Insulin Secretion In Pancreatic Beta-cells Research Articles

RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell function.

Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown. To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice. Stem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes. Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes. Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).

Obesity, heart failure with preserved ejection fraction, and the role of glucagon-like peptide-1 receptor agonists.

Heart failure with preserved ejection fraction (HFpEF) has a high prevalence, affecting more than 50% of patients with heart failure. HFpEF is associated with multiple comorbidities, and obesity is one of the most common. A distinct phenotype has been proposed for obese patients with HFpEF. Recent data show the beneficial role of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) for weight loss in diabetic and non-diabetic patients with obesity or overweight when given as adjunctive therapy to diet and exercise. The mechanisms of action are related to paracrine and endocrine signalling pathways within the gastrointestinal tract, pancreas, and central nervous system that delay gastric emptying, decrease appetite, augment pancreatic beta-cell insulin secretion, and suppress pancreatic glucagon release. These drugs are therefore potentially indicated for treatment of patients with HFpEF and obesity or overweight. Efficacy and safety need to be shown by clinical trials with a first one, Semaglutide Treatment Effect in People with obesity and heart failure with preserved ejection fraction (STEP HFpEF), recently concluded. The aim of the present review is to provide the pathophysiological and pharmacological rationale for GLP-1 RA administration to obese patients with HFpEF.

SAT157 Management Of Spontaneous Hypoglycemia In Dialysis-dependent Chronic Kidney Disease Using Diazoxide

Abstract Disclosure: A. Schledwitz: None. S.S. Habbsa: None. F. Sotomayor Villanueva: None. Y. Kim: None. Background: Patients with end-stage renal disease (ESRD) are at increased risk of spontaneous hypoglycemia due to decreased renal insulin clearance. Due to its inhibitory effect on pancreatic beta cell insulin secretion, oral diazoxide has been used to treat hypoglycemia due to insulinoma or congenital hyperinsulinemic hypoglycemia, but few cases detail the use of diazoxide in the ESRD population. Here, we present 4 cases of spontaneous hyperinsulinemic hypoglycemia in ESRD successfully treated with diazoxide. Clinical Cases: A 66-year-old man with ESRD presented with altered mental status secondary to severe hypoglycemia despite continuous IV 20% dextrose. Labs revealed glucose 50 mg/dL, insulin 7 (0-3 mcIU/mL), C-peptide 7.8 (0-0.6 ng/mL), proinsulin 35.7 (0-5 pmol/L), beta-hydroxybutyrate [bOH] 0.4 (0-2.7 mg/dL), negative sulfonylurea panel, normal cosyntropin stimulation test and undetectable insulin antibodies, without radiographic evidence of insulinoma. He was started on diazoxide 50 mg TID and remained euglycemic for the remainder of his course. On similar readmission 3 months later, he was restarted on diazoxide with resolution of hypoglycemia within 1 day. A 66-year-old male with ESRD and T1DM treated with pancreas transplant presented with toe gangrene and persistent hypoglycemia. Fasting labs revealed insulin 4, C-peptide 4.5, proinsulin 4.5, and bOH 4.5, without radiographic evidence of insulinoma. He maintained euglycemia 4 days after diazoxide 70 mg BID was started. A 67-year-old male with T2DM and ESRD was admitted for osteomyelitis and 2 years of episodic symptomatic hypoglycemia despite dietary adjustments. Labs showed glucose 59, insulin 7, C-peptide 5.2, proinsulin 7.5, bOH 0.04, and unremarkable imaging. Within 1 day of starting diazoxide 100 mg daily, his blood glucose stabilized, and he was discharged. Finally, a 42-year-old male with ESRD presented with 4 years of persistent episodic hypoglycemia, now refractory to continuous IV dextrose. He was found to have glucose 64, insulin 15, C-peptide 11.5, proinsulin 29, and bOH 0.1, with imaging revealing bulky pancreas without a discrete mass. His hypoglycemic episodes resolved within 1 day of starting diazoxide 100 mg daily. He was readmitted 3 months later for catheter site infection and had no episodes of hypoglycemia with continuation of diazoxide. Conclusion: Diazoxide may be a viable option for the treatment of refractory spontaneous hypoglycemia in patients with dialysis dependent ESRD. Presentation: Saturday, June 17, 2023

PSUN278 Factitious hypoglycemia, diagnostic delay due to insulin assay failure to detect insulin analogues.

