Insulin Content Research Articles

231-OR: Reduced NKX2-2 Expression Enhances Insulin Secretion by Primary Human ß-Cells

Pancreatic islet endocrine cell phenotype and function are tightly regulated by transcription factor (TF) regulatory networks. In a mouse model of pancreas organogenesis, the key islet-enriched TF NKX2.2 governs both β and α cell specification. Further, loss of NKX2.2 in adult mouse β cells results in glucose intolerance, reduced insulin content, and polyhormonal β cells, demonstrating its importance in the maintenance of β cell phenotype. To understand the undefined role of NKX2.2 in adult human islets, we utilized adenoviral delivery of CRISPR and shRNA constructs in our primary human pseudoislet system to knockout (gNKX2-2) or knockdown (shNKX2-2) NKX2-2 in islet cells, respectively (n=4-6 donors without diabetes). Immunofluorescence analyses of gNKX2-2 pseudoislets demonstrated loss of NKX2.2 signal across β (81 ± 2.0%; p<0.001), α (58 ± 5.5%; p=0.001), and δ cells (83 ± 2.2%; p<0.001) compared to scrambled controls (gSCR). Surprisingly, functional assessment by dynamic perifusion revealed increased insulin secretion in gNKX2-2 versus gSCR pseudoislets measured as area under the curve in response to 16.7 mM glucose (6.64 ± 1.53 vs. 3.55 ± 1.53 ng/100 islet equivalents (IEQs); p=0.007) and cAMP-evoked potentiation with 16.7 mM glucose + 100 μM isobutylmethylxanthine (IBMX) (5.43 ± 0.50 vs. 3.15 ± 0.59 ng/100 IEQs p=0.003) without changes in total insulin content (gNKX2-2 vs. gSCR: 3.64 ± 0.75 vs. 4.78 ± 0.87 ng/IEQ, p=0.45). Furthermore, these findings were recapitulated in shNKX2-2 versus shSCR human pseudoislets (16.7 mM glucose: 10.17 ± 2.46 vs. 6.50 ± 2.51 ng/100 IEQs, p=0.02; 16.7 mM glucose + IBMX: 8.43 ± 1.31 vs. 4.21 ± 1.33 ng/100 IEQs, p=0.0002; insulin content: 6.18 ± 1.05 vs. 7.53 ± 1.26 ng/IEQ, p=0.36), where we observed a reduction in NKX2.2hi β (65 ± 10%; p=0.008), α (52 ± 5.5%; p=0.003), and δ cells (83 ± 4.6%; p=0.003). Collectively, our data indicate a critical role of NKX2-2 in the maintenance of primary adult human β cell function and suggest differential roles in islet function across species. Disclosure Y.D.Pettway: None. S.Parker: Research Support; Pfizer Inc. A.C.Powers: None. M.Brissova: None. J.Walker: None. C.Dai: None. R.Aramandla: None. A.L.Hopkirk: None. C.Reihsmann: None. C.Davis: None. R.Jenkins: None. L.Sussel: None.

283-OR: ADA Presidents' Select Abstract: An Isogenic hPSC-Based Platform for Functional Interrogation of the Role of Multiple T2D-Associated Genes in Pancreatic Beta-Cell Failure

Type 2 diabetes (T2D) mellitus is a complex polygenic disease. Multiple genome-wide association studies (GWAS) have identified hundreds of T2D-related genetic variants, the vast majority of which are non-coding. Pinpointing the actual causal variants and their target gene transcripts has been challenging. Building on recent advances in CRISPR technology and protocols that allow differentiation into functional pancreatic beta cells, we created isogenic hPSCs encompassing knockout (KO) mutations in 20 GWAS-identified T2D risk genes, and systematically examined their roles in beta cell differentiation, insulin production, glucose response, and apoptosis. As a result, loss of TCF7L2, HNF4A, TLE4, TGFB1, GIPR and COBLL1 severely impaired the differentiation capacities of hPSCs into functional beta cells and over half of the 20 T2D genes shared implications in beta cell dysfunctions including decreased insulin content, impaired insulin secretion and high susceptibility to lipotoxicity. Next, we sorted beta cells from each KO and performed RNA-seq and ATAC-seq assays. An integrative analysis revealed KO of HNF4A, HNF1A, ABCC8, KCNJ11, TLE4 and WDR13 resulted in extensive transcriptomic and epigenetic changes in the beta cells. We identify that KO of HNF4A results in dramatic expressional changes of dozens of known T2D effector genes and additional ones critical to beta cell functions and biology. An analysis of T2D GWAS credible set of SNPs using ATAC-seq and RNA-seq coupled with functional assays, helped us to nominate a single likely T2D causal SNP (rs7132908) at FAIM2 locus. Finally, a correlation analysis of gene expression levels and insulin content across KO lines identified 1,867 differential genes including PCSK1N that likely serves as a hub gene by mediating insulin production in beta cells. This study thus offers a powerful strategy to interrogate shared molecular pathways among multiple risk genes for diabetes. Disclosure D.Xue: None. J.Vandana: None. A.Chong: None. F.S.Collins: None. S.Chen: Consultant; Vesalius Therapeutics, Stock/Shareholder; Oncobeat Theraputics. N.Narisu: None. T.Yan: None. M.Zhang: None. C.Grenko: None. X.Tang: None. J.Zhu: None. L.L.Bonnycastle: None. M.Erdos: None. Funding American Diabetes Association (9-22-PDFPM-06 to D.X.)

