bluuebunny/crossref_metadata_2025 huggingface dataset:
1146-P: Persistent Graft Function after Islet Allotransplantation Into Prevascularized Sernova Cell Pouch Device: Preliminary Results from the University of Chicago
Authors: BACHUL PIOTR J. , GENERETTE GABRIELA S. , PYDA JORDAN S. , BOREK PETER , PEREZ-GUTIERREZ ANGELICA , GOLAB KAROLINA , BASTO LINDSAY , PEREA LAURENCIA , TIBUDAN MARTIN , JUENGEL BRADEN , KUMAR JAYANT , THOMAS CELESTE C. , PHILIPSON LOUIS H. , FUNG JOHN , WITKOWSKI PIOTR
Published: June 2021

Introduction: Pre-vascularized Sernova Cell Pouch (SCP) device was developed to provide an optimal environment for cell/islet engraftment in the extravascular space. A first-in-human pilot study showed SCP safety/islet vascularization; however, conditions did not allow detectable islet function. We present preliminary results of phase I/II study testing safety and efficacy of modified SCPs in islet allotransplantation (ITx).

Material and Methods: 6 patients with longstanding T1DM and multiple severe hypoglycemic events (SHEs) were enrolled. Immunosuppression (Thymoglobulin, Tacrolimus, and Mycophenolate) was initiated 3 weeks after abdominal wall SCP surgical implantation. ITx occurred at 3 weeks and 6 months after implantation. Sentinel SCPs were explanted for histopathological evaluation 3 months after transplant. Islet graft function was assessed based on continuous glucose monitoring (CGM), c-peptide, and insulin requirement.

Results: First patient presented with persistent stimulated serum c-peptide at 6 months after 1st and 2nd ITx into SCP. After 2nd ITx, glucose control improved substantially reaching optimal target values for CGM with only 5% of Time Below Range (TBR). Second patient at 3 months after 2nd ITx had positive stimulated serum c-peptide (0.48 ng/mL) with reduction of HbA1c from 10.6% to 7.6%, decreased insulin requirement from 49 to 28 u/day, improved CGM with TBR <4%, and reduction in Time Above Range (from 75% to 48%). Two patients await ITx and evaluation.

Conclusion: Persistent islet graft function with sustained blood levels of c-peptide, reduction of HbA1c, improved CGM parameters, reduction of SHEs, and decreased total daily insulin requirement was achieved in first 2 patients after ITx into abdominal wall SCPs. Modified approaches, including suspending islets in serum, optimizing islets/dose, and smaller pellet volumes, will likely improve engraftment and outcomes.

Disclosure

P. J. Bachul: None. B. Juengel: None. J. Kumar: None. C. C. Thomas: None. L. H. Philipson: Advisory Panel; Self; Nevro Corp., Research Support; Self; Provention Bio, Inc. J. Fung: None. P. Witkowski: None. G. S. Generette: None. J. S. Pyda: None. P. Borek: None. A. Perez-gutierrez: None. K. Golab: None. L. Basto: None. L. Perea: None. M. Tibudan: None.

Funding

JDRF; Sernova, Corp.
115-LB: Liver-Derived FGF21 Mediates the Promoting Effect of Glucagon Receptor Antagonism on Beta-Cell Regeneration in Type 2 Diabetic Mice
Authors: CUI XIAONA , WEI TIANJIAO , FENG JIN , YANG JIN , YAN HAI , WEI RUI , HONG TIANPEI
Published: June 2021

Objective: Based on the clear role for glucagon in the pathogenesis of diabetes, glucagon-blocking therapies have been pursued as a therapeutic strategy for this disease. Here, we used REMD 2.59, a human antagonistic glucagon receptor (GCGR) monoclonal antibody (mAb), to clarify the effects of GCGR mAb in promoting pancreatic β cell regeneration in type 2 diabetes (T2D), and its potential mechanism.

Methods: GCGR mAb was administrated weekly for 4 weeks in db/db mice and HFD/STZ-induced T2D mice, including different lineage-tracing mice. The plasma insulin and glucagon were determined by using ELISA. The evaluation of islet morphology was performed with immunofluorescent analysis. GCGR mAb was also treated in the systemic Fgf21 gene knockout (Fgf21-/-) and hepatocyte-specific Fgf21 gene knockout (Fgf21Hep-/-) T2D mice.

Results: Treatment with GCGR mAb lowered blood glucose, improved glucose tolerance, and elevated plasma glucagon and insulin levels in both db/db mice and HFD/STZ-induced T2D mice. The β cell dedifferentiation as indicated by β cell lineage-tracing was blocked by GCGR mAb. The β cell proliferation as indicated by insulin+ and BrdU+ cells was boosted by GCGR mAb. The α-to-β cell conversion, as evaluated by glucagon+ insulin+ cells and confirmed by α cell lineage-tracing, was facilitated by GCGR mAb. After GCGR mAb treatment, some insulin+ cells were located in the duct region or even colocalized with duct cell markers, suggestive of duct-derived β cell neogenesis that was also verified by Ngn3+ cell lineage-tracing. Notably, GCGR mAb could still improve glucose tolerance and increase β cell mass, but these effects were attenuated in Fgf21-/- and Fgf21Hep-/- T2D mice compared with WT T2D mice.

Conclusions: GCGR mAb promotes β cell regeneration via inhibiting β cell dedifferentiation and promoting β cell self-replication, α-to-β cell conversion and duct-derived β cell neogenesis in T2D mice, which is partly mediated by liver-derived FGF21.

Disclosure

X. Cui: None. T. Wei: None. J. Feng: None. J. Yang: None. H. Yan: Board Member; Self; REMD Biotherapeutics, Other Relationship; Spouse/Partner; REMD Biotherapeutics. R. Wei: None. T. Hong: Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Novo Nordisk, Sanofi.

