Maintaining Glucose Homeostasis Research Articles

Measurement of gluconeogenesis by 2H2O labeling and mass isotopomer distribution analysis

The gluconeogenesis pathway, which converts nonsugar molecules into glucose, is critical for maintaining glucose homeostasis. Techniques that measure flux through this pathway are invaluable for studying metabolic diseases such as diabetes that are associated with dysregulation of this pathway. We introduce a new method that measures fractional gluconeogenesis by heavy water labeling and gas chromatographic-mass spectrometric analysis. This technique circumvents cumbersome benchwork or inference of positionality from mass spectra. The enrichment and pattern of deuterium label on glucose is quantified by use of mass isotopomer distribution analysis, which informs on how much of glucose-6-phosphate-derived glucose comes from the gluconeogenesis (GNG) pathway. We use an invivo model of the GNG pathway that is based on previously published models but offers a new approach to calculating GNG pathway and subpathway contributions using combinatorial probabilities. We demonstrated that this method accurately quantifies fractional GNG through experiments that perturb flux through the pathway and by probing analytical sensitivity. While this method was developed in mice, the results suggest that it is translatable to humans in a clinical setting.

CREB-Regulated Transcriptional Coactivator 2 Proteome Landscape is Modulated by SREBF1

cAMP response element-binding protein (CREB) regulated transcriptional coactivator 2 (CRTC2) is a critical transcription factor that maintains glucose homeostasis by activating CREB. Energy homeostasis is maintained through multiple pathways; therefore, CRTC2 may interact with other transcription factors, particularly under metabolic stress. CRTC2 liver-specific KO mice were created, and the global proteome, phosphoproteome, and acetylome from liver tissue under high-fat diet conditions were analyzed using liquid chromatography-tandem mass spectrometry and bioinformatics analysis. Differentially regulated proteins (DRPs) were enriched in metabolic pathways, which were subsequently corroborated through animal experiments. The consensus DRPs from these datasets were used as seed proteins to generate a protein-protein interaction network using STRING, and GeneMANIA identified fatty acid synthase as a mutually relevant protein. In an additional local-protein-protein interaction analysis of CRTC2 and fatty acid synthase with DRPs, sterol regulatory element binding transcription factor 1 (SREBF1) was the common mediator. CRTC2-CREB and SREBF1 are transcription factors, and DNA-binding motif analysis showed that multiple CRTC2-CREB–regulated genes possess SREBF1-binding motifs. This indicates the possible induction by the CRTC2–SREBF1 complex, which is validated through luciferase assay. Therefore, the CRTC2–SREBF1 complex potentially modulates the transcription of multiple proteins that fine-tune cellular metabolism under metabolic stress.

The effects of aerobic exercise on liver function, insulin resistance, and lipid profiles in prediabetic and type 2 diabetic mice

Background and aimsThe purpose of the current study was to evaluate the expression of gluconeogenesis and insulin resistance key genes; including insulin receptor substrate 1 (Irs1), a serine/threonine protein kinase (Akt), forkhead box class-O 1 (FoxO1) and phosphoenolpyruvate carboxykinase (Pepck) genes, and lipid profiles following either a standard or a high-fat diet (HFD) and either an aerobic exercise or non-exercise intervention in prediabetic and type 2 diabetic (T2DM) mice. Methods24 male mice were randomly assigned to two groups fed with a normal diet (ND) or a HFD for 12 weeks. The mice in each group were again randomly assigned to two groups to create four groups in total: 1. Prediabetes-exercised (Prediabetes-Exe), 2. Prediabetes-non exercised (Prediabetes-Non exe), 3. Healthy-exercised (Healthy-Exe), and 4. Healthy-non exercised (Healthy-Non exe). Eighteen additional male mice were fed with the HFD for 8 weeks, after which streptozotocin (STZ) was administered. The mice were then fed the HFD for an additional 4 weeks. These T2DM mice were then randomly divided into two groups: 1. Diabetes-exercised (Diabetic-Exe), and 2. Diabetes-Non exe. The three Exe groups all exercised on a treadmill for 8 weeks for 5 sessions/week. After the last training session, liver tissue was extracted, and the expression of Irs1, Akt, FoxO1, and Pepck genes was measured using real time quantitative Polymerase chain reaction tests. Lipid profiles were measured in serum and in the liver. ResultsThe expression of both Irs1 and Akt was significantly increased in the Healthy-Exe, Prediabetes-Exe, and Diabetes-Exe groups as compared to the Healthy-Non exe, Prediabetes-Non exe, and Diabetes-Non exe groups (p < 0.001). Additionally, the expression of FoxO1 (p < 0.05) and Pepck (p < 0.001) decreased significantly in the Prediabetes-Exe, and Diabetes-Exe groups as compared to the Prediabetes-non exe, and Diabetes-Non exe groups. Aerobic exercise did not lead to reductions in FoxO1 or Pepck expression in the Healthy-Exe mice. ConclusionsEight weeks of aerobic exercise (5 sessions/week) significantly increased the expression of key genes that are important for maintaining glucose homeostasis and improving insulin resistance (Irs1 and Akt), and decreased expression of genes that are important for decreasing gluconeogenesis in the liver (FoxO1 and Pepck) in healthy, prediabetic, and T2DM mice. The lipid profiles improved in healthy, prediabetic, and T2DM mice.