292-LB: Cholesterol Accumulation in Islets Exacerbates Insulin Secretory Defects in a Model of Mild ß-Cell Dysfunction

Hypercholesterolemia is common in diabetes. We sought to determine whether cholesterol (Chol) can exacerbate impaired insulin secretion in islets from a mouse model of mild β-cell dysfunction. To induce β-cell dysfunction, C57BL/6J male mice were treated with low-dose (30 mg/kg, i.p.) streptozotocin (STZ) or vehicle (VEH) once daily on 3 consecutive days. Mice were then fed a 9% fat diet for 4 weeks, after which islets were isolated and cultured for 24 h ± Chol prior to endpoint measures. After 4 weeks, body weight (STZ 28.9±1.0 vs VEH 29.1±1.1 g) and fed glucose (STZ 191.2±11.0 vs VEH 177.8±8.2 mg/dl) did not differ. When cultured with 0 mM Chol, isolated islets from STZ-treated mice showed reduced glucose (20 mM)-stimulated insulin secretion (GSIS: 973.9±191.7 vs 1714.8±384.6 pM, n=5, p<0.05) and a trend for lower insulin content (180.6±18.2 vs 230.4±22.9 nM, n=5, p=0.06), compared to islets from VEH-treated mice. Having established STZ-induced β-cell dysfunction, islets from STZ-treated mice were cultured ± 0.5 mM Chol, then Chol content and GSIS measured. Culture in 0.5 mM (vs 0 mM) Chol resulted in increased islet Chol content (58.9 ± 4.3 vs 17.6 ± 3.0 µg/mg protein; n=3, p<0.01). A trend for increased basal insulin secretion (2.8 mM glucose: 185.8±64.7 vs 32.8±3.5 pM, n=5, p=0.09) and reduced GSIS (305.3±60.3 vs 973.9±191.7 pM, n=5, p<0.05) were observed in islets cultured with 0.5 mM Chol. Insulin content was also reduced (125.8±10.8 vs 180.6±18.2 nM, n=5, p<0.01) with 0.5 mM Chol culture. In sum, we have developed a STZ-based, euglycemic mouse model of subclinical β-cell dysfunction. Chol treatment of islets from these mice exacerbates β-cell dysfunction, in keeping with a deleterious effect of Chol. This model now provides the ability to determine whether hypercholesterolemia in vivo results in further impairment of β-cell dysfunction and the development of hyperglycemia, an observation that would be relevant to the progression of prediabetes to diabetes and diabetes per se. Disclosure R. Akter: None. B. Barrow: None. R. L. Hull-meichle: Employee; Veterans Administration, Research Support; Casma Therapeutics, Cystic Fibrosis Foundation, NIH - National Institutes of Health, Veterans Administration. S. Zraika: None. S. E. Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck & Co., Inc., Other Relationship; Novo Nordisk. Funding U.S. Department of Veterans Affairs (I01BX001060); National Institutes of Health (P30DK017047); Diabetes Research Connection (46)

1742-P: Loss of MAFB Compromises a-Cell Identity and Glucagon Secretion in Adult Human Islets

Islet-enriched transcription factors (TFs) are critical regulators of α and β cell development and function. One such TF, MAFB, exhibits a distinct expression pattern between species in that it is restricted to α cells postnatally in rodents, but produced in both developing and adult human α and β cells. While MAFB is essential for insulin production and secretion in human embryonic stem cell models, the impact of MAFB loss in mature α and β cells is unknown. Importantly, both T1D and T2D are associated with dramatic loss of islet MAFB expression. To test the hypothesis that MAFB is a critical regulator in adult human islets, dispersed islet cells were infected with lentiviruses expressing control or MAFB knockdown (MAFBKD) shRNAs, and reaggregated for 6 days to form pseudoislets (PIs; N=6 donors, ages 16-50 years). MAFBKD PIs exhibited a 56.4±4.2% reduction (P<0.001) in MAFB mRNA compared to control. Likewise, glucagon mRNA and hormone content were reduced by 42.5±6.9 and 28.6±7.6% (P<0.05), respectively, whereas insulin expression and content were unchanged. In static hormone secretion experiments, IBMX- and arginine-stimulated glucagon secretion was blunted in MAFBKD PIs (~33% lower than control; P<0.05), an observation that was replicated in α cell-enriched MAFBKD PIs. Insulin secretion, on the other hand, was largely unaffected. Bulk RNA-sequencing analysis of MAFBKD versus control PIs illustrated downregulation of α cell identity genes, upregulation of β, delta, and gamma cell identity genes, and misexpression of PYY, a gut peptide gene. Upregulated genes were also highly enriched in gene sets associated with epithelial mesenchymal transition and transforming growth factor β signaling. Intriguingly, marker genes in both pathways were shown recently to be upregulated in T2D α cells. Our findings suggest that MAFB is a molecular “gatekeeper” of α cell identity through gene activating and repressive functions, with its loss leading to dysregulation of human glucagon secretion. Disclosure K.C.Coate: None. R.Stein: None. X.Tong: None. J.Liu: None. R.Jenkins: None. R.Aramandla: None. N.Dey: None. S.Kim: None. M.Brissova: None. A.C.Powers: None.