Funding

National Natural Science Foundation of China (81830022)
1163-P: Exercise as a Strategy to Decrease Pancreatic ß-Cell Senescence
Authors: CARAPETO PRISCILA , AGUAYO-MAZZUCATO CRISTINA
Published: June 2021

Age increases the prevalence of type 2 diabetes (T2D) and insulin resistance (IR) accelerates the appearance of senescent β-cells, one of the hallmarks of aging. This suggests that senescence could contribute to the development of T2D and be a novel therapeutic target. In addition to improving blood glucose levels, exercise has been shown to attenuate all hallmarks of aging. However, the effects of exercise upon β-cell senescence remain to be determined. Our hypothesis is that exercise can decrease β-cell senescence and we aim to verify the effects of exercise upon senescence prevention and reversibility under control and IR conditions. Retired breeder male C57Bl/6 mice (6-8 months of age) were randomly assigned into groups (n=5/group): sedentary, exercised (treadmill running, 1 h, 5 times per week, for 2 weeks, at 17 cm/s and 5% incline) and IR (S961, 40nM/week for 2 weeks); exercise was performed at the same time, before or after IR to assess prevention and reversibility. ANOVA Brown-Forsythe and Welch were used to compare groups. Under control conditions, exercise decreased β-cell senescent markers (-58.1% p21Cip1 mRNA and -29% β-Galactosidase activity through FACS sorting), improved glucose tolerance (-13.6% in total area under the curve during intraperitoneal glucose tolerance test) and preserved suppression of basal insulin secretion when compared to sedentary (+39.3% insulin). Exercise performed before and during IR decreased mRNA levels of senescence marker and effector p16Ink4a by 41.7% and 80% respectively. Basal insulin secretion was decreased, and glucose-stimulated insulin secretion restored. When performed after IR, exercise decreased p16Ink4a mRNA by 62.4% and restored basal insulin secretion with no change in the stimulation index.

In conclusion, exercise decreased the load of senescent cells in insulin resistant animals. Its effects were more effective before and during IR but it was also able to reverse some of the deleterious effects of IR converting it into a novel β-cell anti-aging strategy.

Disclosure

P. Carapeto: None. C. Aguayo-mazzucato: Consultant; Self; eGenesis.

Funding

Joslin Diabetes Center (to C.A-M.); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (to P.C.)
1165-P: Alterations of Hepatic Mitochondrial Respiration in Murine Models of Diabetes and Nonalcoholic Fatty Liver Disease (NAFLD)
Authors: DEWIDAR BEDAIR , REINADOFUNDO MICHELLE , ENGLISCH CORNELIA , MASTROTOTARO LUCIA , PESTA DOMINIK , RESS CLAUDIA , ESPOSITO IRENE , RODEN MICHAEL
Published: June 2021

Diabetes mellitus (DM) accelerates progression of fatty liver to nonalcoholic steatohepatitis (NASH) and fibrosis. Mitochondrial function is also frequently impaired in DM, but maybe differently regulated in NAFLD. Thus, we examined hepatic mitochondrial respiration during DM and NAFLD development.

Two-days old male C57BL/6j mice received streptozotocin (STZ) or vehicle (placebo, PLC) and then high-fat diet (HFD) or continued regular chow diet (RCD) from week 4 to 12, yielding 4 models (n=8 each): obesity [PLC+HFD], DM [STZ+RCD], NASH [STZ+HFD] and control [PLC+RCD]. Histology and immunohistochemistry were performed to describe NAFLD, including the NAFLD activity score (NAS) derived from steatosis, lobular inflammation and hepatocellular ballooning. High-resolution respirometry and hyperinsulinemic-euglycemic clamps were used to measure mitochondrial respiration and insulin sensitivity.

DM and NASH showed >200% higher blood glucose levels (p<0.0001 vs. control). The NAS (mean±SEM) was not different in obese (1.38±0.32; p>0.99), gradually higher in DM (2.50±0.42; p=0.063) and markedly increased in NASH (3.25±0.45; p<0.01) compared to control (1.00±0.27). Main contributors to NAS were steatosis and hepatocellular ballooning in NASH (p<0.001 and p<0.05 vs. control) and hepatocellular ballooning in DM (p<0.01 vs. control). Maximal uncoupled β-oxidation-associated respiration was 1.6-, 1.7-, and 1.9-fold higher in obesity, DM and NASH compared to control (all p<0.01). Mitochondrial density was decreased in both DM and NASH (p<0.05 vs. control), whereas β-oxidation-dependent mitochondrial leak control ratio was not different between all groups. Insulin-stimulated glucose disposal was 44% and 66% lower in obesity and NASH (both p<0.001 vs. control).

In conclusion, hyperglycemia and high-fat diet feeding associate with higher respiration but lower content of hepatic mitochondria, which may both contribute to progression of NAFLD.

Disclosure

B. Dewidar: None. M. Reinadofundo: None. C. Englisch: None. L. Mastrototaro: None. D. Pesta: None. C. Ress: None. I. Esposito: None. M. Roden: Advisory Panel; Self; Allergan plc, Bristol-Myers Squibb Company, Novo Nordisk A/S, Research Support; Self; Boehringer Ingelheim International GmbH, Danone Nutricia, Sanofi-Aventis Deutschland GmbH.
1168-P: Serum MicroRNA-122 and -192 Are Increased in Adolescents with NAFLD: Potential Role as Biomarkers
Authors: SHORT KEVIN R. , PALLE SIRISH KUMAR , HELLMAN DIANA S. AMAYA , JIANG SHAONING , TRYGGESTAD JEANIE B. , GARCIA ESTEFANIA FEMATT
Published: June 2021

The best available blood biomarker for nonalcoholic fatty liver disease (NAFLD) is alanine aminotransferase (ALT), though it does not reliably distinguish NAFLD severity. Alternate biomarkers have been tested in adults, but fewer in children. We tested serum microRNA-122 (miR-122) as a biomarker for NAFLD in adolescents since it inhibts hepatic lipogenesis in animals and is exported from liver to blood with disease progression. The only prior study of miR-122 in children produced mixed results. We also measured miR-192, -130b, and -155 as they have roles in hepatic lipogenesis. Participants were boys and girls, 12-20 y, with biopsy-confirmed NAFLD (n=23), and peers without NAFLD who were either obese (Ob, n=22), or normal weight (NW, n=24). RNA was extracted from serum from all participants and from liver biopsies from the NAFLD group. Serum miR-122 and -192 were 12- and 10-fold higher (p< 0.01), respectively, in NAFLD patients compared to both Ob and NW controls. Within the NAFLD group, serum abundances of miR-122 and -192 were inversely correlated with their liver values (r = -0.51, p = 0.002 and r = -0.40, p = 0.067, respectively), and positively correlated with histological scores for steatosis (r = 0.39 and 0.28, respectively) and fibrosis (r = 0.56 and 0.54, respectively). mIR-122 and -192 content were also positively correlated with ALT (r = 0.78 for both) and with each other in both serum (r = 0.92) and liver (r = 0.74), suggesting similar regulation. Areas under the receiver operating curve for distinguishing NAFLD was 0.95 for miR-122 and -192. Serum miR-130b, which regulates PGC-1 alpha, was 2.6-fold higher in serum from NAFLD patients versus both control groups, while serum miR-155, which regulates lipogenesis and inflammation, did not differ in NAFLD. Serum miR-130b and miR-155 values were not correlated with their corresponding values in liver biopsies. These results support the potential use of miR-122 and -192 as biomarkers for the presence and severity of NAFLD in adolescents.