Abstract Factitious hypoglycemia may be difficult to diagnose clinically. Hypoglycemia due to insulin self-administration is established by the presence of a low c-peptide and elevated plasma insulin levels. Commercial insulin assays often fail to detect insulin analogs and can create confusion among providers investigating causes of hypoglycemia. A 20 year old female with no significant past medical history presented to an Emergency Room (ER) with hypoglycemia. She was treated with a single dose of octreotide 150 mcg, dexamethasone 10 mg PO and started on a D10W drip at 100ml/hr prior to transfer. Laboratory studies on transfer reported plasma glucose 52ng/dL (RR: 70-100), low C-peptide 0.02 (RR: 0.78-5.19), low insulin level <0.087 uIU/mL, normal IGF-I level 122 ng/mL (RR: 85-370). Normal IGF-II level 401 ng/mL (RR: 333-967), and low Pro-insulin level 1.8 pmol/L (RR: 3.6-22 pmol/L). Sulfonylurea Screen was negative. The patient and her mother both denied exogenous insulin use. The patient and her mother both work in healthcare. The patient's boyfriend has type 1 diabetes mellitus and the patient stated she keeps insulin in her purse for him. The patient was admitted for a 72 hour fast and remained normoglycemic. An aliquot of the admission ER insulin blood sample was sent to second laboratory utilizing an assay able to detect analog insulins. The patient's sample previously reporting an undetectable insulin level (<0.087 uIU/mL) now reported an insulin level 8 uIU/mL. Factitious hypoglycemia is a challenging clinical diagnosis. The term factitious implies an attempt to deceive and creates mistrust between the physician and patient. However, hypoglycemia may be the result of medication errors or administered by a second party with the intent to harm. Analog insulins (glargine, aspart, lispro, glulisine, etc) are genetically modified insulins developed to mimic the physiologic pattern of pancreatic beta cell insulin secretion. The amino acid modifications in analog insulins result in structural variations which alters the ability of highly specific commercial automated immunoassays to accurately quantitate these analog insulins. The variation in lab assay detection may cause confusion when interpreting the results. The DiaSorin Liaison XL platform in our hospital utilizes a chemiluminescence immunoassay which does not detect insulin analogs, but detects regular and NPH insulin as they are structurally identical to endogenous human insulin. The second laboratory uses the Siemens Advia Centaur platform, an immunoassay which reacts with insulin analogs "on a nearly equimolar basis with the analogs insulin aspart, insulin glargine, and insulin lispro. Insulin detemir exhibits approximately 50 percent cross-reactivity. Test reactivity with insulin glulisine is negligible (< 3 percent)". Many commercial insulin assays do not detect analog insulins and none qualitatively distinguish between different insulins. Failure to recognize this detection flaw may result in misdiagnosis, patient safety issues and costly unneeded additional studies Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

COVID-19 associated diabetes mellitus: A review.