236-OR: Pancreatic ß-Cell TRAPδ Deficiency Reduces Insulin Production but Improves Insulin Sensitivity

The endoplasmic reticulum (ER) membrane protein translocon-associated protein-δ (TRAPδ, also known as SSR4) is involved in insulin biosynthesis in pancreatic β cell lines. However, its pathophysiological significance for the maintenance of islet β cell function and glucose homeostasis is unclear. Herein, we generated a mouse line with pancreatic β cell-specific deletion of TRAPδ (TRAPδ βKO), and found that TRAPδ βKO led to decreased circulating insulin in mice fed with either normal chow diet or high fat diet. Multiple independent experiments confirmed that, although TRAPδ deletion decreased insulin content in the islets, it did not affect composition of islet cells, insulin gene expression, insulin/proinsulin ratio, nor the expression and glycosylation of the prohormone enzymes involved in proinsulin processing. These data suggest that TRAPδ does not play a critical role in the transcription of insulin gene and processing of proinsulin. Importantly, untranslocated preproinsulin was significantly increased when the islets were treated with a proteasomal inhibitor, suggesting that TRAPδ deficiency impaired preproinsulin translocation, resulting in a rapid degradation of untranslocated preproinsulin that accounts for the decreased insulin production. Interestingly, the moderate decrease in the circulating insulin level in TRAPδ βKO mice did not cause impaired glucose tolerance and elevated fasting blood glucose, suggesting that compensatory mechanisms may account for maintaining glucose homeostasis. The insulin tolerance tests confirmed improvement of insulin sensitivity accompanied with upregulation of phosphorylated AKT in the peripheral insulin target tissues of TRAPδ βKO mice. Together, these data uncover a critical role of TRAPδ in preproinsulin translocation and insulin biosynthesis. The moderately decreased circulating insulin promotes insulin sensitivity in the insulin target tissues. Disclosure J.Guo: None. Y.Huang: None. M.Liu: None. Y.Yang: None. N.Xu: None. Y.Yang: None. W.Feng: None. Y.Ye: None. X.Li: None. X.Xu: None. J.Cui: None. Funding National Natural Science Foundation of China (81830025, 82220108014, 82270864, 82070854); National Key R&D Program of China (2019YFA0802502, 2022YFE0131400); Tianjin Municipal Health and Health Committee (TJWJ2021ZD00); Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-030A)

1762-P: Increased Glucose Sensing and Insulin Secretion Induced by Verapamil Is Associated with Enhanced Oxygen Consumption Rate in MIN6 ß-Cells

It has been shown recently that oral administration of verapamil in persons with type 1 diabetes (T1D) decreased the dependency on exogenous insulin and improved the β cell function. Additionally, it decreased the frequency of hypoglycemic events. This finding encouraged us to test the effect of verapamil on the function of MIN6 mouse β cell-line. Glucose-Stimulated Insulin Secretion (GSIS) assay was performed using static incubation method. MIN6 cells were pre-treated with 50 μM verapamil for 24 hours, then the levels of total insulin content and secreted insulin were measured before and after GSIS assay using highly sensitive mouse insulin ELISA kit. Glucose sensing was estimated by the percentage of secreted insulin over total insulin content for each concentration of glucose. The measured insulin levels were normalized by the level of total protein. Oxygen consumption rate (OCR) was measured using Seahorse system and metabolic stress kit. MIN6 cells were seeded in Agilent Seahorse XF96 cell culture microplate at a cell density of 4×104 cells/well, under normal (5.6 mM) and high (25.2 mM) glucose concentrations for 24 hours, after which, cells were treated with verapamil (50 μM) or left untreated and incubated for 24 hours. Our results show no significant difference between total insulin content of treated vs. untreated cells. GSIS data show that insulin secretion was significantly higher in verapamil-treated cells at 8.4, 11.2, and 16.8 mM, but not at 2.8 and 5.6 mM glucose. The results suggest that β cells were hypersensitized to glucose when treated with verapamil. In metabolic flux analysis for mitochondrial respiration, treatment with verapamil increased basal and maximal respiration. Taken together, our study showed that verapamil increased the level of glucose sensing and oxidative respiration under conditions of high glucose, which may explain its effect on lowering the frequency of hypoglycemic events in clinical studies. Disclosure H.Arefanian: None. S.E.John: None. M.Alhusayan: None. S.J.Kurian: None. N.A.Abukhalaf: None. S.Shenouda: None. H.Alsaeed: None. S.P.Kochumon: None. M.Abu-farha: None. J.Abubaker: None. A.T.Thangavel: None. F.Bahman: None. R.Ahmad: None. F.Almulla: None. A.Al madhoun: None. F.Alzaid: None. S.T.Sindhu: None. M.R.Williams: None. F.Alrashed: None. R.Nizam: None. S.Jacob: None. Funding Kuwait Foundation for the Advancement of Sciences (RACB-2019-001)

1595-P: Empagliflozin Improves Nephropathy and Heart Failure with Preserved Ejection Fraction Despite Limited Effects on NASH in a Novel Obese Diabetic Hamster Model