Disclosure

K. R. Short: None. S. Palle: None. D. S. Amaya hellman: None. S. Jiang: None. J. B. Tryggestad: None. E. Fematt garcia: None.

Funding

Oklahoma Shared Clinical & Translational Resource
1170-P: Dapagliflozin, a Sodium Glucose Cotransporter 2 Inhibitor, Attenuates the Development of Nonalcoholic Steatohepatitis-Associated Hepatocellular Carcinoma
Authors: LEE MINYOUNG , KIM YOUNG-EUN , LEE SEJEONG , KIM HAE KYUNG , LEE SOOK JUNG , KIM JIWON , KIM SUN-WOO , KIM RYEONG-HYEON , PARK HYUNKI , LEE HAYNGKYU , KANG EUN SEOK
Published: June 2021

Nonalcoholic steatohepatitis (NASH) is the most prevalent chronic liver disease worldwide and increases the risk for hepatocellular carcinoma (HCC) in developed countries. However, no medications have been approved to protect against NASH-driven HCC. Dapagliflozin, a sodium glucose cotransporter-2 (SGLT-2) inhibitor, improves hyperglycemia by promoting glycosuria. We investigated whether dapagliflozin prevents NASH-driven HCC using a mouse model. Diethylnitrosamine (25 mg/kg) was intraperitoneally injected into 2-week-old C57BL6/J male mice, followed by either normal chow diet or 60% high-fat diet (HFD) for 30 weeks with or without dapagliflozin, 0.003% (w/w). HFD-fed mice without dapagliflozin had an increased number of tumor on liver surface compared with chow-fed mice (77.0 vs. 12.5/per mouse; n=6 per group; p=0.01). The number of liver tumors was significantly lower in dapagliflozin treated group (19.5/per mouse in dapagliflozin-treated HFD-fed mice; n=6) than that in HFD-fed mice without dapagliflozin (p=0.03). Dapagliflozin treatment numerically reduced body weight and fasting blood glucose compared with HFD-fed mice without dapagliflozin (41.0 vs. 40.2 g, p=0.94 for body weight, and 196.3 vs. 170.8 mg/dL, p=0.34 for fasting blood glucose). In addition, several human HCC cell lines (Huh 7, HepG2, and Hep3B) expressed SGLT-2 protein. Thus, it is possible that dapagliflozin might directly modulate HCC development via SGLT-2 inhibition.

In conclusion, dapagliflozin attenuates NASH-associated HCC development possibly via its direct effect on HCC.

Disclosure

M. Lee: None. H. Lee: None. E. Kang: None. Y. Kim: None. S. Lee: None. H. Kim: None. S. Lee: None. J. Kim: None. S. Kim: None. R. Kim: None. H. Park: None.
1190-P: Diabetes Activates Pancreatic Stellate Cells through RAGE Signaling in Pancreatic Ductal Adenocarcinoma
Authors: UCHIDA CHIAKI , MIZUKAMI HIROKI , KUDO KAZUHIRO , YAMAMOTO YASUHIKO
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive carcinoma with poor prognosis. Its poor prognosis is ascribed to the extensive fibrotic stroma. Pancreatic stellate cells (PSCs) are major contributors to PDAC fibrosis. Type 2 diabetes (T2D) is an established risk factor for PDAC. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs) and the signaling through receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Nevertheless, the role of RAGE in PSCs activation was not evaluated in PDAC with T2D. First, PSCs were isolated from male obese T2D model, db/db (db) and db/m (C) mice at 12 weeks of age. mRNA expression of the makers for PSCs activation like TGF-β1, αSMA and collagen type1A1 were significantly increased in PSCs isolated from db compared to C (p<0.01, respectively). To evaluate the implication of RAGE, PSCs isolated from C57BL/6 (WT) and RAGE deleted mice (RKO) were stimulated with 12.5-100 μg/ml AGEs. AGEs increased mRNA expression of TGF-β1, αSMA and collagen type1A1 in WT-PSCs with dose dependent manner (p<0.01, respectively) despite no increase in mRNA expressions in RKO-PSCs. Furthermore, PSCs were isolated from high fat diet induced obese WT and RKO for 8 weeks. The mRNA expressions of TGF-β1, αSMA and collagen type1A1 were comparable between two groups. We evaluated surgically resected human PDAC subjects with T2D (PDT2D, 33 cases) and without T2D (PDNT2D, 83cases). αSMA-labeled PSCs significantly surrounded the ducts with dysplasia in PDT2D compared to PDNT2D (p<0.05). The nuclear expression of NF-κB in stromal cells, which was downstream molecules of RAGE, was significantly higher in PDT2D than PDNT2D (p<0.05). Collectively, RAGE signaling was activated in PSCs in T2D, which could be implicated in severe stromal fibrosis in PDAC. Inhibition of RAGE signaling in PSCs may link to a new therapy, in which the stromal fibrosis and cytokine production are reduced in PDAC with T2D.

