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In the article cited above, the affiliations for authors Stephen J. Pandol and Maike Sander were inadvertently transposed. The correct affiliation for Stephen J. Pandol is Department of Gastroenterology, Cedars-Sinai Medical Center, Los Angeles, CA. The correct affiliation for Maike Sander is Department of Pediatrics and Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA. The online version of the article (https://doi.org/10.2337/db22-0942) has been updated with the correct affiliations.

The coronavirus disease 2019 (COVID-19) pandemic led to a rapid expansion in the use of telemedicine for treating many conditions, including type 1 diabetes. In the T1DX-QI, we reported a rapid adoption of telemedicine during the pandemic (2). We described interventions for and barriers to telemedicine practice in type 1 diabetes care and identified access to technology tools as one of the key drivers of adoption (2). After the increased adoption of telemedicine in our clinics, we identified a quality gap in accessing insulin pump data remotely. The Tandem t:slim X2 insulin pump was most used pump used by our patients attending the two clinics. As part of the activities supported by the T1DX-QI, we started a quality improvement (QI) project aimed at increasing the percentage of patients with type 1 diabetes using the t:slim X2 insulin pump who were able to share data remotely. Patients have different options for sharing data when they are not in the clinic, but the process requires a good understanding of the manufacturer’s online data-sharing platform (t:connect) (Supplementary Figure S1). First, patients need a t:connect account. Then, if their account is not linked to the t:connect health care provider (HCP) portal, they must upload their pump data using cables to connect the pump to the computer, generate the report, and send it (e.g., by e-mail) to the clinic. This process is time consuming, requires patients to take several actions, and in our experience, is prone to failure. Instead, if patients’ t:connect account is linked to the HCP portal, uploaded data are readily available on the platform for HCPs to access. Finally, in the ideal scenario, patients can share data through the t:connect mobile app, which became available in August 2020. In this case, no cables are needed and pump data can be easily accessed remotely at any time on the manufacturer platform, as long as a patient’s account is linked to the HCP portal and the t:connect mobile app is open and transferring data. For this project, we sought to increase the percentage of patients with type 1 diabetes using the t:slim X2 insulin pump who were able to share data from a baseline of 40% to 80% in 6 months. The measured primary outcome was the percentage of patients with an established t:connect account linked to the HCP portal, and the secondary outcome was the percentage of patients using the t:connect mobile app.

Male mice lacking the androgen receptor (AR) in islet β cells exhibit blunted glucose-stimulated insulin secretion (GSIS) , leading to hypoinsulinemia and hyperglycemia. We showed that dihydrotestosterone (DHT) activation of an extranuclear AR in β cells potentiates GSIS by amplifying the insulinotropic action of GLP-1 in mouse and human islets. To gain information on DHT-stimulated signaling networks promoting GSIS, we combined functional proteomics with highly selective pharmacological inhibitors in male human islets, and islets from genetic mouse models. We found that DHT-stimulated GSIS in human islets involved a signaling cascade including focal adhesion kinases, the nonreceptor tyrosine kinase SRC, phosphoinositide 3-kinase, and the activation of mTOR complex 2. To define the early network of AR interacting proteins, we performed DHT-stimulated AR immunoprecipitation in INS1 832/3 cells followed by mass spectrometry-based proteomics. We will discuss the AR interactome including: 1) adenylate cyclase-modulating G proteins Gαs and Gαi, cAMP-dependent PKA, and the cAMP-dependent protein LASP1 regulating actin-based cytoskeletal activities; 2) Actin cytoskeleton remodeling proteins promoting actin depolymerization, and 3) Vesicular maturation including the lysosomal vacuolar-type ATPase acidifying insulin secretory granules and the prohormone convertase 2 involved in proinsulin processing. Notably, GLP-1 addition to DHT decreased AR interaction with all these proteins and promoted AR interaction with microtubule motor proteins promoting vesicular transport, proteins involved in exocytic vesicle docking to the plasma membrane, as well as small GTPase activating proteins and guanine nucleotide exchange factors involved in actin remodeling, as well as vesicle trafficking. Together these experiments provide novel insights into the architecture of AR action in enhancing GSIS. Disclosure P.Mota de sa: None. M.Qadir: None. M.Blandino-rosano: None. J.Fan: None. W.Xu: None. E.Bernal-mizrachi: None. F.Mauvais-jarvis: Consultant; Mithra Pharmaceutical. Funding National Institutes of Health (DK107444 and DK074970) U.S. Department of Veterans Affairs Merit Award (BX003725)