Introduction: Sodium-glucose cotransporter 2 inhibitor empagliflozin (EMPA) has shown significant benefits for patients with diabetic nephropathy and heart failure with preserved ejection fraction (HFpEF), but the effects on nonalcoholic steatohepatitis (NASH) and liver fibrosis remain unclear. To further investigate the benefits of EMPA on these comorbidities, we established a novel diet-induced obese diabetic hamster model with human-like NASH and lipoprotein cholesterol metabolism, that also develops nephropathy and HFpEF. Methods: Obesity, NASH, and HFpEF were induced with a 20-week free choice diet, which presents hamsters with a choice between control chow or high fat/cholesterol diet, and normal water or 10% fructose water. After 20 weeks of diet, obese hamsters were intraperitoneally injected with streptozotocin (STZ) to induce diabetes and were then treated orally once daily for 5 weeks with vehicle or EMPA 30mg/kg. Results: Compared with vehicle, EMPA reduced hyperglycemia (vehicle: 373 +/- 43 mg/dL; EMPA: 182 +/- 30 mg/dL, p<0.01), increased pancreatic insulin content (+146%, p<0.05), significantly reduced plasma triglycerides and moderately raised LDL-cholesterol levels, as expected. However, EMPA did not improve NASH (including hepatocyte ballooning score) and liver fibrosis. EMPA reduced glomerular filtration rate and proteinuria (p=0.06 for both vs. vehicle), and significantly reduced urine albumin-to-creatinine ratio (-35%, p<0.01). In addition, EMPA showed significant improvement in HFpEF with reduced E/A and E’/E ratios with no alteration in ejection fraction, as measured by echocardiography. Conclusion: EMPA improves kidney parameters and HFpEF despite limited effects on NASH in obese diabetic hamsters. This preclinical model with multiple comorbidities will be useful to evaluate the combination of EMPA or other antidiabetic therapies with drugs targeting NASH. Disclosure F. Briand: Employee; PHYSIOGENEX. Stock/Shareholder; PHYSIOGENEX. C. Dubroca: Employee; CARDIOMEDEX. T. Sulpice: Employee; PHYSIOGENEX. Stock/Shareholder; PHYSIOGENEX. Employee; CARDIOMEDEX. Stock/Shareholder; CARDIOMEDEX.

28-OR: Characterising a Novel Peptide That Inhibits and Reverses Type 1 Diabetes

Aims: Interventions that regenerate functional pancreatic β-cells are a potential treatment option for type 1 diabetes. D6PV is a small peptide that lowers plasma triglyceride levels. We have found that it also enhances expression of the β-cell survival gene Pdx1 and stimulates insulin production in INS-1E insulinoma cells. This study asks whether D6PV delays diabetes onset and preserves β-cell mass in mice with autoimmune β-cell loss. Methods: Female 8-week-old NOD/ShiLtJ (NOD) mice (n=10-20/group) were treated twice/week for 16 weeks with D6PV (40 mg/kg i.p) or PBS. Diabetes onset was evaluated by measuring blood glucose levels. A separate cohort of female NOD mice were treated with D6PV or PBS twice/week for 4 weeks after they developed diabetes. Glycaemic control was evaluated by glucose tolerance test (1 g/kg i.p). Islet insulin content was quantified by immunostaining of paraffin-embedded, formalin-fixed pancreatic tissue. Expansion of CD8+ T-cells was analysed ex vivo from OTI-TCR transgenic mouse splenocytes and healthy human donors following stimulation via IL-7/15 and incubation ± D6PV. Results: D6PV treatment for 16 weeks decreased fed and fasted blood glucose levels (p<0.05 for both) and delayed diabetes onset in NOD mice (p<0.01). This was accompanied by increased insulin +ve islet area in the D6PV treated, but not the PBS treated animals (p<0.05). D6PV treatment also reduced lymphocyte infiltration into islets but increased lymphocytic migration to the basement membrane (0.2 ± 0.1% vs 1.8 ± 1.7%, p<0.05). When NOD mice with diabetes were treated with D6PV, glucose tolerance improved (AUC 1167±341 vs 543±328, p<0.01). Treatment of both mouse and human cytokine-activated isolated CD8+ T-cells with D6PV suppressed T-cell expansion (p<0.001). Conclusions: D6PV treatment delays diabetes onset, improves glycaemic control in mice with established disease, increases pancreatic insulin expression and inhibits T-cell expansion in NOD mice. D6PV is a potential treatment option for type 1 diabetes. Disclosure T.W.King: None. S.S.Tay: None. F.Colakoglu: None. M.Devalaraja: None. A.Remaley: None. M.Biro: None. B.Cochran: None. K.Rye: None. Funding National Health and Medical Research Council of Australia (RG201951)

1764-P: Assessing Gene Expression Profile and Insulin Secretion in Human Beta Cells (EndoC-BH3) under Glucose Exposure

Maternal diabetes is a risk factor for type 2 diabetes in the offspring, and in utero exposure to high glucose has been shown to affect pancreatic beta cell development in animal models. However, the developmental stage and specific genes which underlie these effects in humans are unknown. We aimed to understand the consequences of high glucose on the fetal pancreas by treating a human beta cell line derived from fetal pancreas, EndoC-BH3, with high, medium and normal glucose (30, 15 and 5.5mM respectively) for 3 weeks during proliferation and maturation and comparing gene expression using whole transcriptome analysis. The top differentially expressed transcripts (P<10−4) and the most abundant transcripts (Log2 unit>10.5) were validated by real-time RT-PCR. KEGG analyses of the top 500 differentially expressed transcripts identified the metabolic pathway as the top pathway. Among individual genes, high glucose exposure during the maturation upregulated EGR1 (58.6-fold), BIRC5 (37.6-fold), GPBAR1 (15.4-fold), G6PC2 (11.8-fold), CHRNA (4.8-fold), GCK (2.7-fold), INS (2.0-fold), CMIP (1.9-fold) and CHGA (1.4-fold) as compared to normal glucose exposure. EGR1 encodes early-growth response 1, a transcription factor that enhances insulin gene expression, while BIRC5 encodes an apoptotic inhibitor that plays a role in beta cell proliferation. High glucose exposure during the proliferation identified similar differentially expressed genes; however, the changes were more modest as compared with the maturation. Methylation has also been implicated in conveying risk for diabetes due to glucose exposure and several key DNA demethylation enzymes; TET1, 2, 3, were also moderately upregulated. Assessment of the effects of glucose exposure on insulin secretion and total insulin content is ongoing. Our data suggest that short term exposure to high glucose may stimulate expression of a set of genes that promote beta cell proliferation and insulin expression and secretion. Disclosure Y.L.Muller: None. K.Wiedrich: None. J.Sutherland: Stock/Shareholder; Pfizer Inc., Bristol Myers. D.Jones: None. L.Baier: None.