Disclosure

C. Uchida: None. H. Mizukami: None. K. Kudo: None. Y. Yamamoto: None.

Funding

Japan Society for the Promotion of Science (JP17K17579)
1193-P: Investigating the Role of Intestinal L-Cell Secretagogin in Circadian GLP-1 Secretion in a Mouse Model of Type 2 Diabetes
Authors: BIANCOLIN ANDREW , BRUBAKER PATRICIA L.
Published: June 2021

The intestinal L-cell hormone Glucagon-like Peptide-1 (GLP-1) increases glucose-dependent insulin secretion and is thus used in the treatment of T2D. GLP-1 secretion from the L-cell exhibits a circadian pattern in rodents and varies by time-of-day in humans. We have shown that KD of the SNARE-regulatory protein, secretagogin (SCGN) prevents time-dependent GLP-1 release. SCGN KD in ß-cells also decreases insulin secretion and SCGN levels are reduced in T2D islets. We thus hypothesized that SCGN is decreased in the L-cell in T2D, causing alterations in the physiological pattern of GLP-1 secretion. High-fat diet, low-dose streptozotocin (HFD-STZ) mice, a model of T2D, exhibited increased fasting glucose (5.5 vs. 15.6 mmol/L; p<0.001) and decreased ß-cell area (by 75%, p<0.001). Unexpectedly, oral glucose tolerance tests conducted in 4-hour intervals through the 24-hr day showed that HFD-STZ mice exhibit a 10x increase in GLP-1 responses (400 vs. 4000 pg/ml*60 min, p<0.001) and exhibit an anti-phasic rhythm to controls. Basal GLP-1 levels remained unchanged between HFD-STZ and controls. Unexpectedly, insulin responses were not reduced in HFD-STZ mice, suggesting that increased GLP-1 levels could act as a compensatory mechanism. HFD-STZ mice also exhibited hyperglucagonemia compared to control, with increased glucagon secretion across timepoints following glucose administration (–243 vs. 800 pg/ml*60 min, p<0.001). RT-qPCR of intestinal mucosa showed an anti-phasic Scgn rhythm in the HFD-STZ model, with a 1.5-fold increase in average expression across timepoints (p<0.05), which could explain the drastically increased GLP-1 secretion. These data show that GLP-1 secretion is both increased and arrhythmic in a murine model of T2D, and that this occurs in association with an unanticipated increase in Scgn expression. Further investigation into the role of SGCN in the L-cell may serve as an avenue for therapeutics aimed at increasing endogenous GLP-1 release.

Disclosure

A. Biancolin: None. P. L. Brubaker: None.

Funding

Canadian Institutes of Health Research (PJT-15308)
1203-P: High-Dose Erythropoietin and Cardiac Function in Mice
Authors: LEE JEEYOUNG , NOGUCHI CONSTANCE T.
Published: June 2021

Erythropoietin (EPO) is the cytokine required for erythropoiesis. EPO stimulated increase in hematocrit elevates blood viscosity that may cause hemodynamic risks in the cardiovascular system, promote ischemic heart disease, and stroke. Herein, we studied the physiological effect of high dose exogenous EPO (i.p. 3000 U/kg, 3 times/week for three weeks) with high fat diet (HFD) on cardiac function using echocardiographic measurements in young wild-type (WT) male and female mice and older obese WT male mice with fat mass >45%. In young male mice, but not in female and older obese male mice, EPO treatment with HFD decreased body weight and fat mass accumulation compared with saline, although all groups showed similar EPO stimulated increases in hematocrit. EPO reduced ejection fraction and fractional shortening of left ventricle (LV) in both young male and female mice. This is consistent with the previous report that mice with chronic overexpression of human EPO have decreased fractional shortening of LV. In contrast, older obese male mice have reduced basal ejection fraction and fractional shortening that remain unchanged with EPO treatment indicating that the changes in basal ejection fraction and fractional shortening with EPO treatment in younger mice were not due to EPO stimulated increase in red cell mass. EPO decreased peak velocity of blood flow in the pulmonary artery in all groups indicative of increased hematocrit promoting higher blood viscosity. In addition, EPO increased LV posterior wall thickness in diastole in older male obese mice. This is consonant with observations that high hematocrit is related to concentric left ventricular hypertrophy in human. These data suggest that high dose EPO treatment may be associated with possible cardiac dysfunction with decreased ejection fraction and fractional shortening of LV, and alteration of left ventricular geometry.

Disclosure

J. Lee: None. C. T. Noguchi: None.
1205-P: Baricitinib Inhibition of Jak/STAT Pathway Changed Immune Composition in Adipose Tissue and Improved Metabolism in Diet-Induced Obese Mice
Authors: LIAN ZEQIN , PERRARD XIAOYUAN , BALLANTYNE CHRISTIE M. , WU HUAIZHU
Published: June 2021

Jak/STAT pathway plays key roles in inflammation and is highly activated in adipose tissue (AT) in obesity. Recently, inhibition of the Jak/STAT pathway with baricitinib, a Jak1/Jak2 inhibitor, has been demonstrated to accelerate clinical improvements in a trial of hospitalized patients with COVID-19, who had a mean BMI of 32. Currently, we aimed to examine effects of long-term treatment with baricitinib on AT inflammation and metabolism in diet-induced obese mice. Starting from 3 months old, male C57BL/6J mice were fed high fat diet (HFD) and concurrently received daily oral gavage of baricitinib (10mg/kg) or vehicle for 12 weeks. Compared to vehicle control, baricitinib treatment significantly improved insulin resistance examined by insulin tolerance test and increased UCP1 (a key browning marker) expression in perigonadal (pAT) and inguinal AT (iAT), but did not affect weight gain and AT mass. Strikingly, total T cells were reduced by ~50% in iAT and pAT in baricitinib group vs. control group. CD4+ IFNγ-expressing Th1 cells, IL5-expressing Th2 cells, and TNFα-expressing T cells were decreased in pAT, but not in iAT, of mice from baricitinib group vs. control group. Unexpectedly, percentage of TNFα-expressing cells in CD8+ T cells were higher in iAT of baricitinib-treated mice than controls. Finally, baricitinib compared to control reduced M1/M2 ratio in pAT, but not iAT, and reduced eosinophils in iAT and pAT. Baricitinib inhibition of Jak/STAT pathway in diet-induced obese mice improved insulin sensitivity and changed immune cells in AT, with dramatic reductions of T cells in particular, indicating an important role of Jak/STAT-mediated inflammation in metabolism in obesity. Given the fact that obesity and insulin resistance are associated with increased severity of COVID-19, our data also point to the need for further studies on AT immune cells, COVID-19, and baricitinib.