CRISPR/Cas9-based double-strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type-Ia.

Glycogen storage disease type-Ia (GSD-Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). Using the G6pc-R83C mouse model of GSD-Ia, we explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with two doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype.

Renal Glucose Release after Unilateral Renal Denervation during a Hypoglycemic Clamp in Pigs with an Altered Hypothalamic Pituitary Adrenal Axis after Late-Gestational Dexamethasone Injection.

Previously, we demonstrated in pigs that renal denervation halves glucose release during hypoglycaemia and that a prenatal dexamethasone injection caused increased ACTH and cortisol concentrations as markers of a heightened hypothalamic pituitary adrenal axis (HPAA) during hypoglycaemia. In this study, we investigated the influence of an altered HPAA on renal glucose release during hypoglycaemia. Pigs whose mothers had received two late-gestational dexamethasone injections were subjected to a 75 min hyperinsulinaemic-hypoglycaemic clamp (<3 mmol/L) after unilateral surgical denervation. Para-aminohippurate (PAH) clearance, inulin, sodium excretion and arterio-venous blood glucose difference were measured every fifteen minutes. The statistical analysis was performed with a Wilcoxon signed-rank test. PAH, inulin, the calculated glomerular filtration rate and plasma flow did not change through renal denervation. Urinary sodium excretion increased significantly (p = 0.019). Side-dependent renal net glucose release (SGN) decreased by 25 ± 23% (p = 0.004). At 25 percent, the SGN decrease was only half of that observed in non-HPAA-altered animals in our prior investigation. The current findings may suggest that specimens with an elevated HPAA undergo long-term adaptations to maintain glucose homeostasis. Nonetheless, the decrease in SGN warrants further investigations and potentially caution in performing renal denervation in certain patient groups, such as diabetics at risk of hypoglycaemia.

A pathway model of glucose-stimulated insulin secretion in the pancreatic β-cell.

The pancreas plays a critical role in maintaining glucose homeostasis through the secretion of hormones from the islets of Langerhans. Glucose-stimulated insulin secretion (GSIS) by the pancreatic β-cell is the main mechanism for reducing elevated plasma glucose. Here we present a systematic modeling workflow for the development of kinetic pathway models using the Systems Biology Markup Language (SBML). Steps include retrieval of information from databases, curation of experimental and clinical data for model calibration and validation, integration of heterogeneous data including absolute and relative measurements, unit normalization, data normalization, and model annotation. An important factor was the reproducibility and exchangeability of the model, which allowed the use of various existing tools. The workflow was applied to construct a novel data-driven kinetic model of GSIS in the pancreatic β-cell based on experimental and clinical data from 39 studies spanning 50 years of pancreatic, islet, and β-cell research in humans, rats, mice, and cell lines. The model consists of detailed glycolysis and phenomenological equations for insulin secretion coupled to cellular energy state, ATP dynamics and (ATP/ADP ratio). Key findings of our work are that in GSIS there is a glucose-dependent increase in almost all intermediates of glycolysis. This increase in glycolytic metabolites is accompanied by an increase in energy metabolites, especially ATP and NADH. One of the few decreasing metabolites is ADP, which, in combination with the increase in ATP, results in a large increase in ATP/ADP ratios in the β-cell with increasing glucose. Insulin secretion is dependent on ATP/ADP, resulting in glucose-stimulated insulin secretion. The observed glucose-dependent increase in glycolytic intermediates and the resulting change in ATP/ADP ratios and insulin secretion is a robust phenomenon observed across data sets, experimental systems and species. Model predictions of the glucose-dependent response of glycolytic intermediates and biphasic insulin secretion are in good agreement with experimental measurements. Our model predicts that factors affecting ATP consumption, ATP formation, hexokinase, phosphofructokinase, and ATP/ADP-dependent insulin secretion have a major effect on GSIS. In conclusion, we have developed and applied a systematic modeling workflow for pathway models that allowed us to gain insight into key mechanisms in GSIS in the pancreatic β-cell.