1734-P: Polyhormonal Cells of the Pancreatic Islets in Congenital Hyperinsulinism

One of the possible causes of insulin deficiency in diabetes is dedifferentiation of β-cells into immature phenotypes with decreased insulin content or into the polyhormonal cells containing both insulin and glucagon. Polyhormonal cells have been detected in the pancreas in diabetic animal models as well as in humans with type 2 diabetes and longstanding type 1 diabetes, suggesting this phenomenon is a consequence of hyperglycemia. However, there is no information about the presence of polyhormonal cells in opposite metabolic conditions such as hypoglycemia in infants with congenital hyperinsulinism (CHI). We examined pancreatic biopsies from 6 infants with diffuse CHI and 5 infants with focal CHI using double immunofluorescence with antibodies to insulin and glucagon. In focal CHI, we found a significant proportion of α-cells containing glucagon and insulin within the focal lesion and in the rest of the tissue. Similarly, in diffuse CHI, we found the majority of α-cells contained glucagon and insulin in all pancreatic islets. The proportion of insulin and glucagon varied in the polyhormonal cells - from cells with equal expression of these hormones to cells with the prevalence of insulin or glucagon. In one diffuse case and in two focal lesions, we observed polyhormonal cells with significantly decreased glucagon expression and normal insulin expression. Outside the latter focal lesions, cells had normal level of glucagon expression. Our results indicate that polyhormonal cells are not exclusive for diabetes and hyperglycemia. We found polyhormonal cells in the pancreas of infants with CHI under hypoglycemic conditions. We suggest that this indicates high degree of the plasticity of the pancreatic cells. The role of plasticity in infants with CHI is yet to be established. Disclosure Y.Krivova: None. A.E.Proshchina: None. D.Gubaeva: None. M.Melikyan: None. D.Otlyga: None.

1752-P: The Role of Translocon-Associated Protein Subunit γ in Insulin Biosynthesis and Glucose Homeostasis

Translocation-associated protein (TRAP) is a four-subunit complex located on the endoplasmic reticulum (ER) membrane, including TRAPα, TRAPβ, TRAPγ, and TRAPδ. TRAPα, TRAPβ and TRAPδ reside on the luminal side of the ER membrane for primary functionality, whereas the extra-membranous portion of TRAPγ is mainly cytosolic. Our recent studies indicate that TRAP complex contributes to the biosynthesis of proinsulin in pancreatic β cell lines and TRAPγ is a rate-limiting element of proinsulin biosynthesis. This might be partially explained by that newly made preproinsulin may first encounter the cytosolic domain of TRAPγ for translocation into the ER. To further understand the role of TRAPγ in β cells and its pathological significance in maintaining glucose homeostasis, we generated a β-cell specific TRAPγ knockout mouse model. We found that TRAPγ deficiency led to a significant reduction of insulin content in islets and a decrease of circulating insulin. TRAPγ βKO mice impaired glucose tolerance with aging, which even further deteriorated with high fat diet. In β cell line, TRAPγ deficiency resulted in less proinsulin synthesis and poor response to high glucose stimulation. Consistently, an acute exposure to high glucose increased expression of TRAPγ and proinsulin protein levels simultaneously in wild-type mouse islets. However, the ability to upregulate proinsulin response to high glucose was markedly compromised in TRAPγ βKO mouse islets. This study uncovers that TRAPγ is critical to insulin synthesis both in vivo and in vitro, which provides insight into the pathogenesis of type 2 Diabetes. Disclosure N.Xu: None. X.Xu: None. J.Hu: None. J.Guo: None. J.Cui: None. M.Liu: None. W.Feng: None. X.Li: None. Funding National Natural Science Foundation of China (81830025, 82220108014, 82100865, 81800733, 81900720, 82000796, 82070854); National Key R&D Program of China (2019YFA0802502, 2022YFE0131400); Tianjin Key Medical Discipline Construction Project (TJYXZDXK-030A)

196-OR: The Long-Chain Fatty-Acid Receptor FFA4 Stimulates Insulin Secretion via Inhibition of Somatostatin Release from the Delta Cell