A significantly higher rate of new-onset diabetes in many coronavirus disease 2019 (COVID-19) patients is a frequently observed phenomenon. The resultant hyperglycemia is known to influence the clinical outcome, thereby increasing the cost of treatment and stay in hospital. This will also affect the post-hospitalization recuperation. It has been observed that new-onset diabetes in COVID-19 patients is associated with considerable increase in morbidity and may be associated with increased mortality in some cases. This mini-review focuses on the possible causes to understand how COVID-19-related diabetes develops, various associated risk factors, and possible mechanism to understand the natural history of the disease process, clinical outcome, associated morbidities and various treatment options in the mana-gement of post COVID-19 diabetes. A literature search was performed in PubMed and other online database using appropriate keywords. A total of 80 articles were found, among which, 53 of the most relevant were evaluated/ analyzed and relevant data were included. The studies show that patients who have had severe acute respiratory syndrome coronavirus 2 infection leading to development of COVID-19 may manifest not only with new-onset diabetes but also worsening of pre-existing diabetes. Cytopathic effect and autoimmune destruction of insulin-secreting pancreatic beta cells, cytokine storm during the active phase of infection causing impaired insulin secretion and resistance, drug-induced hyperglycemia, undetected pre-existing hyperglycemia/diabetic condition, and stress-induced impairment of glucose metabolism are some of the possible potential mechanisms of COVID-19-associated new-onset diabetes mellitus. Many studies published in recent times have found a significantly higher rate of new-onset diabetes mellitus in many COVID-19 patients. Whether it is an inflammatory or immune-mediated response, direct effect of virus or combination of these is unclear. The resultant hyperglycemia is known to influence the clinical outcome and has been associated with considerable increase in morbidity and increased mortality in some cases.

Alterations in Inflammatory Cytokines and Redox Homeostasis in LPS-Induced Pancreatic Beta-Cell Toxicity and Mitochondrial Stress: Protection by Azadirachtin

Inflammation and redox imbalance are hallmarks of cancer, diabetes, and other degenerative disorders. Pathophysiological response to these disorders leads to oxidative stress and mitochondrial dysfunction by alterations and reprogramming in cellular signaling and metabolism. Pancreatic beta cells are very sensitive to the inflammatory and altered nutrient signals and hence play a crucial role in diabetes and cancer. In this study, we treated insulin-secreting pancreatic beta cells, Rin-5F, with the bacterial endotoxin, LPS (1 μg/ml) to induce an inflammatory response in vitro and then treated the cells with a known anti-inflammatory, anticancer and antioxidant phytochemical, azadirachtin (AZD, 25 µM for 24 h). Our results demonstrated lipid peroxidation and nitric oxide production causing increased nitro/oxidative stress and alterations in the activities of anti-oxidant enzymes, superoxide dismutase and catalase after LPS treatment. Pro-inflammatory responses caused by translocation of nuclear factor kappa B and release of inflammatory cytokines were also observed. These changes were accompanied by GSH-dependent redox imbalance and alterations in mitochondrial membrane potential and respiratory complexes enzyme activities leading to mitochondrial respiratory dysfunction, reduced ATP synthesis, and intrinsic caspase-9 mediated apoptosis. Caspase-9 was activated due to alterations in Bcl-2 and Bax proteins and release of cytochrome c into the cytosol. The activities of oxidative stress-sensitive mitochondrial matrix enzymes, aconitase, and glutamate dehydrogenase were also inhibited. Treatment with AZD showed beneficial effects on the recovery of antioxidant enzymes, inflammatory responses, and mitochondrial functions. GSH-dependent redox homeostasis also recovered after the treatment with AZD. This study may help in better understanding the etiology and pathogenesis of inflammation-induced disorders in pancreatic beta cells to better manage therapeutic strategies.

Pancreatic beta cells regeneration.

Insulin-secreting pancreatic beta cells are important for the regulation of glucose metabolism in vivo. Loss of beta cells could lead to diabetes. So far, islet transplantation is an effective way for diabetes therapy, but the shortage of donors limits its extensive application. Therefore, study on beta cell regeneration has great significance for the clinical treatment of diabetes. The major cellular sources of beta cells mainly include endogenous beta cell proliferation, pluripotent stem cell differentiation, and trans-differentiation from other non-beta cells. The existence of endogenous pancreatic stem cells or progenitors in adults remains controversial in the field. In this review, we briefly summarize the research advances of beta cell regeneration and discuss the potential problems of these approaches, to improve our understanding on the mechanisms of beta cell regeneration and provide theoretical information for clinical application.