Loss of CtBP2 may be a mechanistic link between metabolic derangements and progressive impairment of pancreatic β cell function

The adaptive increase in insulin secretion in early stages of obesity serves as a safeguard mechanism to maintain glucose homeostasis that cannot be sustained, and the eventual decompensation of β cells is a key event in the pathogenesis of diabetes. Here we describe a crucial system orchestrated by a transcriptional cofactor CtBP2. In cultured β cells, insulin gene expression is coactivated by CtBP2. Global genomic mapping of CtBP2 binding sites identifies a key interaction between CtBP2 and NEUROD1 through which CtBP2 decompacts chromatin in the insulin gene promoter. CtBP2 expression is diminished in pancreatic islets in multiple mouse models of obesity, as well as human obesity. Pancreatic β cell-specific CtBP2-deficient mice manifest glucose intolerance with impaired insulin secretion. Our transcriptome analysis highlights an essential role of CtBP2 in the maintenance of β cell integrity. This system provides clues to the molecular basis in obesity and may be targetable to develop therapeutic approaches.

ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects oftunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these resultsshowchanges in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

Glycogen storage diseases: An update.

Glycogen storage diseases (GSDs), also referred to as glycogenoses, are inherited metabolic disorders of glycogen metabolism caused by deficiency of enzymes or transporters involved in the synthesis or degradation of glycogen leading to aberrant storage and/or utilization. The overall estimated GSD incidence is 1 case per 20000-43000 live births. There are over 20 types of GSD including the subtypes. This heterogeneous group of rare diseases represents inborn errors of carbohydrate metabolism and are classified based on the deficient enzyme and affected tissues. GSDs primarily affect liver or muscle or both as glycogen is particularly abundant in these tissues. However, besides liver and skeletal muscle, depending on the affected enzyme and its expression in various tissues, multiorgan involvement including heart, kidney and/or brain may be seen. Although GSDs share similar clinical features to some extent, there is a wide spectrum of clinical phenotypes. Currently, the goal of treatment is to maintain glucose homeostasis by dietary management and the use of uncooked cornstarch. In addition to nutritional interventions, pharmacological treatment, physical and supportive therapies, enzyme replacement therapy (ERT) and organ transplantation are other treatment approaches for both disease manifestations and long-term complications. The lack of a specific therapy for GSDs has prompted efforts to develop new treatment strategies like gene therapy. Since early diagnosis and aggressive treatment are related to better prognosis, physicians should be aware of these conditions and include GSDs in the differential diagnosis of patients with relevant manifestations including fasting hypoglycemia, hepatomegaly, hypertransaminasemia, hyperlipidemia, exercise intolerance, muscle cramps/pain, rhabdomyolysis, and muscle weakness. Here, we aim to provide a comprehensive review of GSDs. This review provides general characteristics of all types of GSDs with a focus on those with liver involvement.

Endocrinology for the hepatologist.

Endocrinology for the hepatologist.

The role of GLUT2 in glucose metabolism in multiple organs and tissues.