The long-chain fatty-acid receptor FFA4 exerts beneficial effects on glucose homeostasis and insulin secretion and is considered a potential therapeutic target for type 2 diabetes. FFA4 is expressed in islets, but its precise mechanism of action remains unknown. Previous studies from our group suggest that FFA4 is expressed in delta cells and regulates somatostatin secretion. The objective of this study was to test the hypothesis that FFA4 agonists indirectly stimulate insulin secretion via inhibition of somatostatin release. In 1-h static incubations of isolated, wild-type mouse islets, the FFA4 agonist CpdA dose-dependently potentiated glucose-induced insulin secretion from 2.2±0.2 to 3.4±0.2 % of insulin content at the concentration of 50 μM (n=8; p<0.001), and simultaneously decreased somatostatin secretion from 28.4±2.1 to 10.5±0.9 pM (n=8; p<0.0001). No effect of CpdA on insulin or somatostatin secretion was observed in islets from mice that do not express somatostatin (insulin: 3.2±0.1 vs 2.9±0.4, n=6, NS; somatostatin: 11.4±1.2 vs 11.9±1.2, n=6, NS). No sex differences were observed. Likewise, in delta-cell deficient islets isolated from diphtheria toxin-treated male mice expressing the diphtheria toxin receptor under the somatostatin promoter, no effect of CpdA was observed on insulin or somatostatin release (insulin: 4.9±0.9 vs 4.1±0.8, n=3, NS; somatostatin: 10.1±4.9 vs 9.2±5.3, n=6, NS). We conclude that FFA4 stimulation of insulin secretion is exclusively mediated by inhibition of somatostatin secretion from delta cells. Disclosure L.Reininger: None. M.Ethier: None. C.Tremblay: None. J.Ghislain: None. M.Huising: Consultant; AstraZeneca, Research Support; Crinetics Pharmaceuticals, Inc. V.Poitout: Consultant; AstraZeneca. M.Flisher: None. Funding National Institutes of Health (R01DK132597)

Disrupting of family with sequence similarity 105, member A (Fam105a) deteriorates pancreatic β-cell physiology and insulin secretion in INS-1 cells

The role of “Family with sequence similarity 105, member A” (FAM105A) in pancreatic β-cell function in relation to type 2 diabetes mellitus (T2D) is not fully understood. To address this issue, various molecular and functional experiments were conducted on primary human islets and INS-1 cells. RNA-seq expression analysis showed that FAM105A is highly expressed in human islets and its expression is reduced in diabetic islets compared to healthy islets. FAM105A expression correlated negatively with HbA1c levels and body mass index (BMI). Co-expression analysis showed a significant correlation between FAM105A with PDX1, GCK, GLUT1 and INSR, but not the INS gene. Silencing of Fam105a impaired insulin release, content, glucose uptake, and mitochondria ATP content but did not affect cell viability, reactive oxygen species (ROS) or apoptosis levels. Silencing of Fam105a was associated with reduced Pdx1 and Glut2 expression at mRNA and protein levels. RNA-seq analysis of dysregulated genes in Fam105a-silenced cells showed an overall downregulation of gene expression in β-cells and insulin secretion pathway. Disrupting Pdx1 did not affect Fam105a expression in INS-1 cells. Overall, the results suggest that FAM105A plays an important role in pancreatic β-cells biology and may be involved in the development of T2D.

Metabolic preconditions for the formation andprogression of steatohepatitis of alcoholic,mixed, non-alcoholic aetiology and their comorbidity withobesity and anaemic conditions

Background. The urgency of the problem of comorbidity of alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) with anaemic conditions and the need for their differentiated correction is determined by the significant frequency of these diseases in the world and Ukraine and the presence of the syndrome of the mutual burden. Anaemia is a marker for the progression of steatohepatitis to liver cirrhosis, in which anaemic conditions is often a manifestation of hypersplenism with the increased destruction of red blood cells in the spleen, as well as frequent complications of liver cirrhosis caused by posthemorrhagic anaemia, which occurs due to bleedings from the oesophageal and gastric varices, portal hypertension and decreased biosynthesis of coagulation factors by the liver. The purpose of the study was to find out metabolic prerequisites for the formation and progression of steatohepatitis of alcoholic, mixed and non-alcoholic aetiology and comorbid anaemic conditions. Materials and methods. One hundred and twenty-five patients with steatohepatitis of alcoholic, non-alcoholic and mixed aetiology were examined for comorbidities of anaemic conditions with lipid metabolism and glycemic regulation disorders. Results. The results of investigations demonstrate hyperlipidemia (by 1.35–1.5 times) in patients with steatohepatitis of mixed aetiology and ASH, although in anaemic conditions, the content of blood total cholesterol and low-density lipoprotein (LDL) cholesterol decreased (p<0.05). Increased blood levels of triglycerides (in the range of 1.6–2.2 times) and decreased blood levels of high-density lipoprotein cholesterol (by 1.45–1.55 times) in all groups of patients with steatohepatitis and comorbidity with anaemic conditions deepened (p<0.05). In NASH with anaemic conditions, there is an increase in the blood LDL cholesterol and total cholesterol levels, with the rise of the atherogenic index by 2.8 times (p<0.05). Рatients with NASH and obesity have high values of glucose, insulin and degree of insulin resistance (by 2.6 times, p<0.05), and with anaemic conditions, insulinemia and degree of insulin resistance are increased (by 2.9 and 3.0 times, respectively; p<0.05). In patients with ASH and steatohepatitis of mixed aetiology associated with alcohol consumption, insulin deficiency is formed (by 1.4 and 1.2 times, p<0.05), which develops in addition to anaemic conditions (blood insulin content is below the lower values by 1.8 and 1.6 times). Conclusions. The consequence of metabolic pathology (hyperlipidemia, hyperglycemia, insulin resistance), which developed under the comorbidity of steatohepatitis with anaemic conditions, was an increase in the degree of hepatocyte steatosis (p<0.05).