Disclosure

Z. Lian: None. X. Perrard: None. C. M. Ballantyne: Advisory Panel; Self; Amarin Corporation plc, Arrowhead Pharmaceuticals, Inc., Pfizer Inc., Consultant; Self; Amgen Inc., CSL Behring, Intercept Pharmaceuticals, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk, Regeneron Pharmaceuticals Inc., Research Support; Self; Abbott, Amgen Inc., Ionis Pharmaceuticals, Novartis Pharmaceuticals Corporation, Regeneron Pharmaceuticals Inc., Roche Diagnostic USA. H. Wu: None.
1208-P: Caloric Restriction Modified Factors Regulating Lipid Storage and Apoptosis in Inguinal but Not Epididymal Adipose Tissue of 24-Month-Old Male Rats
Authors: VARSHNEY PALLAVI , SCHLEH MICHAEL W. , AHN CHEEHOON , ZHENG AMY , ARIAS EDWARD B. , CARTEE GREGORY D. , HOROWITZ JEFFREY F.
Published: June 2021

Calorie restriction (CR) and the resultant weight-loss are known to improve metabolic health, but the effects of CR on factors regulating white adipose tissue (WAT) metabolic function and remodeling remain unclear. The purpose of this study was to examine the effects of 8 weeks of CR on markers of lipid storage/release, endoplasmic reticulum (ER) stress, and apoptosis in inguinal and epididymal WAT collected from aged rats. We harvested inguinal and epididymal adipose tissue from 24-month-old male Fischer-344 x Brown Norway rats that were either fed an ad libitum chow diet (AL, n=6) or subjected to CR (consuming 35% below AL intake) of the same diet (n=6) for 8 weeks. Using standard immunoblotting methods, inguinal WAT from CR had a greater abundance of phosphorylated hormone sensitive lipase (pHSL) than AL (11.1±6.6 vs. 4.5±3.3 arbitrary units (AU); P=0.05) indicative of elevated lipase activity in CR, and a lower protein abundance of the esterification marker, diacylglycerol acyltransferase (DGAT; 1.0±0.2 vs. 2.4±0.6 AU; P<0.001). Interestingly, protein abundance of the ER stress marker, CCAAT-enhancer-binding protein homologous protein (CHOP) was also significantly lower in CR vs. AL (0.7±0.3 vs. 1.7±0.5 AU; P<0.01), and the protein abundance of Beclin, a marker of autophagy, tended to be lower in CR vs. AL (1.9±0.4 vs. 2.6±0.7 AU, for CR vs. AL; P=0.08). In contrast to these findings for inguinal WAT, there were no detectable differences in protein abundance for any of these markers in epididymal WAT from CR vs. AL.

In summary, CR-mediated responses in WAT of aged rats appeared to be depot-specific and CR may contribute to important metabolic modifications and may impact cellular remodeling of inguinal adipose tissue.

Disclosure

P. Varshney: None. M. W. Schleh: None. C. Ahn: None. A. Zheng: None. E. B. Arias: None. G. D. Cartee: None. J. F. Horowitz: None.

Funding

National Institutes of Health (R01DK077966, P30DK089503, R01AG010026)
1212-P: Adipose Tissue Mitochondrial Function in Humans with Varying Liver Histology
Authors: PAFILI KALLIOPI , KAHL SABINE , PESTA DOMINIK , STRASSBURGER KLAUS , MASTROTOTARO LUCIA , PUETZER-FURMANCZAK JENNIFER , DEWIDAR BEDAIR , SARABHAI THERESIA , ESPOSITO IRENE , SCHLENSAK MATTHIAS , RODEN MICHAEL
Published: June 2021

Adipose dysfunction may drive the development of insulin resistance, type 2 diabetes (T2D) and nonalcoholic (NA) fatty liver disease (NAFLD), comprising NA fatty liver and steatohepatitis (NAFL and NASH). Possible underlying mechanism comprise impaired lipid storage or mitochondrial oxidation favoring increased fatty acid flux to other tissues. Thus, we examined subcutaneous (SAT) and visceral adipose tissue (VAT) mitochondrial capacity in humans with different degrees of insulin resistance and histologically proven NAFLD. Obese people without NAFL (OBE-CON, n=20, 38±8 years, body mass index 53±6 kg/m2, 10% T2D), with NAFL (OBE-NAFL, n=20, 40±8 years, 51±5 kg/m2, 25% T2D) or NASH (OBE-NASH, n=20, 42±10 years, 51±6 kg/m2, 40% T2D) underwent metabolic phenotyping and tissue biopsies. O2 flux rates from different substrates were measured with high resolution respirometry in SAT and VAT. In VAT, maximal uncoupled respiration was lower in OBE-NAFL (least square means (LSM): 0.46 pmol*mg wet weight–1*s–1 [95% confidence interval 0.21;0.71], p<0.05) and OBE-NASH (LSM: 0.51 [0.26;0.76], p<0.001) compared to OBE-CON. Similar differences were seen for state 3 respiration related to complex I (glutamate) or combined complex I and II activity (succinate). In Pearson correlation analysis, combined complex I and II activity in VAT positively correlated with whole-body insulin sensitivity (M value; r= 0.29, p<0.05). VAT mitochondrial content, as assessed from mitochondrial DNA copy number, was not different between the groups. Protein expression of electron transport chain complexes was similar in VAT and SAT of all groups, except for complex IV that tended to be lower in VAT in OBE-NASH than in OBE-CON (p=0.065). Of note, in SAT, oxidative capacity was comparable between groups despite a higher mitochondrial mass in OBE-NASH than in OBE-CON (p=0.045).

In conclusion, oxidative capacity is downregulated in VAT, but not in SAT of obese humans with NAFLD, which may be related to the degree of insulin resistance.