Lack of healthcare insurance is an important factor for poor glycemic control in persons with diabetes. However, a large proportion of insured people also have inadequate glycemic control. This study was conducted to assess factors associated with poor glycemic control in persons with type 2 diabetics (T2D) who received full healthcare coverage through their employer.The study included employees and spouses with T2D who participated in an annual health assessment in 20 and had medical coverage ≥12 consecutive months prior to the assessment. T2D was defined from ICD codes in claims, self-reported physician diagnosis, or having fasting glucose > 125 mg/dL or HbA1C > 6.4%. A stepwise multinomial logistic regression model was used to assess associations of healthcare engagement, diet, smoking and socioeconomic status with glycemic control. The 2,981 participants included 16% (n=484) with HbA1C > 8.5% and 27% (n=800) with HbA1C 7 to 8.5%. We found that HbA1C > 8.5% was associated with age, sex, years since last physical exam, number of complications, number of medications, and physical activity. The regression model results are shown in the figure. We conclude that poor glycemic control is common among persons with T2D despite good healthcare coverage. We identified factors associated with poor glycemic control in this population that may help design strategies to improve glycemic control. Disclosure C.M.Rowland: Employee; Quest Diagnostics, Stock/Shareholder; Quest Diagnostics. J.Z.Louie: None. D.Shiffman: Employee; Quest Diagnostics, Stock/Shareholder; Quest Diagnostics. R.Garg: None. E.Bernal-mizrachi: None. M.J.Mcphaul: Employee; Quest Diagnostics.

Poor socioeconomic status and inadequate insurance coverage may affect an individual’s ability to utilize healthcare services. This study was conducted to evaluate whether the type of insurance coverage is associated with missed appointments for diabetes care. Data analysis included all patients with diabetes mellitus (DM) managed at a major academic center between Jan 2015 and Dec 2020. Association between insurance coverage and proportion of missed appointments was evaluated using analysis of covariance with adjustments for demographic variables and social determinants of health. The relationship between proportion of missed appointments and glycemic control was also evaluated in multivariate analyses. The final dataset included 30,633 patients, out of which 14,064 (46%) reported commercial insurance, 13,376 (44%) reported Medicare and 3,193 (10%) reported Medicaid coverage. Medicaid group was identified to have the highest proportion of Spanish-speakers, African Americans, women, current smokers and single individuals. Proportion of missed appointments was 18.1 ± 18.1% in Medicaid covered patients as compared to 12.1 ± 15.3% among commercially insured and 10.2 ± 14.1% among Medicare covered patients (p Disclosure R.Radhakrishnan: None. W.H.Cade: None. E.Bernal-mizrachi: None. R.Garg: None.

The ADA recommends an A1C 8.5% (poor control) were included. Patients with improved glycemic control (current A1C ≤8.5%) were compared with those with lack of improvement (current A1C >8.5%) . On multivariate logistic regression analysis including all variables in the model, independent predictors of lack of improvement were: younger age (OR 0.89 per 1-SD [12 years]; 95 % CI 0.79 - 1.00) , female gender (1.30, 1.- 1.56) , presence of hypertension (1.29, 1.- 1.55) , Black race (1.32, 1.- 1.68, White as reference) , low income area code (1.86,1.28 - 2.68, high as reference) and insurance coverage other than Medicare (1.32, 1.- 1.66) . Presence of current smoking was associated with a paradoxical improvement in A1C (0.69, 0.47 - 0.99) . We conclude that socioeconomic factors like income, insurance coverage and neighborhood are associated with lack of improvement in A1C. Further studies need to identify specific modifiable socioeconomic factors. Disclosure R. Garg: None. J.Z. Louie: None. D. Shiffman: Employee; Quest Diagnostics. Stock/Shareholder; Quest Diagnostics. C.M. Rowland: Employee; Quest Diagnostics. Stock/Shareholder; Quest Diagnostics. M.J. McPhaul: Employee; Quest Diagnostics. E. Bernal-Mizrachi: None. N. Kenyon: Research Support; Elodon Phamaceutical, Takeda Pharmaceutical Company Limited.

Elevation of glucagon levels and increase in alpha-cell mass are associated with states of hyperglycemia in diabetes. A better understanding of the molecular mechanisms governing glucagon secretion could have major implications in understanding abnormal responses to hypoglycemia in diabetic patients and provide novel avenues for diabetes management. Stimulation of glucagon secretion in hypoglycemia, or by amino acids, induces hepatic glucose production via cellular mechanisms including suppression of glycogenesis and glycolysis and stimulation of glycogenolysis and gluconeogenesis. The close link between amino acids and the alpha-cell is highlighted by the liver–alpha-cell axis. This axis was identified by the major increase in alpha-cell hyperplasia and hyperglucagonemia in models of reduced glucagon action in hepatocytes genetically or pharmacologically by treatment with glucagon receptor antagonists, which was subsequently attributed to the dramatic rise in amino acids. Induction of mTORC1 by constitutive genetic deletion of TSC2 in alpha-cells (aTSC2KO) recapitulated the effects of chronic hyperaminoacidemia with increases in alpha-cell mass and chronic hyperglucagonemia indicating that mTORC1 mediates amino acids signals in alpha-cells. However, the effects of acute versus chronic induction of alpha-cell mTORC1 signaling on these outcomes have not been explored. Using mice with doxycycline induction of Rheb (activator of mTORC1 signaling) (aRhebTg) and inducible Cre-mediated deletion of the mTORC1 inhibitor, TSC2, in alpha-cells (iaTSC2KO), we showed that induction of hyperglucagonemia is associated with two phases: Acute phase characterized by impaired glucose tolerance because of increased gluconeogenesis and glucose output by the liver. And a chronic phase, characterized by normalization of glucose tolerance due to downregulation of the hepatic glucagon receptor (Gcgr), Pepck, and some genes involved in amino acid metabolism and urea production. Importantly, the consequences of chronic hyperglucagonemia were reversible after normalization of glucagon levels by turning off alpha-cell Rheb expression. These studies uncovered the acute versus chronic effects of hyperglucagonemia on glucose homeostasis and further demonstrate that glucagon resistance is a reversible process. These observations provide insight into the impact of glucagon in the pathogenesis of hyperglycemia and the physiological effects of administration of dual GLP1/Glucagon receptor agonists. Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.