Оцінка адаптаційно-компенсаторних можливостей дівчат із розладами менструальної функції

The duration and regularity of the menstrual cycle are influenced by many factors, including psychosocial stress, which can lead to activation of the hypothalamic-pituitary-adrenal system. Purpose - to assess the levels of stress hormones cortisol, insulin (IRI), prolactin, and the cortisol/insulin (K/In) index in adolescent girls with menstrual disorders. Materials and methods. 177 adolescent girls aged 12–17 years with menstrual disorders were examined (87 with abnormal uterine bleeding (АUB), 90 - with oligomenorrhea (OM)). The comparison group consisted of 57 girls of the same age with normal menstrual cycles examined in the early follicular phase. The level of cortisol, insulin, prolactin (PRL) was measured in blood serum on an empty stomach using the immunoenzyme method to determine the level of organism’s adaptive capabilities. All hormones were defined according to the instructions for the kits. The ratio of K/In was calculated. Results. In girls with menstrual disorders, three types of reaction from the adrenal glands were found, which were expressed in increased levels of cortisol above 75 and 90 percentile, a decrease (below 10 percentile) and its fluctuations within physiological values. At cortisol values above the 90 percentile, the average level of glucose, IRI, and HOMA in teenage girls with OM did not differ from the normative values, and in patients with AUB these indicators were significantly higher than in adolescents with OM. The K/In ratio in girls with OM by 1.8 times exceeded the similar indicator of adolescents with AUB (p<0.04) and was slightly higher than in the comparison group, which indicates a satisfactory reaction of stress tolerance. With AUB, this coefficient decreased, which may indicate exhaustion of the body’s adaptive capabilities. The same situation was observed when cortisol increased above the 75 percentile. When the cortisol level was within physiological values, the content of glucose and IRI also did not go beyond the limits of normative fluctuations. The stress ratio (K/In) had no significant difference depending on the type of violations and was significantly reduced. The absence of changes in the content of cortisol and IRI in the blood serum in response to the onset of menstrual disorders may indicate a certain instability in the development of the body's protective reactions in teenage girls with menstrual disorders. Conclusions. The revealed changes in the content of cortisol, insulin, K/In and PRL as markers of a non-specific stress reaction in the structure of the adaptive response in disorders of menstrual function carry additional information about impaired adaptation to stress and the state of compensatory capabilities of the girl's body, expand the understanding of pathogenetic mechanisms these disorders and can be used both in the assessment of the state of adaptation and in the development of appropriate preventive strategies. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Discovery of anthraquinones as DPP-IV inhibitors: Structure-activity relationships and inhibitory mechanism

Dipeptidyl peptidase IV (DPP-IV) is an integrated type II transmembrane protein that reduces endogenous insulin contents and increases plasma glucose levels by hydrolyzing glucagon-like peptide-1 (GLP-1). Inhibition of DPP-IV regulates and maintains glucose homeostasis, making it an attractive drug target for the treatment of diabetes II. Natural compounds have tremendous potential to regulate glucose metabolism. In this study, we examined the DPP-IV inhibitory activity of a series of natural anthraquinones and synthetic structural analogues on DPP-IV using fluorescence-based biochemical assays. The inhibitory efficiency differed among anthraquinone compounds with different structures. Alizarin (7), aloe emodin (11), emodin (13) emerged the outstanding inhibitory potential for DPP-IV with IC50 values lower than 5 μM. To clarifying the inhibitory mechanism, inhibitory kinetics were performed, which showed that alizarin red S (8) and 13 were effective non-competitive inhibitors of DPP-IV, while alizarin complexone (9), rhein (12), and anthraquinone-2-carboxylic acid (23) were mixed inhibitors. Emodin was determined as inhibitor with the strongest DPP-IV-binding affinity determined via molecular docking. Structure-activity relationship (SAR) demonstrated that hydroxyl group at C-1 and C-8 sites and hydroxyl, hydroxymethyl or carboxyl group at the C-2 or C-3 site were very essential for DPP-IV inhibition, replacement of hydroxyl group with amino group at C-1 could led to an increase of the inhibitory potential. Further fluorescence imaging showed that both compounds 7 and 13 significantly inhibited DPP-IV activity in RTPEC cells. Overall, the results indicated that anthraquinones would be a natural functional ingredient for inhibiting DPP-IV and provided new ideas for searching and developing potential antidiabetic compounds.

Glucose-stimulated insulin secretion depends on FFA1 and Gq in neonatal mouse islets

