to\nindividual reviewer comments.\nReviewer #1 (Remarks to the Author):\nIn the manuscript entitled “The focal adhesion kinases regulate leptin action and the weight reducing effect\nof HDAC6 inhibition”, Hadley and colleagues follow up on their recent published work demonstrating that\npharmacological HDAC6 inhibition by TubA leads to peripheral production of a yet unidentified factor that\nincreases hypothalamic leptin sensitivity and reduces body weight in Diet induced obese mice. In the\npresent study they find that the increase in hypothalamic leptin sensitivity in this model requires the focal\nadhesion kinases FAK and Pyk2 and that these kinases sensitize leptin signaling by interacting and\nphosphorylating Stat3, the main downstream regulator of leptin action.\nSpecifically:\n- Using comparative in silico analysis of hypothalamic transcriptome profiles obtained experimentally to\nthose of the Connectivity Map database, the authors identify FAK and Pyk2 as candidates for mediating the\nleptin sensitizing effect of Tub-A treatment.\n- Using pharmacological inhibition of focal kinases and genetic deletion of Pyk2 the authors demonstrate\nthat the central action of these focal adhesion kinases is required for the anti-obesity effect of TubA.\n- In cell culture assays, the authors confirm that leptin can activate Stat3 in a Jak2 independent manner and\nthat Stat3 activation (ie phosphorylation) can be mediated by FAK or Pyk2.\n- Finally, in further in vivo studies the authors determine that FAK or Pyk2 are expressed in LepR\nexpressing neurons, that Pyk2 expression is increased by fasting in an ARC Lepr neuron population, that\npharmacological inhibition of focal kinases or genetic ablation of Pyk2 attenuates pharmacological leptin\ninduced activation of stat3 and its short-term anorectic effects in a refeeding paradigm.\nIn general, the study is well constructed and logically presented and within the confine of my expertise I find\nno major issues with both the in vivo and in vitro experimental approaches and data.\nSome clarifications and modifications in the presentation would benefit the manuscript:\n1) The initial model should be better described in the abstract and introduction. As currently written the\nreader is led to believe that inhibitors of the cytosolic deacetylase HDAC6 increase central leptin sensitivity\ndirectly, which is not what the authors have demonstrated in their previous publication nor what they are\nactually proposing here. The authors should make it much clearer that the manuscript is about the\nhypothalamic effects of an unknown peripheral factor induced by HDAC6 inhibition by TubA and NOT by\nHDAC6 inhibition by TubA directly on the hypothalamus. Maybe adding a schematic representation of the\nmodel as a first panel in figure 1 or as a supplemental figure could be useful.\nThe discussion should also be clearer in that respect and address this upfront rather than in a convoluted\nway in the last paragraph. As written, the discussion seems to imply that the study was motivated by the\nsearch of a non-canonical JAK2- independent but STAT3-dependent leptin signaling mechanisms when\ninstead it was initially motivated by understanding the leptin sensitizing effect of a pharmacologically\ninduced peripheral factor, leading to the identification of a JAK2- independent but STAT3-dependent leptin\nsignaling mechanisms of potential physiological relevance.\nResponse: The manuscript was revised to reflect these points. Specifically, the revised abstract and\nintroduction clearly state that the effect of HDAC6 inhibitors on leptin sensitization and weight loss are\nindirect, through their primary action in the periphery, and the leptin sensitization occurs by an unknown\nsystemic factor. The revised discussion also emphasizes this two-site-of-action mechanism of HDAC6\ninhibitors in the beginning. The discussion is further revised to reflect how the study was motivated in that\nthe hypothalamic transcriptional profile of TubA and leptin were used to identify focal adhesion kinases as\npotential regulators of leptin action. In the revised manuscript, we further discuss the faulty conclusion of a\nrelated recent publication on the direct action of HDAC6 in leptin receptor signaling (discussed further\nbelow). Finally, as suggested by the reviewer, we added a schematic illustration of the main conclusion of\nthe manuscript: HDAC6 inhibition in the periphery induces the expression of the focal adhesion kinases in\nthe hypothalamus which acts as STAT3 kinases downstream of leptin receptor.