The glucose transporter family has an important role in the initial stage of glucose metabolism; Glucose transporters 2 (GLUTs, encoded by the solute carrier family 2, SLC2A genes) is the major glucose transporter in β-cells of pancreatic islets and hepatocytes but is also expressed in the small intestine, kidneys, and central nervous system; GLUT2 has a relatively low affinity to glucose. Under physiological conditions, GLUT2 transports glucose into cells and allows the glucose concentration to reach balance on the bilateral sides of the cellular membrane; Variation of GLUT2 is associated with various endocrine and metabolic disorders; In this study, we discussed the role of GLUT2 in participating in glucose metabolism and regulation in multiple organs and tissues and its effects on maintaining glucose homeostasis.

Reciprocal Regulation of Hepatic TGF-β1 and Foxo1 Controls Gluconeogenesis and Energy Expenditure.

Hepatic TGF-β1 levels are increased in obese humans and mice. Hepatic TGF-β1 maintains glucose homeostasis in lean mice and causes glucose and energy dysregulations in obese and diabetic mice. Hepatic TGF-β1 exerts an autocrine effect to promote hepatic gluconeogenesis via cAMP-dependent protein kinase-mediated Foxo1 phosphorylation at serine 273, endocrine effects on brown adipose tissue action, and inguinal white adipose tissue browning (beige fat), causing energy imbalance in obese and insulin-resistant mice. TGF-β1→Foxo1→TGF-β1 looping in hepatocytes plays a critical role in controlling glucose and energy metabolism in health and disease.

Natural products as novel anti-obesity agents: insights into mechanisms of action and potential for therapeutic management.

Obesity affects more than 10% of the adult population globally. Despite the introduction of diverse medications aimed at combating fat accumulation and obesity, a significant number of these pharmaceutical interventions are linked to substantial occurrences of severe adverse events, occasionally leading to their withdrawal from the market. Natural products serve as attractive sources for anti-obesity agents as many of them can alter the host metabolic processes and maintain glucose homeostasis via metabolic and thermogenic stimulation, appetite regulation, pancreatic lipase and amylase inhibition, insulin sensitivity enhancing, adipogenesis inhibition and adipocyte apoptosis induction. In this review, we shed light on the biological processes that control energy balance and thermogenesis as well as metabolic pathways in white adipose tissue browning, we also highlight the anti-obesity potential of natural products with their mechanism of action. Based on previous findings, the crucial proteins and molecular pathways involved in adipose tissue browning and lipolysis induction are uncoupling protein-1, PR domain containing 16, and peroxisome proliferator-activated receptor-γ in addition to Sirtuin-1 and AMP-activated protein kinase pathway. Given that some phytochemicals can also lower proinflammatory substances like TNF-α, IL-6, and IL-1 secreted from adipose tissue and change the production of adipokines like leptin and adiponectin, which are important regulators of body weight, natural products represent a treasure trove for anti-obesity agents. In conclusion, conducting comprehensive research on natural products holds the potential to accelerate the development of an improved obesity management strategy characterized by heightened efficacy and reduced incidence of side effects.

1746-P: The Role of Cell–Cell Junction Associated Mediator in Glucose-Stimulated Insulin Secretion of Pancreatic Islets

Glucose-stimulated insulin secretion from β cells is essential in maintaining glucose homeostasis. Cell-cell communication within the pancreatic islets, mediated by cell-cell junctions and their mediators, regulates insulin secretion dynamics and glucose homeostasis. It has been reported that Nephrin-mediated intercellular junction between β cells is implicated in the regulation of insulin secretion, however, the underlying mechanisms are not fully understood. Here, we investigated the effects of Girdin, a cell-cell junction associated mediator, on insulin release. We found that Girdin was expressed in mouse pancreatic β cells and mediated glucose-stimulated insulin secretion, especially for the second phase. Next, we demonstrated that glucose-stimulated tyrosine phosphorylation of Girdin mediated Nephrin phosphorylation by recruiting Src kinase, which leads to the endocytosis of Nephrin. Furthermore, we observed the glucose-induced Girdin/Nephrin/Src signaling induced downstream Akt phosphorylation and activated Rac1-mediated cytoskeleton reorganization, allowing insulin secretory granules to access the plasma membrane for the second-phase secretion. Finally, we found that Girdin is downregulated in the diabetic islets, and rescue of Girdin restored impaired glucose-stimulated insulin secretion. Taken together, these data provide evidence that the cell-cell junction associated mediator play important roles in fusion of insulin secretory vesicles within the cell and establish a role for Girdin in regulating this process. Disclosure H.Wang: None. Funding National Natural Science Foundation of China