Myostatin Deletion Preserves Cardio‐Metabolic Function in Type 1 diabetes

Type 1 Diabetes Mellitus (T1DM) is a disease characterized by the destruction of insulin‐secreting pancreatic beta cells and results in hyperglycemia, muscle wasting, and vascular dysfunction. Patients afflicted with T1DM suffer from increased morbidity and early mortality, largely driven by an inability to appropriately maintain glucose homeostasis. Skeletal muscle is the body’s largest metabolic reservoir, absorbing significant amounts of glucose from the bloodstream. The myokine myostatin is a potent negative regulator of muscle growth and is upregulated in T1DM patients but downregulated following regular exercise. Physical exercise is also known to improve cardiovascular health and increase insulin sensitivity of muscle, but many T1DM patients are unable to exercise at a level that conveys benefit due to muscle atrophy. Thus, directly targeting skeletal muscle, independent of exercise, may prove beneficial for T1DM therapy. Our hypothesis is that genetic deletion of myostatin will preserve glucose homeostasis, maintain muscle function, and protect against vascular dysfunction and cardiometabolic effects in a mouse model of T1DM.T1DM was induced via streptozotocin (STZ) in adult male mice with (WT) and without myostatin (MyoKO). Multiple variables were assessed including glucose homeostasis (plasma glucose, HbA1c, IGTT), fluid dynamics, muscle function (in vivo plantarflexion), and vascular function (ex vivo pressure myography of gracilis arteriole).Myostatin deletion inhibited STZ‐induced increases in plasma glucose, preserved fluid dynamics, and prevented decreases in muscle function, independent of insulin. Further, endothelial function was protected with myostatin deletion. Taken together, this data suggests that myostatin inhibition may be a target for effective treatment and management of the cardiometabolic and skeletal muscle dysfunction that occurs with T1DM.

The Calcium Channel Subunit Gamma-4 as a Novel Regulator of MafA in Pancreatic Beta-Cell Controls Glucose Homeostasis.

Impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) are high-risk factors of diabetes development and may be caused by defective insulin secretion in pancreatic beta-cells. Glucose-stimulated insulin secretion is mediated by voltage-gated Ca2+ (CaV) channels in which the gamma-4 subunit (CaVγ4) is required for the beta-cell to maintain its differentiated state. We here aim to explore the involvement of CaVγ4 in controlling glucose homeostasis by employing the CaVγ4−/− mice to study in vivo glucose-metabolism-related phenotypes and glucose-stimulated insulin secretion, and to investigate the underlying mechanisms. We show that CaVγ4−/− mice exhibit perturbed glucose homeostasis, including IFG and IGT. Glucose-stimulated insulin secretion is blunted in CaVγ4−/− mouse islets. Remarkably, CaVγ4 deletion results in reduced expression of the transcription factor essential for beta-cell maturation, MafA, on both mRNA and protein levels in islets from human donors and CaVγ4−/− mice, as well as in INS-1 832/13 cells. Moreover, we prove that CaMKII is responsible for mediating this regulatory pathway linked between CaVγ4 and MafA, which is further confirmed by human islet RNA-seq data. We demonstrate that CaVγ4 is a key player in preserving normal blood glucose homeostasis, which sheds light on CaVγ4 as a novel target for the treatment of prediabetes through correcting the impaired metabolic status.

The Therapeutic Effects of Magnesium in Insulin Secretion and Insulin Resistance.

Insulin resistance (IR) is a chronic pathological condition that is related to reduce the rates of glucose uptake, especially in the liver, muscle, and adipose tissue as target tissues. Metabolic syndrome and type 2 diabetes mellitus can occur following progression of the disease. The majority of prior research has applied that some cations such as magnesium (Mg2+) have important physiological role in insulin metabolism. Mg2+ is the fourth most abundant mineral in the human body that gets involved as a cofactor of various enzymes in several metabolic events, such as carbohydrate oxidation, and it has a fundamental role in glucose transporting mechanism of the cell membrane. This cation has numerous duties in the human body such as regulation of insulin secretion in pancreatic beta-cells and phosphorylation of the insulin receptors in target cells and also gets involved in other downstream signal kinases as intracellular cation. On this basis, intracellular Mg2+ balancing is vital for adequate carbohydrate metabolism. This paper summarizes the present knowledge about the therapeutic effects of Mg2+ in reducing IR in liver, muscle, and pancreases with different mechanisms. For this, the search was performed in Google Scholar, PubMed, Scopus, and Web of Science by insulin resistance, skeletal muscle, liver, pancreases, magnesium, Mg2+, and inflammation keywords.