\n2) There is no method section for the in silico analysis. The first paragraph of the result describing this\nanalysis should be more concise and the details of the analysis were moved to the methods section.\nResponse: The results section describing the in-silico analysis was revised to be more concise and the\ndetails of the analysis were moved to the methods section.\nReviewer #2 (Remarks to the Author):\nThe authors examined potential roles of Fak and Pyk2 in central leptin actions, independent of JAK2\npathway. Both in vitro and in vivo evidence was provided to try to support the possibility that Fak/Pyk2 is a\nnovel downstream signal of leptin, contributes to stat3 phosphorylation in leptin-responsive neurons, and\nmediates leptin-induced anorexia. While the hypothesis or model, if proven true, is quite significant and\nnovel, the evidence as presented in the current manuscript falls short and is not as convincing. A number of\nissues were noted as detailed below.\n1. A major concern is the study did not use a cell type-specific (e.g. LepR+ neurons) Fak or Pyk2 deletion\nmodel to really examine the functional involvement of these factors in central leptin actions. Data obtained\nfrom the global Pyk2 KOmice could not exclude the possibility that Pyk2 in peripheral tissues are somehow\ninvolved.\nResponse: In order to directly test the role of central focal adhesion kinases in leptin receptor signaling,\nwe conducted new experiments using a genetic and a pharmacological approach. We now provide new\ndata showing that hypothalamus-specific knockdown of PYK2 and FAK in wild type mice impedes leptin\nsignaling, results in hyperphagia and obesity, and blocks the anti-obesity effect of two structurally unrelated\nHDAC6 inhibitors (Fig. 6 and Supplementary Fig. 7). Furthermore, we provide new results in Fig. 5c\nshowing that central infusion of the dual FAK/PYK2 inhibitor to lean wild type mice significantly reduces\nleptin-induced STAT3 phosphorylation in the arcuate nucleus, dorsomedial hypothalamus and the\nventromedial hypothalamus. Because of the redundant role and expression of PYK2 and FAK, we designed\nAVV vectors that knockdown both genes in the hypothalamus. Likewise, we used the dual inhibitor\nthroughout our studies. The individual contribution of PYK2 versus FAK to the leptin receptor signaling is\nstudied in our cell culture studies reported in Fig. 3h and Supplementary Fig3 e-h.\n2. Fig. 2a-b is missing important control groups: vehicle+vehicle, and vehicle+PF.\nResponse: We included the missing controls in the revised Figure 2a, b.\n3. For all body weight data, absolute body weight values should be presented, instead of the % change. The\ndifferent %change could be due to different baseline body weight when the treatment started.\nResponse: We now report absolute body weight values in all graphs and include % change in body weight\nto account for variability across cohorts. Differences in initial body weights can lead to misleading\nstatistics—for instance, a 10% loss represents a 5g reduction in a 50g mouse but only a 4g loss in a 40g\nmouse. In mixed cohorts (e.g., 50g and 40g mice), this variability increases the standard error, potentially\nobscuring significant weight changes despite comparable relative weight loss (~10%).\n4. Pyk2 KO mice showed similar body weight as WT mice after a 24-week HFD feeding, while a previous\nstudy (ref#28) reported a dramatic weight increase in Pyk2 KO mice after a 8-week HFD feeding compared\nto WT controls. The authors should show the body weight curves for the entire 24-week HFD study. The\nlack of body weight phenotype in HFD-fed mice suggests a minor role of Pyk2 in leptin signaling, which\nactually does not support the hypothesis.\nResponse: We now report the growth curves of the WT and PYK2 KO cohorts on HFD in the revised\nFig. 2c. Yu et al. previously reported that PYK2 KOs gained significantly more weight on HFD. In their study,\nYu et al. exposed the mice to HFD at ages ranging 5-9 week-of-age whereas we weaned mice on to HFD\nwhen they are 4-week-old. Whether this difference explains the varying results in the growth curves of the\ncohorts is unknown. We did not observe a difference in the overall body weights of the WT versus PYK2 KO\nmice, and we think one potential explanation for this phenotype is compensation of the absence of PYK2 by\nFAK, which is ubiquitously expressed.