Disclosure

K. Pafili: None. M. Schlensak: None. M. Roden: Advisory Panel; Self; Allergan plc, Bristol-Myers Squibb Company, Novo Nordisk A/S, Research Support; Self; Boehringer Ingelheim International GmbH, Danone Nutricia, Sanofi-Aventis Deutschland GmbH. S. Kahl: None. D. Pesta: None. K. Strassburger: None. L. Mastrototaro: None. J. Puetzer-furmanczak: None. B. Dewidar: None. T. Sarabhai: None. I. Esposito: None.
1222-P: Adipose Tissue Antilipolytic Insulin Sensitivity Protects against Whole-Body Insulin Resistance and Lowers Acylcarnitine Accumulation in Adults with Obesity
Authors: SCHLEH MICHAEL W. , RYAN BENJAMIN J. , AHN CHEEHOON , LUDZKI ALISON , VARSHNEY PALLAVI , GILLEN JENNA B. , HOROWITZ JEFFREY F.
Published: June 2021

The aims of this study were; 1) to assess clinical and subclinical factors associated with insulin-stimulated glucose uptake and 2) compare skeletal muscle lipid profile (untargeted lipidomics) in a sub-cohort of insulin sensitive vs. insulin resistant subjects. 66 obese adults (F=46 [BMI=34±3kg/m2], M=20 [BMI=33±2.9 kg/m2]) with homogeneous body composition completed a 2h hyperinsulinemic-euglycemic clamp, and stable isotope dilution methods to assess glucose rate of disappearance (Glucose Rd: index of insulin sensitivity), fatty acid rate of appearance (FA Ra), and hepatic glucose production (Glucose Ra) under basal and hyperinsulinemic conditions. We also performed an array of metabolic measurements (e.g., hepatic and visceral fat accumulation [via MRI], fat oxidation, lipid profile, circulating cytokines). Covariate analysis (LASSO) identified insulin-mediated suppression of FA Ra, basal glucose Ra, and fat oxidation as the cluster of measurements best predicting Glucose Rd (r=0.76, p<0.01). Insulin-mediated suppression of FA Ra was also the strongest independent predictor of Glucose Rd (r=0.51; p<0.01). As a result of greater adipose tissue anti-lipolytic response to insulin, lower FA delivery to skeletal muscle may improve insulin-mediated glucose uptake via lower muscle lipid accumulation. Importantly, we found the accumulation of several long-chain acylcarnitine species (measure of incomplete β-oxidation: C18:0,18:1,20:1,20:3) to be lower (p<0.05) in muscle samples from an insulin sensitive sub-cohort (Glucose Rd > 8.5 µmol/(min•FFM•[Insulin]); n=10) compared with an insulin resistant sub-cohort (< 7.2 µmol/(min•FFM•[Insulin]); n=8).

In conclusion, our findings indicate that greater adipose tissue anti-lipolytic response to insulin is an important predictor of insulin-mediated glucose uptake - and acylcarnitine accumulation in skeletal muscle may mediate this effect.

Disclosure

M. W. Schleh: None. B. J. Ryan: None. C. Ahn: None. A. Ludzki: None. P. Varshney: None. J. B. Gillen: None. J. F. Horowitz: None.

Funding

National Institutes of Health (R01DK077966, P30DK089503, T32DK007245, F32DK117522); Canadian Institutes of Health Research (DFS146190, 338735)
1223-P: Effects of Bariatric Surgery on Quality of Life, Body Image, and Sex Life in Obese Women
Authors: DI GIOIA LUDOVICO , PERRINI SEBASTIO , BRAUN ANTONIO , ROSSI ERICA , GIORDANO FIORELLA , GUARINI FRANCESCA , SICILIANO EDOARDO , CIGNARELLI ANGELO , LEONARDINI ANNA , NATALICCHIO ANNALISA , LAVIOLA LUIGI , BERTOLINO ALESSANDRO , GIORGINO FRANCESCO
Published: June 2021

Bariatric surgery and consequent weight loss improve metabolic and anthropometric parameters in obese subjects. However, a detailed assessments about the impact of bariatric surgery on quality of life (QoL), body image perception, and sex life is lacking. The aim of this study was to assess the QoL, body image satisfaction and sexual function in 389 female patients with obesity (age 40±10 years, pre-surgery weight 120.4±17.7 kg) before and at least 6 months after bariatric surgery (60% sleeve gastrectomy, 40% gastric bypass). All participants were asked to complete the following online self-report questionnaires seven days before and six months after bariatric surgery: the Body Uneasiness Test (BUT) to assess self-perception of body image, the Short Form-12 (SF-12) to QoL as per the physical (PCS) and mental (MCS) domains, and the Female Sexual Function Index (FSFI) to screen for sexual dysfunctions in women. After bariatric surgery, patients lost 44.5±16.5 kg (p<0.001), with an increase of the PCS (53.3±7.2 vs. 31.9±21.4, p<0.001) and MCS (50.5±8.8 vs. 39.6±12.5, p<0.001) scores, that fell within the normal range of healthy individuals. All patients showed a significant improvement of the global score index domain of the BUT (1.9±1.1 vs. 3.7±0.9, p<0.001). The youngest patients (18-39 years old) who lost at least 40% of the baseline weight obtained a normalization of their body image concern domain results, reaching the reference range for healthy non-obese subjects. After bariatric surgery, patients also had a significant improvement of the FSFI (26.8±9.7 vs. 19.6±8.8, p<0.001), with an increase of non-dysfunctional, sexually active women from 26.2% to 78.2%. Significant correlations were found between the extent of weight loss and the improvement of all the psychometric parameters. Bariatric surgery is an efficient and safe treatment for obesity, and the physical gains related to weight loss are also mirrored by significant improvements in mental and physical health and sexual function.