Exenatide improves antioxidant capacity and reduces the expression of LDL receptors and PCSK9 in human insulin-secreting 1.1E7 cell line subjected to hyperglycemia and oxidative stress

Abstract Introduction GLP-1 receptor agonists (e.g., exenatide) are novel drugs used in the treatment of diabetes. These drugs, working with other mechanisms of action, improve glycemic control by increasing secretion of insulin and improving survival of pancreatic islet beta cells. Alterations in the oxidative stress level or the expression of proteins associated with cholesterol uptake might be responsible for those findings. Currently, there are few in vitro studies on the impact of exenatide antioxidant capacity in human islet beta cell lines and none that assess the influence of exenatide on LDL receptors and PCSK9 under hyperglycemia and oxidative stress. Therefore, we evaluated the impact of exenatide on antioxidant capacity, insulin secretion, and proteins involved in cholesterol metabolism. Materials and Method An in vitro culture of insulin-secreting cells 1.1E7 was subjected to hyperglycemia and oxidative stress. Assessment was made of the expression of enzymes associated with oxidative stress (NADPH oxidase, catalase, glutathione peroxidase, superoxide dismutase, iNOS) and cholesterol uptake (LDL receptors, PCSK9). Additionally, insulin and nitrite levels in culture media were quantified. Results We showed that exenatide improves expression of catalase and reduces the amount of nitrite in cell cultures in a protein kinase A–dependent manner. Those results were accompanied by a drop in the expression of LDL receptors and PCSK9. Insulin secretion was modestly increased in the culture condition. Conclusions Our findings show potential protective mechanisms exerted by exenatide in human insulin-secreting pancreatic beta cell line (1.1E7), which may be exerted through increased antioxidant capacity and reduced accumulation of cholesterol.

In vitro effect of DDE exposure on the regulation of B-TC-6 pancreatic beta cell insulin secretion: a potential role in beta cell dysfunction and type 2 diabetes mellitus

Organochlorine compounds (OC) include synthetic insecticides previously used throughout the world before being banned for their adverse effects and environmental persistence; DDT (dichlorodiphenyltrichloroethane) was one of the most widely used. Epidemiological evidence suggests that higher levels of some OC, including metabolites of DDT, such as dichlorodiphenyldichloroethylene (DDE), are associated with type 2 diabetes mellitus (T2D). DDE exposure may affect pancreatic cellular functions associated with glucose control and possibly cause beta cell dysfunction. The in vitro effect of DDE exposure on pancreatic beta cell insulin secretion was investigated using Beta-Tumor Cell-6 (B-TC-6) murine pancreatic beta cells. DDE exposure significantly increased insulin secretion suggesting a role for DDE in altering insulin synthesis and secretion. Reactive oxygen species (ROS) levels were not significantly increased indicating that oxidative stress is not responsible for the DDE-induced insulin secretion. Pancreatic and duodenal homeobox factor-1 (PDX-1) levels were not significantly increased suggesting that DDE exposure does not alter insulin transcription, but prohormone convertase (PC) levels were increased suggesting a role for DDE in altering insulin translation. Based on these in vitro results, DDE may play a role in beta cell dysfunction by affecting mechanisms that regulate insulin secretion but it is not likely to be the major mechanism behind the DDE/T2D epidemiological association.