\nOur new results obtained in wild-type adult mice where we knocked down both genes specifically in the\nhypothalamus shows that focal adhesion kinases are required for body weight regulation. Knocking down\nthe hypothalamic expression of PYK2 and FAK attenuated leptin signaling and resulted in hyperphagic\nobesity on mice consuming regular diet or HFD. These results are in agreement with the central hypothesis\nof our manuscript that focal adhesion kinases mediate leptin action.\n5. The protein interactions observed in vitro should be also examined in vivo using mouse hypothalamic\ntissues.\nResponse: In revised Fig. 3d, we show that PYK2 and FAK interact with STAT3 in mouse total\nhypothalamic lysates in agreement with the in vitro results presented in Fig. 2c. Furthermore, the interaction\nof either kinase with STAT3 significantly increased upon leptin treatment.\n6. Fig. 4c, f, and i are not convincing at all to support the statement that the majority of LepR+ cells at least express either\nFak or Pyk2.\nResponse: We now provide new images documenting the colocalization of LepR with FAK or PYK2 in\nthe hypothalamus. The images in the first version of manuscript were not visually compelling because of the\nrelative expression difference of the transcripts in different regions. For example, PYK2 expression is much\nstronger in the DMH and more abundant than LepR. However, in the ARC, the signal from LepR is stronger\nthan PYK2. Because we presented the images acquired at the same settings for each probe, the strong\nsignals from, eg., LepR in the ARC masked the relatively weaker PYK2 signal. In the revised images, we\npresented signal from single channels and merged images and clearly identified the colocalization of signals\nfrom different transcripts. We also marked LepR+ neurons that also express FAK and PYK2 in all three\nregions analyzed (ARC, VMH, and DMH).\n7. In Fig. 5c, PF pretreatment did not blunt leptin-induced stat3 phosphorylation. It is probably due to the short (30 min) leptin\naction. In Fig. 5h, Pyk2 KO mice only blunted leptin-induced stat3 phosphorylation at 120 min time point. The same time\npoints should have been checked in Fig. 5c. This also brings up another possibility that is worth examining: whether JAK2\nand FAK/Pyk2 pathways mediate leptin signaling at different time points. Given the focus of this manuscript, such a question\nshould be addressed to further enhance the significance of the study.\nResponse: In order to address this question and also study the neuroanatomy of leptin-induced STAT3\nphosphorylation, we conducted new experiments where central vehicle or PF (dual PYK2/FAK inhibitor)\ninfusion was followed by PBS or leptin injection in lean wild-type mice. We analyzed the P-STAT3 signal 30,\n60, and 120 minutes after leptin injection. New data in revised Fig. 5c document that inhibition of FAK/PYK2\nactivity leads to time and region-specific attenuation of leptin-induced STAT3 phosphorylation. For example,\nwe detected decreased pSTAT3 signal as early as 30min after leptin treatment in the ARC and VMH in PF-\npretreated mice. These results agree with the data presented in Fig. 5b, 5g, and 5h obtained from total\nhypothalamic lysates. PF-induced attenuation of leptin-induced hypothalamic STAT3 phosphorylation is the\nlikely cause of the resistance to leptin action as presented in Fig. 5a. Finally, the new data in Fig. 6 provides\ngenetic support for the role of focal adhesion kinases in leptin signaling.\n8. The study seems to be exclusively done in male animals without examining similar phenotypes in female\nmice. At the very least, this limitation should be acknowledged.\nResponse: We appreciate the reviewer’s concern on the lack of evaluation of sex as a biological\nfactor. In the revised discussion, we acknowledge this limitation and expand the discussion on the\nexpression profile of FAK and PYK2 in male and female hypothalamus. The revised manuscript now reads:\n“Our study has utilized male mice for all the experiments and therefore has the limitation of not directly\naddressing the role of sex as a biological factor. FAK or PYK2 do not display a significant expression\ndifference in male versus female human hypothalamus samples in the Genotype-Tissue Expression (GTEx)\nPortal (p=0.091 and 0.59, for PYK2 and FAK, respectively by Wilconox test), and we previously showed that\nHDAC6 inhibition induces weight loss also in female DIO mice47, suggesting that the role for focal adhesion\nkinases in leptin signaling is independent of sex. However, future research will reveal the potential\nexpression difference and role of FAK or PYK2 in LepRb positive neurons of male and female animals.”