Aims/hypothesisAfter birth, the neonatal islets gradually acquire glucose-responsive insulin secretion, a process that is subjected to maternal imprinting. Although NEFA are major components of breastmilk and insulin secretagogues, their role for functional maturation of neonatal beta cells is still unclear. NEFA are the endogenous ligands of fatty acid receptor 1 (FFA1, encoded by Ffar1 in mice), a Gq-coupled receptor with stimulatory effect on insulin secretion. This study investigates the role of FFA1 in neonatal beta cell function and in the adaptation of offspring beta cells to parental high-fat feeding.MethodsWild-type (WT) and Ffar1−/− mice were fed high-fat (HFD) or chow diet (CD) for 8 weeks before mating, and during gestation and lactation. Blood variables, pancreas weight and insulin content were assessed in 1-, 6-, 11- and 26-day old (P1–P26) offspring. Beta cell mass and proliferation were determined in P1–P26 pancreatic tissue sections. FFA1/Gq dependence of insulin secretion was evaluated in isolated islets and INS-1E cells using pharmacological inhibitors and siRNA strategy. Transcriptome analysis was conducted in isolated islets.ResultsBlood glucose levels were higher in CD-fed Ffar1−/− P6-offspring compared with CD-fed WT P6-offspring. Accordingly, glucose-stimulated insulin secretion (GSIS) and its potentiation by palmitate were impaired in CD Ffar1−/− P6-islets. In CD WT P6-islets, insulin secretion was stimulated four- to fivefold by glucose and five- and sixfold over GSIS by palmitate and exendin-4, respectively. Although parental HFD increased blood glucose in WT P6-offspring, it did not change insulin secretion from WT P6-islets. In contrast, parental HFD abolished glucose responsiveness (i.e. GSIS) in Ffar1−/− P6-islets. Inhibition of Gq by FR900359 or YM-254890 in WT P6-islets mimicked the effect of Ffar1 deletion, i.e. suppression of GSIS and of palmitate-augmented GSIS. The blockage of Gi/o by pertussis toxin (PTX) enhanced (100-fold) GSIS in WT P6-islets and rendered Ffar1−/− P6-islets glucose responsive, suggesting constitutive activation of Gi/o. In WT P6-islets, FR900359 cancelled 90% of PTX-mediated stimulation, while in Ffar1−/− P6-islets it completely abolished PTX-elevated GSIS. The secretory defect of Ffar1−/− P6-islets did not originate from insufficient beta cells, since beta cell mass increased with the offspring’s age irrespective of genotype and diet. In spite of that, in the breastfed offspring (i.e. P1–P11) beta cell proliferation and pancreatic insulin content had a genotype- and diet-driven dynamic. Under CD, the highest proliferation rate was reached by the Ffar1−/− P6 offspring (3.95% vs 1.88% in WT P6), whose islets also showed increased mRNA levels of genes (e.g. Fos, Egr1, Jun) typically high in immature beta cells. Although parental HFD increased beta cell proliferation in both WT (4.48%) and Ffar1−/− (5.19%) P11 offspring, only the WT offspring significantly increased their pancreatic insulin content upon parental HFD (5.18 µg under CD to 16.93 µg under HFD).Conclusions/interpretationFFA1 promotes glucose-responsive insulin secretion and functional maturation of newborn islets and is required for adaptive offspring insulin secretion in the face of metabolic challenge, such as parental HFD.Graphical

Trimetazidine Preconditioning Potentiates the Effect of Mesenchymal Stem Cells Secretome on the Preservation of Rat Pancreatic Islet Survival and Function In Vitro.

Islet transplantation offers improved glycemic control in individuals with type 1 diabetes mellitus. However, in vitro islet culture is associated with islet apoptosis and eventually will lose their functionality prior to transplantation. In this study, we examined the effects of mesenchymal stem cells (MSCs) secretome preconditioned with diazoxide (DZ) and trimetazidine (TMZ) on rat islet cells during pre-transplant culture. With and without preconditioned hAD-MSCs' concentrated conditioned media (CCM) were added to the culture medium containing rat islets every 12h for 24 and 48h, after testing for selected cytokine concentrations (interleukin (IL)-4, IL-6, IL-13). Insulin content, glucose-stimulated insulin secretion, islet cell apoptosis, and mRNA expression of pro-apoptotic (BAX, BAK-1, and PUMA) and anti-apoptotic factors (BCL-2,BCL-xL, and XIAP) in rat islets were assessed after 24 and 48h of culture. The protein level of IL-6 and IL-4 was significantly higher in TMZ-MSC-CM compared to MSC-non-CM. In rat isolated islets, normalized secreted insulin in the presence of 16.7mM glucose was significantly higher in treated islet groups compared to control islets at both 24 and 48h cultivation. Also, the percentage of apoptotic islet cells TMZ-MSC-CCM-treated islets was significantly lower compared to MSC-CM and MSC-CCM-treated islets in both 24 and 48h cultivation. Consistent with the number of apoptotic cells, after 24h culture, the expression of BCL-2 and BCL-xL genes in the control islets was lower than all treatment islet groups and in 48h was lower than only TMZ-MSC-CM-treated islets. Also, the expression of the XIAP gene in control islets was significantly lower compared to the TMZ-MSC-CCM-treated islets at both at 24 and 48h. In addition, mRNA level of the BAX gene in TMZ-MSC-CCM-treated islets was significantly lower compared to other groups at 48h. Our findings revealed that TMZ proved to be more effective than DZ and could enhance the potential of hAD-MSCs-CM to improve the function and viability of islets prior to transplantation.

RyR2 regulates store-operated Ca2+ entry, phospholipase C activity, and electrical excitability in the insulinoma cell line INS-1.

The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 μM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Cav current density and SK channel activation.

The Proliferative and Functional Roles of CEBP/A in Ins-1 832/13 Cells and Primary Rat Islets

The beta cell transcription factor Nkx6.1, when overexpressed in primary rodent and human islets, is sufficient to enhance beta cell proliferation, increase insulin secretion and enhance cell survival. We have sought to define the transcriptional targets of Nkx6.1 that allow for its ability to increase functional beta cell mass. We have shown that Nkx6.1 induces expression of the transcription factor CEBP/A. CEBP/A overexpression is sufficient to induce proliferation of Ins-1 832/13 cells and primary rat islets. CEBP/A overexpression enhances glucose stimulated insulin secretion from primary rat islets, while decreasing total insulin content. Finally, CEBPa overexpression protects Ins-1 832/13 cells from thapsigargin and glucolipotoxicity induced cell death but fails to protect against etoposide and camptothecin induced cell death. These data suggest that CEBP/A may play a critical role in Nkx6.1 mediated expansion of functional beta cell mass through a endoplasmic reticulum stress-mediated pathway. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.