338-OR: Reversal of Insulin Resistance by Resveratrol and Hesperetin Combination in Overweight and Obese Subjects Correlates with Decrease in Expression of Thioredoxin Interacting Protein

Trans-Resveratrol and hesperetin combination (tRES-HESP) in randomized, double-blind, placebo-controlled crossover study in overweight and obese subjects corrected insulin resistance; healthy aging through functional food study (HATFF, Clinicaltrials.gov identifier NCT02095873). tRES-HESP is an optimized supplement for induction of expression of glyoxalase 1 (Glo1) via activation of transcription factor Nrf2. Increased Glo1 expression decreases reactive metabolite, methylglyoxal (MG), contributing to insulin resistance through glycation-linked unfolded protein formation and activation of the unfolded protein response (UPR). Herein, we report further analysis of study variables and follow-up reverse translational studies. With tRES-HESP treatment, plasma MG correlated negatively with peripheral blood mononuclear cell (PBMC) quinone reductase activity - a marker of Nrf2 activation (r = - 0.68, P<0.01). For change from baseline of PBMC gene expression with tRES-HESP treatment (assessed by the Nanostring method), change in area-under-the-curve plasma glucose (ΔAUGg) during oral glucose tolerance tests correlated negatively with change in Glo1 expression (r = - 0.56, P<0.05) and correlated positively with change in expression of thioredoxin interacting protein (TXNIP) (r = 0.59, P<0.05). In reverse translational studies, we found tRES-HESP decreased TXNIP expression by 39 ± 1% (P<0.001) in human hepatocyte-like HepG2 cells and 14 ± 3% in BJ fibroblasts in vitro (P<0.01). tRES-HESP may decrease TXNIP expression by decreasing the MG-driven UPR and by direct Nrf2-driven down regulation. TXNIP is a mediator of insulin resistance in liver and skeletal muscle and impaired pancreatic beta-cell insulin secretion. tRES-HESP is suitable for further evaluation for prevention and treatment of type 2 diabetes where decreased TXNIP may contribute to the therapeutic response. Disclosure M. Xue: None. M. Weickert: None. N. Rabbani: None. P. Thornalley: None. Funding Qatar Foundation; Qatar University

Expanding the spectrum of endocrinopathies identified in Schaaf-Yang syndrome - A case report and review of the literature

Schaaf-Yang syndrome is a genetic disorder caused by mutations in the paternal allele of the MAGEL2 gene. Developmental delay, feeding difficulties, joint contractures and a high prevalence of autism spectrum disorders are characteristic of the syndrome. Endocrine abnormalities include mostly various pituitary hormonal deficiencies, presenting as hypoglycemia in 48% of reported cases. Persistent hyperinsulinism was only described in two siblings and responded to diazoxide treatment. We describe a unique case of an infant with Schaaf-Yang syndrome that presented with persistent hyperinsulinism unresponsive to diazoxide. Furthermore, we conducted a literature review of the endocrine abnormalities described in MAGEL2 related disorders. The case presented expands the clinical phenotype of Schaaf-Yang syndrome and emphasizes the importance of endocrine follow-up in these patients. Further investigation into the role of MAGEL2 in the regulation of pancreatic beta-cell insulin secretion, will improve our understanding of the abnormalities in glucose regulation in this syndrome.

Changes in the effect of HDL on pancreatic beta-cell insulin secretion following metabolic surgery

Changes in the effect of HDL on pancreatic beta-cell insulin secretion following metabolic surgery

Role of mitochondria in pathogenesis of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is global health problem. An estimated 425 million people in the world had diabetes in 2017. It is a major cause of morbidity and mortality worldwide. Although, pathogenesis of T2DM and its complications have been focus of medical research for long, much remains to be learned. A better understanding of molecular pathogenesis is essential for more effective preventive and therapeutic interventions. Role of mitochondria in pathogenesis of metabolic problems such as obesity, metabolic syndrome, and T2DM is the focus of many recent research studies. Mitochondrial dysfunction contributes to the oxidative stress and systemic inflammation leading to insulin resistance (IR). Mitochondria are also essential for pancreatic beta cell insulin secretion. Hence, mitochondria are important players in the pathogenesis of T2DM. In this article, pathogenesis of T2DM is examined from a mitochondrial perspective.