\nReviewer #3 (Remarks to the Author):\nHadley et al. used a combination of mouse genetics and in vitro cell culture systems to examine whether\nfocal adhesion kinases (FAK and Pyk2) mediate leptin action in body weight regulation. This study appears\nto be a continuation of the authors' previous study that revealed improved leptin action as a mechanism\nunderlying the body weight reducing effect of HDAC6 inhibition. Physiologically, the current studies\npresented data supporting that body weight reducing effect of HDAC6 inhibition and food intake reducing\neffect were both blunted by pharmacological inhibition of FAK and Pyk2, and by genetic knock out of Pyk2.\nAdditional extensive cell culture assays showed that FAK and Pyk2 interacted with STAT3 and were\nrequired for leptin action on activating pSTAT3. Although the study on potential mediators of leptin signaling\nis important and of the effect of HDAC6 inhibition is significant, there appear to be some major concerns on\nthe consistency issues regarding the proposal mechanism and the observed results.\n1) If FAK and Pyk2 represent real important mediators of leptin induced pSTAT3 action, then Pyk2 KO or\npharmacological inhibition of FAK and Pyk2 should produce increased feeding and body weight. Loss of\nfunction of leptin signaling is known to produce robust effects on hyperphagia and obesity as previously\nshown in hypothalamic STAT3 KO, leptin receptor point mutation causing specific defective STAT3\nsignaling etc. However, the data presented in this study with Pyk2 KO and inhibition of FAK and Pyk2 both\nfailed to produce any effect on feeding or body weight, which argue against the key hypothesis the authors\nproposed that FAK and Pyk2 mediate leptin action on pSTAT3 signaling.\nResponse: The leptin response tests in PYK2 KO mice and PF-treated wild type mice were\nconducted under acute conditions where we observed attenuated leptin-induced STAT3 phosphorylation\nand suppression of feeding. Lack of PYK2 is known to be compensated by FAK, and FAK is more\nubiquitously expressed throughout the hypothalamus compared to PYK2 (Fig. 4). Therefore, we targeted\nthe expression of FAK and PYK2 in the adult hypothalamus. In the revised manuscript, we provide new data\ndocumenting that hypothalamic knockdown of PYK2 and FAK ablates leptin action and leads to\nhyperphagic obesity (Fig. 6). These results are in agreement with other data in the manuscript showing that\nacute central inhibition of focal adhesion kinases also ablate leptin-induced STAT3 phosphorylation and\nleptin’s anorectic response. Taken together, the results strongly argue for an indispensable role for\nPYK2/FAK in leptin receptor signaling and central regulation of body weight. please also see our response\nto reviewer #2, question 4 above.\n2) The colocalization data on FAK and PyK2 with leptin receptors are also not convincing. To bolster\nthe authors' claim, it is necessary to produce additional in vivo data on hypothalamic or leptin receptor\nspecific KO of FAK and Pyk2 (baseline body weight as well as the effect of leptin action) that can be used\nto support the claim.\nResponse: We now provide new colocalization data in revised Fig. 4 showing the co-expression of\nthe kinases with leptin receptor in the hypothalamic feeding centers. Depending on the sub-hypothalamic\nnucleus analyzed the relative expression of the transcripts highly varied. For example, PYK2 is highly\nexpressed in the DMH compared to its expression in the arcuate nucleus, where LepR expression is more\nabundant. Therefore, in the colocalization images presented in the first version of the manuscript the strong\nsignal from the LepR in the ARC, e.g., masked the relatively weaker signal from PYK2 and FAK. In the new\nimages provided, we clearly labelled individual neurons where co-expression of different transcripts were\nlabelled.\nWe also conducted the experiment requested by the reviewer and knocked down FAK and PYK2\nexpression in the hypothalamus of adult mice (Fig. 6), which led to hyperphagic obesity as expected of\nablation of leptin signaling. Please also see our response to the question #1.