Identifying the mechanisms behind the b-cell adaptation-to-failure is important to develop strategies to manage type 2 diabetes (T2D). Using db/db mice at early stages of the disease process, we took advantage of unbiased RNAseq to identify genes/pathways regulated by insulin resistance in b-cells. We demonstrate herein that islets from 4-week-old non-obese and non-diabetic leptin-receptor deficient db/db mice exhibited downregulation of several genes involved in cell-cycle regulation and DNA repair. We identified the transcription factor Yin Yang 1 (YY1) as a common gene between both pathways. The expression of YY1 and its targeted genes was decreased in the db/db islets. We confirmed the reduction in YY1 expression in b-cells from diabetic db/db mice, mice fed high fat diet (HFD) and individuals with T2D. ChIP-seq profiling in EndocBH1 cells, a human pancreatic b-cell line, indicated that YY1 binding regions regulate cell-cycle control, DNA damage recognition and repair. We then generated mouse models with constitutive and inducible YY1 deficiency in b-cells. YY1 deficient mice developed diabetes early in life due to b-cell loss. b-cells from these mice exhibited higher DNA damage, cell cycle arrest and cell death as well as decreased maturation markers. Tamoxifen-induced YY1 deficiency in mature b-cells impaired b-cell function and induced DNA damage. In summary, we identified YY1 as a critical factor for b-cell DNA repair and cell-cycle progression.

Maternal low-protein diet (LP) throughout gestation affects pancreatic β-cell fraction of the offspring at birth, thus increasing their susceptibility to metabolic dysfunction and type 2 diabetes in adulthood. The present study sought to strictly examine the effects of LP during the last week of gestation (LP12.5) alone as a developmental window for β-cell programming and metabolic dysfunction in adulthood. Islet morphology analysis revealed normal β-cell fraction in LP12.5 newborns. Normal glucose tolerance was observed in 6- to 8-wk-old male and female LP12.5 offspring. However, male LP12.5 offspring displayed glucose intolerance and reduced insulin sensitivity associated with β-cell dysfunction with aging. High-fat diet exposure of metabolically normal 12-wk-old male LP12.5 induced glucose intolerance due to increased body weight, insulin resistance, and insufficient β-cell mass adaptation despite higher insulin secretion. Assessment of epigenetic mechanisms through microRNAs (miRs) by a real-time PCR-based microarray in islets revealed elevation in miRs that regulate insulin secretion (miRs 342, 143), insulin resistance (miR143), and obesity (miR219). In the islets, overexpression of miR143 reduced insulin secretion in response to glucose. In contrast to the model of LP exposure throughout pregnancy, islet protein levels of mTOR and pancreatic and duodenal homeobox 1 were normal in LP12.5 islets. Collectively, these data suggest that LP diet during the last week of pregnancy is critical and sufficient to induce specific and distinct developmental programming effects of tissues that control glucose homeostasis, thus causing permanent changes in specific set of microRNAs that may contribute to the overall vulnerability of the offspring to obesity, insulin resistance, and type 2 diabetes.

AIMS/HYPOTHESIS: Islet vascular fibrosis may play an important role in the progression of type 2 diabetes, but there are no mouse models allowing detailed mechanistic studies to understand how a dysfunctional islet microvasculature contributes to diabetes pathogenesis. Here we report that the transgenic AktTg mouse, unlike other mouse strains, shows an increased deposition of extracellular matrix (ECM) proteins in perivascular regions, allowing us to study the cellular mechanisms that lead to islet vascular fibrosis. METHODS: Using immunohistochemistry, we labelled the islet microvasculature and ECM in pancreas sections of AktTg mice and human donors and performed lineage tracing to follow the fate of islet pericytes. We compared islet microvascular responses in living pancreas slices from wild-type and AktTg mice. RESULTS: We found that vascular pericytes proliferate extensively, convert into profibrotic myofibroblasts and substantially contribute to vascular fibrosis in the AktTg mouse model. The increased deposition of collagen I, fibronectin and periostin within the islet is associated with diminished islet perfusion as well as impaired capillary responses to noradrenaline (norepinephrine) and to high glucose in living pancreas slices. CONCLUSIONS/INTERPRETATION: Our study thus illustrates how the AktTg mouse serves to elucidate a cellular mechanism in the development of islet vascular fibrosis, namely a change in pericyte phenotype that leads to vascular dysfunction. Because beta cells in the AktTg mouse are more numerous and larger, and secrete more insulin, in future studies we will test the role beta cell secretory products play in determining the phenotype of pericytes and other cells residing in the islet microenvironment under physiological and pathophysiological conditions.