249-LB: Effects of Oxidative Phosphorylation Complex Defects on Beta-Cell Biology

Background: The insulin-secreting pancreatic beta cell is a highly metabolic cell type relying on mitochondrial metabolism for stimulus-secretion coupling. Specifically, the ATP-producing oxidative phosphorylation (OXPHOS) pathway is central to insulin secretion. Although disruptions in ATP production occur in beta cell dysfunction, the exact mechanisms remain unknown. We have generated beta cell specific OXPHOS complex inducible knock out mouse models of Complex I, III, and IV to determine the direct effects OXPHOS complexes on beta cell function. We hypothesize that individual complexes contribute distinctly to beta cell function. Methods: Eight-week-old, male OXPHOSf/f - MIPcreERT mice were administered tamoxifen (150mg/kg) or vehicle by I.P. injection for 5 consecutive days. Non-fasting glycemia and body weight were measured one week prior and once per week up to 5 weeks after induction. Glucose tolerance was assessed by glucose tolerance tests (IP-GTT) 1 week before and 3 weeks after induction for all groups. Mice were anesthetized with ketamine/xylazine and perfused with sterile PBS followed by 4% PFA. Pancreata were dissected, embedded in OCT, and cut at 40µM sections for immunofluorescent staining of insulin, glucagon, and somatostatin. Results: No changes in body weight were observed throughout the experimental period for any group. Complex III knockout animals developed severe hyperglycemia and significant glucose intolerance at 3 weeks after induction. Although the other models display a minor trend towards hyperglycemia starting at 5 weeks after induction, they retain their ability to regulate glucose at 3 weeks as seen by IP-GTT. Interestingly, no changes in islet cytoarchitecture are observed in any of the groups at the 3-week timepoint. Conclusions: Mice with Complex III deficient beta cells show a robust diabetic phenotype compared to Complex I and IV models. These results support the hypothesis that specific OXPHOS complexes contribute differentially to beta cell function. Disclosure A.L. Lang: None. A. Caicedo: None. Funding National Institute of Environmental Health Sciences (R21ES025673-01)

A genetic screen identifies Crat as a regulator of pancreatic beta-cell insulin secretion

ObjectivesGlucose-stimulated insulin secretion is a critical function in the regulation of glucose homeostasis, and its deregulation is associated with the development of type 2 diabetes. Here, we performed a genetic screen using islets isolated from the BXD panel of advanced recombinant inbred (RI) lines of mice to search for novel regulators of insulin production and secretion. MethodsPancreatic islets were isolated from 36 RI BXD lines and insulin secretion was measured following exposure to 2.8 or 16.7 mM glucose with or without exendin-4. Islets from the same RI lines were used for RNA extraction and transcript profiling. Quantitative trait loci (QTL) mapping was performed for each secretion condition and combined with transcriptome data to prioritize candidate regulatory genes within the identified QTL regions. Functional studies were performed by mRNA silencing or overexpression in MIN6B1 cells and by studying mice and islets with beta-cell-specific gene inactivation. ResultsInsulin secretion under the 16.7 mM glucose plus exendin-4 condition was mapped significantly to a chromosome 2 QTL. Within this QTL, RNA-Seq data prioritized Crat (carnitine O-acetyl transferase) as a strong candidate regulator of the insulin secretion trait. Silencing Crat expression in MIN6B1 cells reduced insulin content and insulin secretion by ∼30%. Conversely, Crat overexpression enhanced insulin content and secretion by ∼30%. When islets from mice with beta-cell-specific Crat inactivation were exposed to high glucose, they displayed a 30% reduction of insulin content as compared to control islets. We further showed that decreased Crat expression in both MIN6B1 cells and pancreatic islets reduced the oxygen consumption rate in a glucose concentration-dependent manner. ConclusionsWe identified Crat as a regulator of insulin secretion whose action is mediated by an effect on total cellular insulin content; this effect also depends on the genetic background of the RI mouse lines. These data also show that in the presence of the stimulatory conditions used the insulin secretion rate is directly related to the insulin content.