\n3) It is not clear what is the functional relationship between FAK and Pyk2. The role of FAK is purely\nestimated from relative difference in the degree of body weight reduction of HDAC6 inhibition between Pyk2\nand TubA pharmacology, which may not be reliable at all given the potential differences between mouse\ngenetics and pharmacology. Pharmacological effects of PF between control and Pyk2 KO mice related to\nfeeding and body weight may be able to tease out the effect of PF inhibition of FAK.\nResponse: We agree with the reviewer’s concern on this point. Accordingly, to directly assess the\nrole of hypothalamic FAK and PYK2, we knocked down both genes in adult mouse hypothalamus. New\nresults presented in Fig. 6 show that these kinases are indispensable regulators of leptin signaling and body\nweight (Please also see our response to the question #1). Notably, knockdown of these kinases ablated the\nweight reducing effect of two different HDAC6 inhibitors, TubA and ACY775 (Fig. 6l-o and Supplementary\nFig. 8). Collectively, these data are in agreement with the pharmacological findings showing that dual\ncentral inhibition of focal adhesion kinases also block TubA-induced weight loss (Fig. 2a, b).\n4) Supplementing the data presented in Fig 5, alternative data on pSTAT3 immunostaining of the\nhypothalamic section will reveal details of changes in pSTAT3 responses, which will add more confidence to\nthe observed findings.\nResponse: We analyzed the time course response of lean wild-type mice to leptin by conducting P-\nSTAT3 immunostaining in hypothalamic sections. The new data in revised Fig. 5c show that central dual\ninhibition of FAK and PYK2 block leptin-induced STAT3 phosphorylation in leptin responsive hypothalamic\nregions including the DHM, VMH, and ARC. These data are in agreement with the rest of the\npharmacological and genetic intervention data obtained from cell culture and mice and point to the role of\nfocal adhesion kinases in leptin receptor signaling. Please also see our response to reviewer #2, question 7.\n5) It will be more helpful if the authors could provide more information on the background and function of\nFAK and Pyk2 as well as speculation on how these kinases can be involved in leptin signaling.\nResponse: We revised the introduction section to incorporate more background on the focal\nadhesion kinases. We cited the literature on STAT3 being a substrate for FAK and PYK2 while noting that\nthese interactions were demonstrated in cell lines and contexts not related to leptin signaling.\nReviewer #4 (Remarks to the Author):\nThis is a very interesting manuscript, original and well achieved. However, there several key questions and\nsome improvement to deal with before accepting the manuscript\n1) PYK2 and FAK came out from the transcriptomic in DIO mice treated with TubA, however, it is unclear\nwhether the level of the two proteins increased with respect of Stat3 activation. Confirmation that the Pyk2\nand Fak proteins and activity increased with Phospho-Stat3 in hypothalamic neurons of DIO-induced mice\ntreated with TubA is required. This can be done by Immunohistochemistry for example.\nResponse: To respond the reviewer’s request, we conducted immunostaining against FAK and\nPYK2 using sections from the mouse hypothalamus. Unfortunately, none of the antibodies we tested\nworked for immunohistochemistry: In order to assure that the signals we detected were specific, we\ncompared the sections from mice that received AAV-shScr to the ones from mice that received AAV-\nshFAK/PYK2. None of the antibodies we tested showed a decreased signal in the shFAK/PYK2 groups,\nalthough these hairpins effectively knock-down the expression of these transcripts. The staining signals\nwere diffuse and broad for FAK, and fiber-like staining was detected for PYK2, despite the expected\nintracellular and predominantly membrane-proximal staining profile. Furthermore, the IHC staining pattern\ndid not match the signals obtained from in situ hybridization. For example, while PYK2 expression is much\nhigher in the DMH compared to ARC or VMH (e.g., Fig. 4b), the antibodies we tested did not reveal such a\ndifference. We concluded therefore that the signals from these antibodies were not specific (Figure 1).\nFurthermore, we noticed that the phospho-PYK2 antibody was detecting a band at the expected molecular\nweight even in PYK2 KO mice, questioning its validity at least in the detection of the protein in the\nhypothalamus.