G protein–coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gβ5–R7, a protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit β 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M–stimulated glucose–stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5(−/−) islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K(+)-evoked membrane depolarization. These results indicate that Gβ5–R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gβ5–R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Mammalian target of rapamycin (mTOR) kinase is an essential hub where nutrients and growth factors converge to control cellular metabolism. mTOR interacts with different accessory proteins to form complexes 1 and 2 (mTORC), and each complex has different intracellular targets. Although mTORC1's role in β-cells has been extensively studied, less is known about mTORC2's function in β-cells. Here, we show that mice with constitutive and inducible β-cell-specific deletion of RICTOR (; βRicKO; and i; βRicKO; mice, respectively) are glucose intolerant due to impaired insulin secretion when glucose is injected intraperitoneally. Decreased insulin secretion in βRicKO islets was caused by abnormal actin polymerization. Interestingly, when glucose was administered orally, no difference in glucose homeostasis and insulin secretion were observed, suggesting that incretins are counteracting the mTORC2 deficiency. Mechanistically, glucagon-like peptide-1 (GLP-1), but not gastric inhibitory polypeptide (GIP), rescued insulin secretion in vivo and in vitro by improving actin polymerization in; βRicKO; islets. In conclusion, mTORC2 regulates glucose-stimulated insulin secretion by promoting actin filament remodeling.; NEW & NOTEWORTHY; The current studies uncover a novel mechanism linking mTORC2 signaling to glucose-stimulated insulin secretion by modulation of the actin filaments. This work also underscores the important role of GLP-1 in rescuing defects in insulin secretion by modulating actin polymerization and suggests that this effect is independent of mTORC2 signaling.

The dynamic regulation of autophagy in b-cells by cycles of fasting-feeding and its effects on insulin secretion are unknown. In b-cells mTORC1 is inhibited while fasting, and is rapidly stimulated during refeeding by a single amino acid, leucine, and glucose. Stimulation of mTORC1 by nutrients inhibited the autophagy initiator ULK1 and the transcription factor TFEB, thereby preventing autophagy when b-cells are continuously exposed to nutrients. Inhibition of mTORC1 by Raptor knockout mimicked the effects of fasting and stimulated autophagy while inhibiting insulin secretion, whereas moderate inhibition of autophagy under these conditions rescued insulin secretion. These results show that mTORC1 regulates insulin secretion through modulation of autophagy under different nutritional situations. In the fasting state, autophagy is regulated in an mTORC1-dependent manner and its stimulation is required to keep insulin levels low, thereby preventing hypoglycemia. Reciprocally, stimulation of mTORC1 by elevated leucine and glucose, which is common in obesity, may promote hyperinsulinemia by inhibiting autophagy.

Type 2 diabetes (T2D) is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in β-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs (snRNAs and snoRNAs) and is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in β-cells and glucose homeostasis had not been explored. Here we show that TGS1 is upregulated by insulin and upregulated in islets from mice exposed to a high-fat diet and in human β-cells from T2D donors. Using mice with conditional (βTGS1KO and βTGS1Het) and inducible (MIP-CreERT-TGS1KO) TGS1 deletion, we determine that TGS1 regulates β-cell mass and function. Unbiased approaches allowed us to identify a link between TGS1 and ER stress and cell cycle arrest and how TGS1 regulates β-cell apoptosis. Deletion of TGS1 results in an increase in the unfolded protein response by increasing XBP-1, ATF-4, and the phosphorylation of eIF2α, and several changes in cell cycle inhibitors and activators such as p27 and Cyclin D2. This study establishes TGS1 as a key player regulating β-cell mass and function as well as playing a role in the adaptive β-cell function to a high-fat diet. These observations can be used as a stepping-stone for the design of novel strategies using TGS1 as a therapeutic target for the treatment of diabetes.

O-GlcNAc transferase (OGT), a nutrient sensor sensitive to glucose flux, is highly expressed in the pancreas. However, the role of OGT in the mitochondria of β-cells is unexplored. In this study, we identified the role of OGT in mitochondrial function in β-cells. Constitutive deletion of OGT (βOGTKO) or inducible ablation in mature β-cells (iβOGTKO) causes distinct effects on mitochondrial morphology and function. Islets from βOGTKO, but not iβOGTKO, mice display swollen mitochondria, reduced glucose-stimulated oxygen consumption rate, ATP production, and glycolysis. Alleviating endoplasmic reticulum stress by genetic deletion of Chop did not rescue the mitochondrial dysfunction in βOGTKO mice. We identified altered islet proteome between βOGTKO and iβOGTKO mice. Pancreatic and duodenal homeobox 1 (Pdx1) was reduced in in βOGTKO islets. Pdx1 overexpression increased insulin content and improved mitochondrial morphology and function in βOGTKO islets. These data underscore the essential role of OGT in regulating β-cell mitochondrial morphology and bioenergetics. In conclusion, OGT couples nutrient signal and mitochondrial function to promote normal β-cell physiology.

Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in β-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in β-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human β-cells from type 2 diabetes donors. Using mice with conditional (βTGS1KO) and inducible (MIP-Cre

The dynamic regulation of autophagy in β-cells by cycles of fasting-feeding and its effects on insulin secretion are unknown. In β-cells, mechanistic target of rapamycin complex 1 (mTORC1) is inhibited while fasting and is rapidly stimulated during refeeding by a single amino acid, leucine, and glucose. Stimulation of mTORC1 by nutrients inhibited the autophagy initiator ULK1 and the transcription factor TFEB, thereby preventing autophagy when β-cells were continuously exposed to nutrients. Inhibition of mTORC1 by Raptor knockout mimicked the effects of fasting and stimulated autophagy while inhibiting insulin secretion, whereas moderate inhibition of autophagy under these conditions rescued insulin secretion. These results show that mTORC1 regulates insulin secretion through modulation of autophagy under different nutritional situations. In the fasting state, autophagy is regulated in an mTORC1-dependent manner, and its stimulation is required to keep insulin levels low, thereby preventing hypoglycemia. Reciprocally, stimulation of mTORC1 by elevated leucine and glucose, which is common in obesity, may promote hyperinsulinemia by inhibiting autophagy.

Elevation of glucagon levels and increase in a-cell mass are associated with states of hyperglycemia in diabetes. Our previous studies have highlighted the role of nutrient signaling via mTOR Complex 1 (mTORC1) regulation that controls glucagon secretion and a-cell mass. The current studies investigated the effects of activation of nutrient signaling by conditional deletion of the mTORC1 inhibitor, TSC2, in a-cells (aTSC2KO). We showed that activation of mTORC1 signaling is sufficient to induce chronic hyperglucagonemia as a result of a-cell proliferation, cell size and mass expansion. Hyperglucagonemia in aTSC2KO was associated with an increase in glucagon content and enhanced glucagon secretion. This model allowed us to identify the effects of chronic hyperglucagonemia on glucose homeostasis by inducing insulin secretion and resistance to glucagon in the liver. Liver glucagon resistance in aTSC2KO mice were characterized by reduced expression of the glucagon receptor (GCGR), phosphoenolpyruvate carboxykinase (PEPCK) and genes involved in amino acid metabolism and urea production. Glucagon resistance in aTSC2KO mice was associated with improved glucose levels in Streptozotocin (STZ)-induced β-cell destruction and HFD-induced glucose intolerance. These studies demonstrate that chronic hyperglucagonemia can improve glucose homeostasis by inducing glucagon resistance in the liver.

We previously demonstrated that exposing mouse dams to metformin during gestation results in increased beta-cell mass at birth and increased beta-cell insulin secretion in adult male offspring. Given these favorable changes after a gestational maternal metformin exposure, we wanted to understand the long-term metabolic impact on offspring after exposing dams to metformin during the postnatal window. The newborn period provides a feasible clinical window for intervention and is important for beta-cell proliferation and metabolic tissue development. Using a C57BL/6 model, we administered metformin to dams from the day of birth to postnatal day 21. We monitored maternal health and offspring growth during the lactation window, as well as adult glucose homeostasis through in vivo testing. At necropsy we assessed pancreas and adipocyte morphology using histological and immunofluorescent staining techniques. We found that metformin exposure programmed male and female offspring to be leaner with a higher proportion of small adipocytes in the gonadal white adipose tissue (GWAT). Male, but not female, offspring had an improvement in glucose tolerance as young adults concordant with a mild increase in insulin secretion in response to glucose in vivo. These data demonstrate long-term metabolic programming of offspring associated with maternal exposure to metformin during lactation.

Insulin receptor (Insr) protein can be found at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Ins1cre allele was used to delete Insr specifically in β-cells of both female and male mice. Experimental mice were compared to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells revealed transcriptomic consequences of Insr loss, which differed between female and male mice. Action potential and calcium oscillation frequencies were increased in Insr knockout β- cells from female, but not male mice, whereas only male βInsrKO mice had reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female βInsrKO and βInsrHET mice exhibited elevated insulin release in perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr did not alter β-cell area up to 9 months of age, nor did it impair hyperglycemia-induced proliferation. Based on our data, we adapted a mathematical model to include β-cell insulin resistance, which predicted that β-cell Insr knockout would improve glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance was significantly improved in female βInsrKO and βInsrHET mice when compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We did not observe improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia was associated with mildly reduced fasting glucose and increased body weight. We further validated our main in vivo findings using the Ins1-CreERT transgenic line and found that male mice had improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that loss of β-cell Insr contributes to glucose-induced hyperinsulinemia, thereby improving glucose homeostasis in otherwise insulin sensitive dietary and age contexts.