Disclosure

L. Di gioia: None. A. Natalicchio: Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Sanofi. L. Laviola: Advisory Panel; Self; A. Menarini Diagnostics, Abbott, AstraZeneca, Lilly Diabetes, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis, Speaker’s Bureau; Self; Boehringer Ingelheim International GmbH, Medtronic. A. Bertolino: None. F. Giorgino: Consultant; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Research Support; Self; Lilly Diabetes, Roche Diabetes Care. S. Perrini: None. A. Braun: None. E. Rossi: None. F. Giordano: None. F. Guarini: None. E. Siciliano: None. A. Cignarelli: None. A. Leonardini: None.
1234-P: Impact of Treatment Adherence to Observed Weight Loss in the GLOW Study
Authors: URBAN LORIEN E. , APOVIAN CAROLINE , ARONNE LOUIS , LEIDER HARRY
Published: June 2021

Adherence to weight management treatments is suboptimal. The Gelesis Loss Of Weight (GLOW; NCT02307279) pivotal trial data were used to assess treatment adherence impact on weight loss. The primary objectives of the GLOW study were to assess the effects of an oral superabsorbent hydrogel (OSH) on weight loss (targeted difference of ≥ 3%) and ≥ 5% weight loss responder status in subjects with a body mass index of 27-40 kg/m2. A total of 436 subjects were randomized to 3 capsules twice per day of OSH or placebo taken with 500 mL (16 oz) of water before lunch and dinner while on a hypocaloric diet (-300 kcal/day) for 24 weeks. There were 172 OSH- and 152 placebo-treated subjects who completed the study. Capsules counted at each study visit every 2-4 weeks were used to calculate overall percent treatment adherence (PTA) after the final visit. The PTA impact on weight loss was assessed using ANCOVA models with baseline BMI, fasting glucose and sex as covariates. A logit model with the same covariates was also used to assess the impact of PTA ≥ 95% or < 95% in subjects who achieved ≥ 10% or < 10% weight loss at 24 weeks. In the OSH group, mean PTA for was 97.61% ± 4.68% (SD) ranging from 70% to 111%, and all but 1 subject had PTA ≥ 80%. A significant interaction of ≥ 5% weight loss responder status and PTA was found (P=0.039); therefore, separate ANCOVA models were fit. PTA predicted weight loss at week 24 among the OSH group ≥ 5% responders, but not the ≥ 5% non-responders. Also, a PTA increase of 1 day/week predicted an additional 3.5% weight loss (β = 0.-25 ± 0.11 SE, P=0.024). PTA did not predict weight loss in the placebo group. OSH-treated subjects with ≥ 95% PTA were 4 times more likely to achieve ≥ 10% weight loss compared to those with < 95% PTA (P=0.027, odds ratio=4.27, 95%CI=1.18 to 15.41). These findings suggest that OSH has a binary impact on the treated subjects rather than a uniform effect. Increased PTA during OSH treatment predicted increased weight loss in patients responding to therapy with ≥ 5% weight loss, and not in non-responders or the placebo group.

Disclosure

L. E. Urban: Employee; Self; Gelesis. C. Apovian: Board Member; Self; Abbott, Allergan plc, Gelesis, Novo Nordisk, Nutrisystem, Tivity Health, Xeno Bioscience, Zafgen, Inc. L. Aronne: Other Relationship; Self; Allurion, Eisai & Janssen Pharmaceuticals, ERX Pharmaceuticals, Gelesis, Jamieson Wellness, Myos Corp, Intellihealth Corp, Novo Nordisk, Pfizer Foundation, Real Appeal, Research Support; Self; Astrazeneca, Aspire Bariatrics. H. Leider: Employee; Self; Gelesis.
1241-P: Inhibition of MiR-146a-5p Prevents Mitochondrial Dysfunction and ß-Cell Apoptosis, and Improves Insulin Secretion
Authors: KRISHNAN PREETHI , SYED FAROOQ , WEAVER STACI A. , LEE CHIH-CHUN , EVANS-MOLINA CARMELLA
Published: June 2021

MicroRNAs (miRNAs) are small non-coding RNAs that function as modulators of gene expression. We previously identified upregulation of miR-146a-5p in pancreatic islets treated with proinflammatory cytokines and in human pancreatic sections from individuals with type 1 diabetes. While other studies have associated overexpression of miR-146a-5p with β cell apoptosis and defective insulin secretion, the molecular mechanisms through which these effects are mediated remain elusive. To this end, we transduced MIN6 cells with lentiviral vectors (i) overexpressing and (ii) inhibiting the expression of miR-146a-5p and developed stable cell lines resistant to puromycin. Monoclonal cell populations were developed by single clone selection and then treated with/without proinflammatory cytokines (IL-1β, IFN-γ and TNF-α) for 24 hours. Further characterization of the cells was performed using mRNA sequencing, western blot, RT-PCR of cognate targets and Seahorse mitochondrial functional assays. Overexpression of miR-146a-5p led to increased expression of cleaved caspase-3 and reduced insulin secretion under basal and cytokine-treated conditions, whereas inhibition of miR-146a-5p reversed these effects. Similarly, miR-146a-5p inhibition increased mitochondrial copy number, mitochondrial membrane potential, basal respiration rate, maximal respiration rate, spare respiratory capacity, and ATP production, suggesting a protective effect on the mitochondria, even upon exposure to proinflammatory cytokines. Consistent with these findings, bioinformatic analysis of sequencing data and validation of targets such as MAPT, MYRIP, NRG1, GPX1 also showed enrichment of pathways related to insulin secretion, apoptosis, mitochondrial function. Overall, findings from our study show for the first time that miR-146a-5p upregulation may alter β cell health through reduced mitochondrial function.

Disclosure

P. Krishnan: None. F. Syed: None. S. A. Weaver: None. C. Lee: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc.
1245-P: Characteristic Distribution of GRP78 in Islets and Intraislet Ductal Cells of Diabetic C414A-CRY1 Transgenic Mice
Authors: OKANO SATOSHI , YASUI AKIRA , KANNO SHIN-ICHRO , SATOH KENNICHI , IGARASHI MASAHIKO , NAKAJIMA OSAMU
Published: June 2021

Our preceding studies demonstrated that cysteine414 (zinc-binding site of mCRY1) - alanine mutant mCRY1 transgenic mice (TG mice) exhibit diabetes characterized by the reduction of β-cell proliferation caused by senescence-associated secretory phenotype (SASP)-like characters of β-cells. Not only pancreatic intraepithelial neoplasias (PanINs) with pancreatic duct glands (PDGs) but also intra-islet ducts emerged age-dependently in TG mice (Okano S. et al., 2019). Reportedly GRP78 (also called BiP) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and in lesions of acinar-to-ductal metaplasia (ADM) which is known as precursors of PanIN. As for the islet, GRP78 has been intensively studied with regard to the unfolded protein response (UPR) in the β cells. However, the details of in vivo distribution of GRP78, particularly in endocrine cells outside β cells, are still remain elusive. In this study, we conducted staining experiments for GRP78 in the pancreatic sections of well-matured mice to explore the relation between endocrine cells and intra-islet ducts. The results showed that the expression of GRP78 was higher in PanINs and PDGs than in normal ductal cells in TG mice, suggesting that GRP78 plays important roles in the maintenance of the atypical ductal lesions. In both wild-type and TG mice, GRP78 was observed in the whole islet. The expression of GRP78 was higher in β cells than in alpha and delta cells in both mice. Also, GRP78 was highly expressed in primary intra-islet ducts. Some of the GRP78 high-expressing primary intra-islet ductal cells were co-expressed with chromogranin A, which is known as an endocrine marker. Taken together, our results suggest that the direct trans-differentiation of endocrine cells (possibly β cells) to ductal cells is involved in the generation of the intra-islet ductal cells.