\nIn order to address the reviewer’s question, we include the data on the qPCR results documenting\nthat TubA administration results in increased hypothalamic expression of focal adhesion kinases in the\nrevised manuscript.\n2, 6, 7) How does cytoplasmic HDAC6 inhibition links the increase of the Pyk2 and Fak mRNA level? What\nis the working hypothesis? The authors focused on the roles of the kinases on Stat3 activation, but the\nmain finding that HDAC6 inhibition results in the change of the mRNA levels remains unmet. This\nlink can be confirmed in in vitro experiments using Pyk2 and Fak cell lines expressing that are exposed to\nTubA or Leptin. Are Pyk2 and Fak mRNA directly or indirectly regulated by HDAC6? What are the HDAC6\ntargets modified by TubA that modulate mRNA expression. Does it happen at transcription level or at\nposttranscription level? Effect of TubA in the presence and absence of Actinomycin D in any adequate cell\nmodel as suggested above would help in unraveling this hypothesis.\n6) In Fig. 3, experiments using ShRNA against HDAC6 are required to confirm the effect of Leptin on the\nupregulation of FAK and PYK2.\n7) In the literature, some articles reveal that HDAC6 directly interacts with Stat3. If so, this confirms the link\nbetween HDAC6 and Leptin receptor signaling, and that, in anorexigen neurons, leptin may regulate Stat3\nvia association/dissociation of HDAC6 with Stat3. This mechanism can synergize with the upregulation of\nFAK and PYK2. This may also explain the similarities between TubA and Leptin\nResponse: We apologize for the confusion on the mechanism of anti-obesity effect of HDAC6\ninhibitors. HDAC6 inhibition leads to leptin sensitization and weight loss in a cell non-autonomous manner.\nThat is, HDAC6 inhibition primarily in the adipose tissue induces leptin sensitization in the hypothalamus\nwhich in turn triggers weight loss. We previously showed that there is a protein-based hormonal signal that\nis secreted into the plasma in response to HDAC6 inhibition that in turn potentiates leptin signaling1. We are\ncurrently working to identify this factor, which is beyond the scope of the work presented here.\nHDAC6 does not regulate leptin receptor signaling cell autonomously as we have previously\npublished1. In order to bolster this conclusion, we conducted new experiments presented in Supplementary\nFigures 9 and 10. Inhibition of endogenous HDAC6 (we tested three different HDAC6 inhibitors) or\noverexpression of HDAC6 in LepR expressing cells do not alter leptin receptor signaling1 (Supplementary\nFig. 9). Furthermore, infusion of TubA directly into the brain of DIO mice potently inhibits brain HDAC6\nactivity but does not alter food intake or reduce obesity1. These results collectively suggest that the effect of\nHDAC6 inhibition on leptin sensitization is indirect. We apologize for the confusing language used in the first\nversion of the manuscript, which is now made explicit in the introduction and discussion of the revised\nversion, as also requested by reviewer 1.\nA recent report suggested that HDAC6 inhibits and the acetyltransferase P300 activates leptin\nreceptor signaling2. We repeated the core experiments in that manuscript and now show that those findings\nwere results of an artifact (false positives): The authors claimed that leptin receptor acetylation by the\nacetyltransferase P300 increased leptin receptor activity. We identified that P300 or the related\nacetyltransferase CBP increase the expression of other genes when they are co-transfected\n(Supplementary Fig. 10). The authors have never tested the LepRb gene expression when LepRb was co-\ntransfected with P300. P300 (and also CBP) appear to increase leptin-induced STAT3 phosphorylation\nsimply because there is more LepRb when it is co-transfected with P300. The revised manuscript now\nreads:\n“During the revision of our manuscript, another group also showed that HDAC6 inhibitors reduce\nobesity by improving central leptin sensitivity but proposed a cell-autonomous mechanism of leptin\nsensitization52. The authors suggested that leptin receptor is a substrate of HDAC6 and that acetylation of\nleptin receptor increased leptin-induced STAT3 phosphorylation. We repeated some of the key experiments\nproposed by this group. In direct contrast to the authors’ findings, we did not detect an effect of HDAC6\noverexpression in leptin-induced STAT3 phosphorylation. In accordance with our previous findings30, none\nof the three different