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&amp;D Program of China (2019YFA0802502, 2022YFE0131400); Tianjin Key Medical Discipline Construction Project (TJYXZDXK-030A)

1639-P: Meroterpenoid-Rich Fraction of the Ethanol Extract from Sargassum Serratifolium Suppressed Muscle Atrophy Induced by Dexamethasone in C2C12 Myotubes and C57BL/6 Mice

Decline in muscle mass and strength impairs the ability to perform normal daily functions and increases the risk of falls and fractures, significantly reducing the quality of life. In addition, as the muscle is considered a major regulator of systemic glucose homeostasis, maintaining muscle mass is important for glycemic control. Although dexamethasone (DEX), a synthetic glucocorticoid, is a well-known anti-inflammatory drug, high doses or long-term use induces side effects such as skeletal muscle atrophy. Sargassum serratifolium is a brown alga that contains high concentrations of meroterpenoids and exhibits antioxidant, anti-inflammatory, anti-obesity, anti-atherosclerosis, and hepatoprotective activities. However, its effects on DEX-induced muscle atrophy remain unknown. Here, we investigated the beneficial effects of the meroterpenoid-rich fraction of the ethanol extract from S. serratifolium (MES) on DEX-induced muscle atrophy in C2C12 myotubes and C57BL/6 mice. In the in vitro study using C2C12 myotubes, DEX (5 μM) treatment reduced cell viability, myotube diameter, and glucose uptake, whereas MES treatment reversed these effects. In addition, in DEX-stimulated C2C12 myotubes, MES treatment not only increased the phosphorylation of Akt and p706SK but also decreased the mRNA and protein expression of protein degradation factors such as MAFbx/Atrogin-1, MuRF1, and FoxO3a. In the in vivo study, MES suppressed DEX-induced decrease in body weight, ability to exercise, lean body mass, calf thickness, muscle weight, and cross-sectional area of the gastrocnemius muscle. Taken together, MES ameliorated DEX-induced muscle atrophy and maintained glucose homeostasis. Therefore, MES can be used as a potential therapeutic agent for the treatment of muscle atrophy. Disclosure M.Lee: None. H.Ryu: None. H.Jeong: None. B.Lee: None.

233-OR: The Beta-Cell Specific Deletion of Complement 1q-Like-3 Secreted Protein Improves Beta-Cell Function and Glucose Homeostasis

Using bioinformatics network analysis, we identified complement-1q like-3 (C1ql3) as a secreted protein that affects pancreatic islet function in obese mice. Previously, we showed that the recombinant C1ql3 protein inhibits insulin secretion stimulated by exendin-4 (an agonist for GLP-1 receptor) from human and mouse islets. Currently, we are elucidating the role of C1ql3 in pancreatic β-cell function by using mice with β-cell-specific deletion of the C1ql3 (βKO) gene on a standard chow diet. C1ql3βKO mice showed significantly improved glucose clearance after oral glucose challenge than the control mice, whereas no difference in insulin sensitivity was observed. Moreover, the plasma insulin levels were significantly increased at 15 min after the glucose challenge in the C1ql3 βKO vs. control mice. However, islets isolated from C1ql3βKO significantly increased insulin secretion stimulated by cAMP and Exendin-4 than control islets. These results suggest that the loss of C1ql3 in β-cells increases insulin secretion to improve glucose tolerance. Adenovirus-mediated overexpression of C1ql3 in islets showed increased ER stress and decreased expression of genes involving β-cell function and maintenance. In pathophysiological conditions, increased C1ql3 expression coupled to secretion was observed from both T2D donor islets and obese mouse islets. Interestingly, reduction in C1ql3 secretion is inversely correlated with increased glucose-stimulated insulin secretion from islets of C1ql3βKOLeptinob/ob compared to Leptinob/ob control mice. These findings suggest that the loss of β-cell C1ql3 maintains ER stress and increases insulin secretion to maintain glucose homeostasis. Our work shows that an autocrine regulation of the C1ql3-signaling pathway contributes to β-cell dysfunction in obesity and type 2 diabetes. Disclosure M.Rahman: None. H.Alsharif: None. A.Michl: None. S.Bhatnagar: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK120684-01)