Mechanistic target of rapamycin complex 1 (mTORC1) deficiency or chronic hyperactivation in pancreatic β-cells leads to diabetes. mTORC1 complexes with La-related protein 1 (LARP1) to specifically regulate the expression of 5′ terminal oligopyrimidine tract (5′TOP) mRNAs which encode proteins of the translation machinery and ribosome biogenesis. Here we show that LARP1 is the most expressed LARP in mouse islets and human β-cells, being 2–4-fold more abundant than LARP1B, a member of the family that also interacts with mTORC1. Interestingly, β-cells from diabetic patients have higher LARP1 and LARP1B expression. However, specific deletion of Larp1 gene in β-cells (β-Larp1KO mice) did not impair insulin secretion and glucose metabolism in male and female mice. High fat or high branched-chain amino acid (BCAA) diets did not disturb glucose homeostasis compared to control littermates up to 8 weeks; BCAA diet slightly impaired glucose tolerance in the β-Larp1KO mice at 16 weeks. However, no differences in plasma insulin levels, non-fasting glycemia and β-cell mass were observed in the β-Larp1KO mice. In conclusion, LARP1 is the most abundant LARP in mouse islets and human β-cells, and it is upregulated in diabetic subjects. However, genetically disruption of Larp1 gene did not impact glucose homeostasis in basal and diabetogenic conditions, suggesting no major role for LARP1 in β-cells.

Mechanistic target of rapamycin complex 1 (mTORC1) is a cellular rheostat linking nutrient availability and growth factor to cellular protein translation. In pancreatic insulin secreting β-cells, mTORC1 deficiency or chronic hyperactivation leads to diabetes. mTORC1 complexes with La-related protein 1 (LARP1) to specifically regulate the expression of 5’ terminal oligopyrimidine tract (5’TOP) mRNAs which encode proteins of the translation machinery and ribosome biogenesis. We aimed to investigate the role played by LARP1 in β-cells in vivo. Here we show that LARP1 is the most expressed LARP in mouse islets and human β-cells, being 2-4-fold more abundant than LARP1B, a member of the family that also interacts with mTORC1. Interestingly, β-cells from diabetic patients have higher LARP1 and LARP1B expression suggesting greater protein translation. These studies led us to generate a conditional LARP1 knockout mouse in β-cells (β-Larp1KO mice). These mice exhibit normal levels of all LARP family members including Larp1B, Larp4, Larp6 and Larp7. We did not observe any difference between control and β-Larp1KO male mice in body weight gain, glucose levels and glucose tolerance at 8, 14 and 44 weeks of age. Female β-Larp1KO mice also performed normally during the glucose tolerance test. We then challenged the β-Larp1KO mice with high fat (HFD) or high branched-chain amino acid (BCAA) diets. During the course of 8 weeks in HFD, β-Larp1KO and control mice had similar weight gain and did not show alterations in glucose homeostasis compared to control littermates. BCAA did not impair glucose metabolism up to 8 weeks of diet challenge. However, glucose tolerance was slightly impaired in the β-Larp1KO mice at 16 weeks under BCAA diet. In conclusion, LARP1 is the most abundant LARP in mouse islets and human β-cells and it is upregulated in diabetic subjects. While the lack of LARP1 specifically in β-cells did not alter glucose homeostasis in basal conditions, long-term high branched-chain amino acid feeding could impair glucose tolerance.

mTORC1 signaling is a central regulator of autophagy and altered autophagic activity has been implicated in the beta-cells of patients with type 2 diabetes, and in the beta-cells of obese mice. The importance of mTORC1 signaling and Raptor in beta-cells has been recently demonstrated using raptor knock-out models (βraKO mice). These studies have shown that complete deletion of Raptor impairs beta-cell proliferation and survival of the beta-cell, and both in function and insulin processing. However, nothing is known whether reduced mTORC1 levels impair beta-cell adaptation to diabetogenic conditions. Therefore, we decided to study mice with haploinsufficiency of Raptor in beta-cells (βraHET). Mice with heterozygous deletion of Raptor in beta-cells were generated by crossing raptorf/f with Rip-Cre mice (βraHET). βraHET and control mice were fed regular chow (RC) or high-fat diet (HFD). Mice fed with RC showed no difference at the metabolic level, islets morphology, or beta-cell function. βraHET mice fed HFD for eight weeks showed an impaired Intraperitoneal Glucose Tolerance Test, fed insulin secretion and a decreased beta-cell mass, compared to control mice. Isolated islets from βraHET mice fed HFD for eight weeks showed impaired insulin secretion either measured by perifusion or static incubation compared to control islets. Next, we tested whether isolated islets from βraHET mice are more susceptible to beta-cell stressors. Beta-cell death determined by TUNEL and cleavage Caspase 3 was increased (50 and 60%) in the βraHET islets after 24 h exposure to palmitate. In addition, autophagy markers such as LC3 and p62 were increased in islets from βraHET treated with palmitate. The increase in cell death and autophagy is specific to lipotoxicity since there βraHET islets exhibited no increase in cleavage Caspase 3 in TUNEL when they were treated with proinflammatory cytokines or ER Stress inducers. In conclusion, we demonstrate that adequate levels of Raptor are critical in the adaptation of the beta-cell to HFD. Disclosure M. Blandino-Rosano: None. E. Bernal-Mizrachi: None.