HDAC6 inhibitors we tested increased leptin receptor signaling. Notably, we noticed\nthat the authors’ finding that the acetyltransferase p300 potentiated leptin-induced STAT3 phosphorylation\nwas due to an artifact of the overexpression system used. While p300 seems to potentiate leptin-induced\nSTAT3 phosphorylation (Supplementary Fig. 10), we noticed that p300 overexpression led to increased\nexpression of LepRb from the co-transfected plasmid. This effect was not restricted to LepRb as p300\noverexpression also increased GFP and LacZ gene expressions (Supplementary Fig. 10), suggesting that\nthe effect of p300 overexpression on leptin-induced STAT3 phosphorylation is an artifact of this in vitro\nsystem. In agreement with the cell nonautonomous effect of HDAC6 inhibition on leptin sensitization and\nweight loss, we previously showed that central infusion of HDAC6 inhibitors to DIO mice strongly inhibit\nHDAC6 activity but does not reduce body weight. These results combined with the observations that\nneuronal30 or AgRP-specific52 HDAC6 deletion does not prevent obesity whereas adipose specific HDAC6\nKOs are resistant to weight gain and hyperglycemia30 strongly supports our two-site-of-action model, where\nperipheral HDAC6 inhibition is coupled to central leptin sensitization though a yet unidentified factor.”\n3) In the Fig. 2a, mice were under HFD for the treatment with TubA. However, it is unclear whether the diet\nstarted with the TubA treatment at the same time or whether mice were previously under HFD for several\nweeks or days before starting the TubA (Veh or PF) treatment. The procedure is not well described and\nshould be indicated in the material and. methods section and in the legend.\nResponse: These mice were under HFD to induce obesity for several weeks prior to the start of the\nTubA treatment. We now provide the absolute body weights of these cohorts in Supplementary Fig. 2a. The\nrevised methods and figure legend now clearly explains the experimental protocol and states that these\nmice were DIO wild type mice kept on HFD.\n4) TubA restores leptin sensitivity via FAK and PYK2. However, it is unclear if LepR is required for Leptin to\ninduce FAK and PYK2. The authors must treat db/db mice with TubA (with or without PF) and monitor the\nweight changes overtime as\nachieved in the Fig. 2a. In vitro experiments, should confirm the role of Leptin in the effect of LepR\nResponse: We previously showed that ob/ob or db/db mice are resistant to the anti-obesity effect of\nHDAC6 inhibitors including TubA (Cakir et al, Nat Metab . 2022 Jan;4(1):44-59.) In order to understand\nwhether TubA treatment increased focal adhesion kinase expression in a LepR dependent manner, we\ntreated db/db mice with vehicle or TubA and anlayzed the hypothalamic FAK and PYK2 expressions by\nqPCR. The results show that TubA did not significantly alter the expression of either transcript in the\nhypothalamus (FAK: 1.00 ± 0.045 Veh vs. 1.00 ± 0.027 TubA, p=0.92; PYK2: 1.00 ± 0.049 Veh vs. 1.16 ±\n0.117 TubA, p=0.26 by t-test) suggesting a leptin dependent mechanism. The revised discussion of the\nmanuscript now reads:\n“Following TubA administration, the hypothalamic expression of FAK and PYK2 increases suggesting a\npotential transcriptional regulation. Furthermore, this regulation appeared to be leptin dependent as\nTubA failed to increase the hypothalamic expression of either kinase in db/db mice (FAK: 1.00 ± 0.045\nVeh vs. 1.00 ± 0.027 TubA, p=0.92; PYK2: 1.00 ± 0.049 Veh vs. 1.16 ± 0.117 TubA, p=0.26 by t-test)\nwhich helps explain why HDAC6 inhibition does not exert weight loss in db/db mice.”\n5) Why did PYK2 KO have normal food intake?\nResponse: Normal food intake and body weight of the PYK2 KO mice, compared to the WT mice, is\npossibly due to embryonic compensation by FAK, which is ubiquitously expressed throughout the\nhypothalamus including the PYK2 positive cells; only 1-2 % of the LepR+ neurons were FAK negative\n(Supplementary Fig. 4). However, upon dual hypothalamic knockdown of both kinases in adult mice, we\nobserved loss of response to leptin and hyperphagic obesity (Fig. 6), documenting the central role of these\nenzymes in the regulation of feeding and body weight. Please also see our response 4 to Reviewer 2 and\nresponse 1 to Reviewer 3 above.\n8) A figure showing a schema summarizing the mechanism unraveled is useful.