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&amp;D Program of China (2019YFA0802502, 2022YFE0131400); Tianjin Municipal Health and Health Committee (TJWJ2021ZD00); Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-030A)

269-LB: Modeling Alpha-Cell Dysfunction Using Stem Cell–Derived Alpha Cells

Type 1 Diabetes (T1D) therapies have focused on restoring beta cell function via exogenous insulin or islet transplant. While promising, these approaches fail to account for the role of alpha cells and intra-islet communication in disease etiology. Alpha cells secrete glucagon and maintain effective paracrine signaling with beta cells to maintain glucose homeostasis and metabolic control. Mounting evidences suggest that impaired alpha cell function is a significant contributor to disease progression. Understanding triggers of alpha cell dysfunction and the resulting effects will be crucial for restoring both beta and alpha cell function in T1D. Lack of human alpha cell models have limited progress towards this goal. We recently reported a protocol to generate human alpha cells from stem cell sources (SC-alpha). Using these SC-alpha cells, we have established a T1D disease model to identify the triggers that induce alpha cell dysfunction. Importantly, we have identified defects in proglucagon processing that mimic those observed in diabetes and have identified conditions that modulate proglucagon processing and secretion. Here we demonstrate that exposure of SC-alpha cells to diabetogenic stressors such as hyperglycemia, hyperlipidemia, and ER stress impacts the processing of proglucagon and ultimately results in alpha cell dysfunction. We show that upon exposure to these stressors, SC-alpha cells secrete elevated glucagon compared to the control conditions and show a loss of glucose responsiveness. We also observed changes in the secretion of alternate proglucagon processing products, GLP-1, GLP-2, and glicentin. Interestingly, glicentin levels were affected by ER stress induction, but were unaffected by other stressors, suggesting that ER stress plays a direct role in the disruption of proglucagon processing, leading to alpha cell dysfunction. Identifying triggers of alpha cell dysfunction and assessing impacts in proglucagon processing will be a step towards restoring islet paracrine signaling. Disclosure S. Shrestha: None. Q. P. Peterson: None.

290-OR: FoxK1-Associated Gene Regulatory Network—A New Transcriptional Regulator of Hepatic Insulin Action

Insulin acts on the liver to maintain glucose homeostasis, and disruption of insulin action in liver is central in the pathogenesis of diabetes and metabolic syndrome. While most previous studies of insulin action on gene expression in liver have focused on FoxO1, we have found that Forkhead box protein K1 (FoxK1) is another important regulator of hepatic gene expression which acts in a complementary manner. Thus, upon insulin stimulation, FoxK1 translocates from the cytoplasm to nucleus while FoxO1 translocates from nucleus to cytoplasm. Chromatin immunoprecipitation (ChIP) sequencing shows that FoxK1 binds to &amp;gt;1000 gene regulatory elements in the proximal promoters and enhancers in hepatic chromatin, with TGTTTAC being the major FoxK1 DNA binding motif. De novo motif analysis suggested that HNF4A is also one of the major co-regulators for FoxK1 in mediating gene expression. Insulin stimulated binding of FoxK1 to promoters was observed in up-regulation of genes involved in steroid biosynthesis and cell cycle, and down-regulation of genes involved in autophagy. FoxK1 is also a potential transcription factor for insulin receptor (IRβ) mediated gene regulation as evidenced by its binding to LARS1 and TIMM22 promoters. Both FoxK1 and IRβ share regulation of genes involved in rRNA processing, mitotic cell cycle and nonsense-mediated decay. Interestingly, gene promoters of genes in glucose metabolism (PCK1, G6PC and PKM) are bound by FoxK1, but not by IRβ in response to insulin. FoxK1 can also work with FoxO1 in regulation of genes involved in mitochondrial translation, and with both IRβ and FoxO1 in insulin regulation of AHCY, PPRC1, and CDKN1A, i.e., genes involved in cell senescence, amino acid metabolism and mitochondrial biogenesis. Thus, FoxK1 is a potential factor priming the response to insulin and helps with the recruitment of IRβ or FoxO1 in regulation of gene expression in liver in metabolism and other important biological pathways. Disclosure P.Allu: None. H.Pan: None. J.Dreyfuss: None. C.Kahn: None.