Diabetes is characterized by loss of β cell mass and function. mTORC1 has an important role in the control of insulin secretion, β cell proliferation and death. YY1 is important in development, apoptosis, metabolism and growth in several tissues. In muscle, mTORC1 regulates the expression of insulin signaling genes by controlling YY1 binding to the promoter region. However, YY1 role in β cells was not addressed so far. Here we observed that diabetic and diet-induced obese mice exhibited lower levels of YY1 in the β cells. To understand the role of YY1 in β cell, we developed a new β cell YY1 depleted mouse model (β-YY1KO) by crossing our floxed YY1 mouse with a Ins-cre mouse. Haploinsufficiency of YY1 (β-YY1HET) did not impair fed or fasting glycemia, blood insulin levels and glucose tolerance. Even after 10 weeks in HFD, β-YY1HET did not differ from littermate controls in terms of glucose tolerance and glucose-stimulating insulin secretion. However, β-YY1KO mice exhibited a profound diabetic phenotype. While glucose and insulin levels in newborn β-YY1KO were normal, blood glucose begun to rise in the β-YY1KO mice at 2 weeks. All β-YY1KO became diabetic at 3 weeks presenting fed hyperglycemia and lower fed insulin levels, suggesting decreased β cell function and/or β cell loss. Remarkably, β cell mass is reduced at 2 and 3 weeks. To understand the decrease in β cell mass, we assessed β cell proliferation and death by IHF for Ki67 and flow cytometry for active Casp 3, respectively. Although β cell proliferation was normal at 2 weeks, there was a reduction Ki67+ β cell at 3 weeks. However, active casp 3 was already increased at 2 weeks. One striking role of YY1 is to regulate mitochondrial function and structure, therefore, we evaluated mitochondrial morphology by electron microscopy. We found that β-YY1KO mice presented less mitochondrial area and abnormal structure. Altogether, these results show that YY1 is important for appropriate control of glucose homeostasis and β-cell maintenance. Disclosure F.M. Peçanha: None. E. Bernal-Mizrachi: None.

Elevation of glucagon levels and increase in α-cell mass are associated to states of hyperglycemia in diabetes. However, little is known about the mechanisms that control glucagon secretion and α-cell mass expansion in normal or diabetogenic conditions. The current studies investigated the effects of activation of nutrient signaling by conditional deletion of the mTORC1 inhibitor, TSC2, in α-cells (αTSC2KO), we showed that activation of mTORC1 signaling is sufficient to induce chronic hyperglucagonemia as a result of α-cell proliferation, cell size and mass expansion. Hyperglucagonemia in αTSC2KO was associated to increase in glucagon content, enhanced glucagon secretion and defective adaptation to fasting. This model allowed us to identify the beneficial effects of chronic hyperglucagonemia in glucose homeostasis by inducing insulin secretion and resistance to glucagon in the liver. Liver glucagon resistance in αTSC2KO mice was characterized by reduced expression of the glucagon receptor (GCGR), phosphoenolpyruvate carboxykinase (PEPCK) and genes involved in amino acid metabolism and urea production. Surprisingly, hyperglucagonemia in αTSC2KO mice was associated to improved glucose levels in models of Streptozotocin (STZ)-induced β-cell destruction and high fat diet-induced glucose intolerance. Contrary to our current understanding of glucagon action, these studies demonstrate that endogenous chronic hyperglucagonemia improve glucose homeostasis by augmenting insulin secretion and by inducing glucagon resistance in the liver. These novel in vivo findings support the concept that enhancing glucagon action could be used as an alternative strategy to treat hyperglycemia in diabetes. Disclosure C. Lubaczeuski: None. N. Bozadjieva: None. M. Blandino-Rosano: None. E. Bernal-Mizrachi: None. Funding U.S. Department of Veterans Affairs (IBX002728A); Diabetes Research Connection