Arf6 Heterozygosity Results in Impaired Glucose Tolerance Due to Abberant Hepatic Gluconeogenesis

Adenosine diphosphate (ADP) ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in numerous cellular processes including proliferation and membrane trafficking. Arf6 has been found to be dysregulated in diseases such as diabetic retinopathy and tumor metastasis. Furthermore, genetic insufficiency or pharmacological inhibition of Arf6 is protective in the aforementioned diseases, suggesting that Arf6 may be a potential therapeutic target. Here, we assessed the metabolic consequences of a reduction in Arf6 expression by performing glucose (GTT: 2 g/kg, ip), insulin (ITT: 1 U/kg, ip) and pyruvate (PTT: 2 g/kg, ip) tolerance tests in 3–4 mo old whole body Arf6 heterozygote (Arf6+/−) and wildtype (Arf6+/+) littermate control mice. Both protein and gene expression of Arf6 in the liver were 40–50% lower (p<0.05) in Arf6+/− mice compared to Arf6+/+. Body mass was higher in normal chow fed Arf6+/− compared to Arf6+/+ mice (30.3±0.9 vs 36.4±2.0 g, p<0.05). Blood glucose at 15, 30, and 60 min after injection during the GTT were 20–30% higher in Arf6+/− mice compared to Arf6+/+ (each p<0.05). Likewise, the area under the GTT time response curve was ~15% higher in Arf6+/− compared to Arf6+/+ (p<0.05), indicating impaired glucose metabolism. To examine the underlying cause of this glucose intolerance, we performed an ITT and assessed insulin‐stimulated suppression of plasma free fatty acids 15, and 30 min after insulin injection. We found no groups differences between the ITT time response curves (p>0.05) and no difference in the percent decrease in plasma FFAs from fasted between Arf6+/− and Arf6+/+ mice (~50% vs ~50% p>0.05). Likewise, fasted and glucose‐stimulated (2g/kg, ip) serum insulin were not different between the groups (2.44±0.58 vs 1.73±0.28, fasted; 4.54±1.74 vs 4.03±1.68 insulin stimulated; both p>0.05), suggesting that reductions in Arf6 expression do not impact pancreatic beta cell insulin secretion. Collectively, these measures suggest that neither peripheral insulin resistance nor impaired pancreatic beta cell function underlie glucose intolerance in Arf6+/− mice. To determine the contribution of hepatic gluconeogenesis to the observed glucose intolerance in the Arf6+/− mice, we performed a PTT and found that Arf6+/− mice exhibited a 15–25% increase in blood glucose over time compared to Arf6+/+ mice (p<0.05), suggesting a role for elevated hepatic gluconeogenesis. Taken together, this study demonstrates that Arf6 heterozygosity impairs glucose metabolism that is associated with aberrant hepatic gluconeogenesis but is independent of peripheral insulin resistance or pancreatic beta cell dysfunction.Support or Funding InformationNIA R01 AG048366, R01 AG050238 and K02 AG045339, R01 AG060395 and US Department of Veterans Affairs I01 BX002151, I01 BX004492.

Exposure of Pancreatic β-Cells to Excess Glucose Results in Bimodal Activation of mTORC1 and mTOR-Dependent Metabolic Acceleration.

SummaryChronic exposure of pancreatic β-cells to excess glucose can lead to metabolic acceleration and loss of stimulus-secretion coupling. Here, we examined how exposure to excess glucose (defined here as concentrations above 5 mM) affects mTORC1 signaling and the metabolism of β-cells. Acute exposure to excess glucose stimulated glycolysis-dependent mTORC1 signaling, without changes in the PI3K or AMPK pathways. Prolonged exposure to excess glucose led to hyperactivation of mTORC1 and metabolic acceleration, characterized by higher basal respiration and maximal respiratory capacity, increased energy demand, and enhanced flux through mitochondrial pyruvate metabolism. Inhibition of pyruvate transport to the mitochondria decelerated the metabolism of β-cells chronically exposed to excess glucose and re-established glucose-dependent mTORC1 signaling, disrupting a positive feedback loop for mTORC1 hyperactivation. mTOR inhibition had positive and negative impacts on various metabolic pathways and insulin secretion, demonstrating a role for mTOR signaling in the long-term metabolic adaptation of β-cells to excess glucose.