Disclosure

S. Okano: None. A. Yasui: None. S. Kanno: None. K. Satoh: None. M. Igarashi: None. O. Nakajima: None.

Funding

Japan Society for the Promotion of Science (19K07498); Tohoku University
125-LB: Differential Influences of Gastric Bypass and Sleeve Gastrectomy on Glucose-Reducing Effects of DPP-4 Inhibitor
Authors: HONKA HENRI , RAYAS MARIA S. , DEFRONZO RALPH A. , GASTALDELLI AMALIA , SALEHI MARZIEH
Published: June 2021

The two most commonly performed bariatric surgical procedures, gastric bypass (GB) and sleeve gastrectomy (SG), induce diabetes remission before any significant weight loss. While the role of entero-insular axis after SG remains unknown, weight-independent glycemic effects of GB have been attributed to larger systemic appearance of glucose and incretin effect. We examined the effect of increasing endogenous incretins 2-3 fold with a DPP-4 inhibitor on glycemic effects of GB and SG. Glucose and islet-cell secretory responses were measured in 4 subjects with a history of GB, 4 with SG and 4 non-surgical controls (CN) during 50-gram oral glucose ingestion with and without 200 mg sitagliptin orally on two separate days. GB, SG, and CN groups were matched for BMI, FFM, HbA1c, age, fasting glucose and insulin levels; GB and SG were matched for weight loss and time postsurgery; none had diabetes. Compared to SG-treated subjects and controls, subjects with prior GB had lower prandial glycemia (AUCGlucose 3hr) (P < 0.001), and higher AUCGlucagon 3hr (P < 0.0001). The expected differences in prandial insulin response pattern to glucose ingestion among surgical and controls was observed; however, AUCInsulin 3hr and AUCC-peptide 3hr were similar between groups. Sitagliptin did not change AUCGlucose 3hr in GB-treated subjects, whereas the prandial glucose response was reduced by 21 ± 13 % in SG-treated and non-surgical controls by sitagliptin. Enhanced endogenous incretins diminished early glucagon (AUCGlucagon 1hr) in all 3 groups (P < 0.05), improved β-cell glucose sensitivity in CN (P < 0.05), but it had no effect on beta-cell function in GB or SG subjects. Sitagliptin had no effect on insulin clearance or insulin sensitivity in fasting or fed states. These findings indicate that glucose metabolism after SG, similar to non-operated controls, is enhanced by incretins, which are at maximum in GB group and thus not influenced by DPP-4 inhibition.

Disclosure

H. Honka: None. M. S. Rayas: Research Support; Self; National Center for Advancing Translational Sciences. R. A. Defronzo: Other Relationship; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Novo Nordisk, Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Speaker’s Bureau; Self; AstraZeneca, Novo Nordisk. A. Gastaldelli: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Consultant; Self; Eli Lilly and Company, Inventiva Pharma, Research Support; Self; Gilead Sciences, Inc., Speaker’s Bureau; Self; Novo Nordisk. M. Salehi: None.

Funding

National Institutes of Health (TR002646); National Institute of Diabetes and Digestive and Kidney Diseases (DK105379)
1251-P: Pancreatic ß-Cell Specific Deletion of Complement 1q Like-3 Secreted Protein Improves Glucose Tolerance by Increasing Insulin Secretion
Authors: RAHMAN MOSTAFIZUR , ALSHARIF HAIFA , TROMBLEY JOHN E. , PATHAK ASMITA , BHATNAGAR SUSHANT
Published: June 2021

Secreted proteins are important metabolic regulators in the healthy and disease states. Using network biology approaches, our laboratory identified that complement-1q like-3 (C1ql3) secreted protein has a role in affecting pancreatic islet function. We demonstrated that the recombinant C1ql3 protein inhibits insulin secretion in response to stimulation by exendin-4 (an agonist for the stimulatory Gs-coupled GLP-1 receptor) from human and mouse islets. C1ql3 is expressed in β-cells but not in α- or δ-cells of mouse islets. Its expression is also conserved in human islets. Our goal is to investigate the role of β-cell C1ql3 in whole-body glucose homeostasis. We generated mice with β-cell-specific deletion of C1ql3 (βKO) by crossing C1ql3fl/fl and RIP-Cre+ mice. Preliminary data show that C1ql3 βKO mice have increased body weight with no change in fasting plasma glucose levels than the control mice. The glucose clearance was significantly improved after oral glucose challenge, whereas no difference in insulin action was observed in the C1ql3 βKO vs. control mice. Moreover, the plasma insulin levels were significantly increased at 15 min after the glucose challenge in the C1ql3 βKO vs. control mice. These results suggest that the loss of C1ql3 in β-cells increases insulin secretion to improve glucose tolerance. The expression-coupled secretion of C1ql3 from islets is increased with obesity. Interestingly, reduction in C1ql3 secretion is inversely correlated with increased insulin secretion from islets of C1ql3 βKO Leptinob/ob compared to Leptinob/ob control mice. These findings identify that C1ql3 in an autocrine manner inhibits insulin secretion from β-cells to regulate whole-body glucose homeostasis. Our work identifies a novel C1ql3-signaling pathway that may contribute to the observed β-cell dysfunction in obesity and type 2 diabetes.

Disclosure

M. Rahman: None. H. Alsharif: None. J. E. Trombley: None. A. Pathak: None. S. Bhatnagar: None.