Summary Diabetic kidney disease (DKD) increases the risk for mortality and is the leading cause of end-stage renal disease. Treatment with sodium-glucose cotransporter 2 inhibitors (SGLT2i) attenuates the progression of DKD, especially in patients with advanced kidney disease. Herein, we show that in diabetes, mTORC1 activity is increased in renal proximal tubule cells (RPTCs) along with enhanced tubule-interstitial fibrosis; this is prevented by SGLT2i. Constitutive activation of mTORC1 in RPTCs induces renal fibrosis and failure and abolishes the renal-protective effects of SGLT2i in diabetes. On the contrary, partial inhibition of mTORC1 in RPTCs prevents fibrosis and the decline in renal function. Stimulation of mTORC1 in RPTCs turns on a pro-fibrotic program in the renal cortex, whereas its inhibition in diabetes reverses the alterations in gene expression. We suggest that RPTC mTORC1 is a critical node that mediates kidney dysfunction in diabetes and the protective effects of SGLT2i by regulating fibrogenesis., Graphical Abstract, Highlights • In diabetes, mTORC1 activity is increased in renal proximal tubule cells (RPTCs) • Diabetes and SGLT2i regulate mTORC1 by modulating nutrient transport to RPTCs • Inhibition of mTORC1 in RPTCs prevents fibrosis and the decline in renal function • RPTC mTORC1 mediates renal fibrosis in diabetes and the beneficial effects of SGLT2i, Kogot-Levin et al. show that treatment with sodium-glucose cotransporter 2 inhibitors (SGLT2i) attenuates the progression of diabetic kidney disease (DKD), which is the leading cause of end-stage renal disease. The nutrient sensor mTORC1 is a critical node that mediates kidney dysfunction in diabetes and the protective effects of SGLT2i by regulating fibrogenesis.

Deoxyhypusine synthase (DHPS) utilizes the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly-defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that enabled the inducible, postnatal deletion of Dhps specifically in postnatal islet β cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces β-cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation and protein secretion, reduced production of the cell cycle molecule cyclin D2, impaired β-cell proliferation, and induced overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-ζ and was required for c-Myc-induced proliferation. Our studies reveal a requirement for DHPS in β cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis.

Regulation of glucose homeostasis by insulin depends on β-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in β-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in β-cells. We examined glucose homeostasis and β-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in β-cell function without alterations in β-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their β-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and β-cell insulin secretion defect but showed no changes in β-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to β-cell growth but is involved in regulating β-cell function in normal and diabetogenic conditions.

MicroRNA 199 (miR-199) negatively impacts pancreatic β-cell function and its expression is highly increased in islets from diabetic mice as well as in plasma of diabetic patients. Here we investigated how miR-199 expression is regulated in β-cells by assessing expression of miR-199 precursors (primiR-199a1, primiR-199a2, and primiR-199b) and mature miR-199 (miR-199-3p and miR-199-5p) and promoter transcriptional activity assays in mouse islets and mouse insulinoma cells (MIN6) under different stimuli. We found that mouse islets equally express miR-199-3p and miR-199-5p. However, the primiRNA expression levels differed; although primiR-199a1 expression was about 30% greater than that of primiR-199a2, primiR-199b is barely detected in islets. We observed a 2-fold increase in primiR-199a1 and primiR-199a2 mRNA levels in mouse islets cultured in 10 mm glucose compared with 5.5 mm glucose. Similar responses to glucose were observed in MIN6 cells. Exposure to 30 mm KCl to induce membrane depolarization and calcium influx increased expression of primiR-199a2 but not of primiR-199a1 in MIN6 cells, indicating that calcium influx was involved. Transcriptional activity studies in MIN6 cells also revealed that primiR-199a2 promoter activity was enhanced by glucose and reduced by 2-deoxy-D-glucose–induced starvation. KCl and the potassium channel blocker tolbutamide also stimulated primiR-199a2 promoter activity. Calcium channel blockade by nifedipine reduced primiR-199a2 promoter activity in MIN6 cells, and diazoxide-mediated calcium influx inhibition blunted glucose up-regulation of miR-199-3p in islets. In conclusion, we uncover that glucose acutely up-regulates miR-199 family expression in β-cells. Glucose metabolism and calcium influx are involved in primiR-199a2 expression but not primiR-199a1 expression.

Branched-chain amino acid (BCAAs: leucine, isoleucine, and valine) contribute to the development of obesity-associated insulin resistance in the context of consumption of a high-fat diet (HFD) in humans and rodents. Maternal diet is a major determinant of offspring health, and there is strong evidence that maternal HFD alters hypothalamic developmental programming and disrupts offspring energy homeostasis in rodents. In this study, we exposed pregnant and lactating C57BL/6JB female mice to either HFD, HFD with supplemented BCAA (HFD+BCAA), or standard diet (SC), and we studied offspring metabolic phenotypes. Both maternal HFD and HFD supplemented with BCAA had similar effect rendering the offspring metabolic imbalance and impairing their ability to cope with HFD when challenged during aging. The metabolic effects of HFD challenge were more profound in females, worsening female offspring ability to cope with an HFD challenge by activating hypothalamic inflammation in aging. Moreover, the sex differences in hypothalamic estrogen receptor α (ER-α) expression levels were lost in female offspring upon HFD challenge, supporting a link between ER-α levels and hypothalamic inflammation in offspring and highlighting the programming potential of hypothalamic inflammatory responses and maternal nutrition.