\nResponse: We present an illustration summarizing the main conclusion of the manuscript. In summary,\nHDAC6 inhibition in the periphery induces expression of the focal adhesion kinases which in turn\npotentiates LepRb-STAT3 signaling.\n9) Were Genuine Western Blotting (uncropped and with molecular weights) deposited in repository? Can\nthe authors provide the original raw data?\nResponse: Uncropped immunoblots will be deposited as supporting figures as required by Nature\nCommunication guidelines."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All my comments have been addressed adequately.", "reviewer_2": "The authors largely addressed my previous comments with new data. A few minor issues rise in the new data as presented\nwhich should be addressed.\n1. Fig. 3c is missing scale bars.\n2. Fig. 6d, the calorie intake was only measured during week 3, and the calories consumed by control chow mice and control\nHFD mice are very similar which is unusual.", "reviewer_3": "The authors have largely addressed the concerns raised.\nTwo minor points if addressed would further improve the manuscript.\n1) the pictures presented in Fig. 5C are not matched, and since Arc sections at various anterior to posterior levels exhibit\ndramatic differences in pSTAT3 response to leptin, it is strongly suggested that the authors use matched sections to\nconvincingly demonstrate the difference.\n2) the authors argue that the single Pyk2 KO didn't show obesity is due to developmental compensation from FAK;\nhowever, if this is the case, why this KO was still able to produce resistance to the weight-reducing effect of TupA?\nIn principle, these two effects should be consistent with each other.", "reviewer_4": "The authors have addressed all of the reviewer’s comments"}, "author_response": "Response To Reviewer Comments\nReviewer #2 (Remarks to the Author):\nThe authors largely addressed my previous comments with new data. A few minor issues rise in the new\ndata as presented which should be addressed.\n1. Fig. 3c is missing scale bars.\nResponse: The missing scale bars are added to the corresponding figures.\n2. Fig. 6d, the calorie intake was only measured during week 3, and the calories consumed by control chow\nmice and control HFD mice are very similar which is unusual.\nResponse: We note the reviewer’s comment that the mice exposed to HFD consume more calories\nthan mice on control chow. For example, we reported earlier1 that after 16 weeks the difference in the total\ncaloric intake of mice is 184.7±1.9 vs. 218.1±7.6 kcal (chow vs. HFD, mean±SEM), which is only about 18%\ndifference. While acute 24h food intake measurement in home cages may not readily reflect this difference\n(as is reported in what is now Figure 7d -Fig. 6d in the earlier version), which is contributed by among other\nfactors control chow crumbs, the mice exposed to HFD cumulatively consumed more calories contributing\nto their weight gain.\nReviewer #3 (Remarks to the Author):\nThe authors have largely addressed the concerns raised.\nTwo minor points if addressed would further improve the manuscript.\n1) the pictures presented in Fig. 5C are not matched, and since Arc sections at various anterior to posterior\nlevels exhibit dramatic differences in pSTAT3 response to leptin, it is strongly suggested that the authors\nuse matched sections to convincingly demonstrate the difference.\nResponse: We updated the corresponding figure with matching brain sections.\n2) the authors argue that the single Pyk2 KO didn't show obesity is due to developmental compensation\nfrom FAK; however, if this is the case, why this KO was still able to produce resistance to the weight-\nreducing effect of TupA? In principle, these two effects should be consistent with each other.\nResponse: We argued that the normal body weight of global Pyk2 KO mice might be due to\ncompensation by FAK. In diet-induced obese wild-type mice, our data suggest that TubA treatment\nincreases the hypothalamic expression of both PYK2 and FAK to bolster leptin signaling, while in Pyk2 KO\nmice display only FAK expression can increase, which explains the partial resistance of the Pyk2 KO DIO\nmice to TubA treatment. This result is supported by more robust attenuation of leptin signaling by dual\nknowndown of Pyk2 and Fak compared to the effect of their individual knock down.\nReferences\n1. Çakır, I. et al. Histone deacetylase 6 inhibition restores leptin sensitivity and reduces obesity. Nat Metab\n4, 44–59 (2022)."}]

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