Sox9 regulates alternative splicing and pancreatic beta cell function.

Despite significant research, mechanisms underlying the failure of islet beta cells that result in type 2 diabetes (T2D) are still under investigation. Here, we report that Sox9, a transcriptional regulator of pancreas development, also functions in mature beta cells. Our results show that Sox9-depleted rodent beta cells have defective insulin secretion, and aging animals develop glucose intolerance, mimicking the progressive degeneration observed in T2D. Using genome editing in human stem cells, we show that beta cells lacking SOX9 have stunted first-phase insulin secretion. In human and rodent cells, loss of Sox9 disrupts alternative splicing and triggers accumulation of non-functional isoforms of genes with key roles in beta cell function. Sox9 depletion reduces expression of protein-coding splice variants of the serine-rich splicing factor arginine SRSF5, a major splicing enhancer that regulates alternative splicing. Our data highlight the role of SOX9 as a regulator of alternative splicing in mature beta cell function.

Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans.

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

Combined Deletion of Free Fatty-Acid Receptors 1 and 4 Minimally Impacts Glucose Homeostasis in Mice.

The free fatty-acid receptors FFAR1 (GPR40) and FFAR4 (GPR120) are implicated in the regulation of insulin secretion and insulin sensitivity, respectively. Although GPR120 and GPR40 share similar ligands, few studies have addressed possible interactions between these 2 receptors in the control of glucose homeostasis. Here we generated mice deficient in gpr120 (Gpr120KO) or gpr40 (Gpr40KO), alone or in combination (Gpr120/40KO), and metabolically phenotyped male and female mice fed a normal chow or high-fat diet. We assessed insulin secretion in isolated mouse islets exposed to selective GPR120 and GPR40 agonists singly or in combination. Following normal chow feeding, body weight and energy intake were unaffected by deletion of either receptor, although fat mass increased in Gpr120KO females. Fasting blood glucose levels were mildly increased in Gpr120/40KO mice and in a sex-dependent manner in Gpr120KO and Gpr40KO animals. Oral glucose tolerance was slightly reduced in male Gpr120/40KO mice and in Gpr120KO females, whereas insulin secretion and insulin sensitivity were unaffected. In hyperglycemic clamps, the glucose infusion rate was lower in male Gpr120/40KO mice, but insulin and c-peptide levels were unaffected. No changes in glucose tolerance were observed in either single or double knock-out animals under high-fat feeding. In isolated islets from wild-type mice, the combination of selective GPR120 and GPR40 agonists additively increased insulin secretion. We conclude that while simultaneous activation of GPR120 and GPR40 enhances insulin secretion ex vivo, combined deletion of these 2 receptors only minimally affects glucose homeostasis in vivo in mice.

HB-EGF Signaling Is Required for Glucose-Induced Pancreatic ß-Cell Proliferation in Rats.

The molecular mechanisms of ß-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced ß-cell proliferation in rats is dependent on epidermal growth factor receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the ß-cell proliferative response to glucose, a ß-cell mitogen and key regulator of ß-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates ß-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks ß-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in ß-cells in response to glucose in a carbohydrate-response element-binding protein (ChREBP)-dependent manner. In addition, chromatin immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced ß-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in ß-cell compensation to increased metabolic demand.

Heterogeneous Nuclear Ribonucleoprotein F Mediates Insulin Inhibition of Bcl2-Modifying Factor Expression and Tubulopathy in Diabetic Kidney.

We investigated the molecular mechanism(s) by which insulin prevents Bcl2-modifying factor (Bmf)-induced renal proximal tubular cell (RPTC) apoptosis and loss in diabetic mice. Transgenic mice (Tg) mice specifically overexpressing human BMF in RPTCs and non-Tg littermates were studied at 10 to 20 weeks of age. Non-diabetic littermates, diabetic Akita mice +/- insulin implant, Akita Tg mice specifically overexpressing heterogeneous nuclear ribonucleoprotein F (hnRNP F) in their RPTCs and immortalized rat renal proximal tubular cells (IRPTCs) were also studied. BMF-Tg mice exhibited higher systolic blood pressure, urinary albumin/creatinine ratio, RPTC apoptosis and urinary RPTCs than non-Tg mice. Insulin treatment in Akita mice and Akita mice overexpressing hnRNP F suppressed Bmf expression and RPTC apoptosis. In hyperinsulinemic-euglycemic wild type mice, renal Bmf expression was down-regulated with up-regulation of hnRNP F. In vitro, insulin inhibited high glucose-stimulation of Bmf expression, predominantly via p44/42 mitogen-activated protein kinase (MAPK) signaling. Transfection of p44/42 MAPK or hnRNP F small interfering RNA (siRNA) prevented insulin inhibition of Bmf expression. HnRNP F inhibited Bmf transcription via hnRNP F-responsive element in the Bmf promoter. Our results demonstrate that hnRNP F suppression of Bmf transcription is an important mechanism by which insulin protects RPTCs from apoptosis in diabetes.

The regulator of G-protein signaling RGS16 promotes insulin secretion and ß-cell proliferation in rodent and human islets.

OBJECTIVE: G protein-coupled receptor (GPCR) signaling regulates insulin secretion and pancreatic ß cell-proliferation. While much knowledge has been gained regarding how GPCRs are activated in ß cells, less is known about the mechanisms controlling their deactivation. In many cell types, termination of GPCR signaling is controlled by the family of Regulators of G-protein Signaling (RGS). RGS proteins are expressed in most eukaryotic cells and ensure a timely return to the GPCR inactive state upon removal of the stimulus. The aims of this study were i) to determine if RGS16, the most highly enriched RGS protein in ß cells, regulates insulin secretion and ß-cell proliferation and, if so, ii) to elucidate the mechanisms underlying such effects. METHODS: Mouse and human islets were infected with recombinant adenoviruses expressing shRNA or cDNA sequences to knock-down or overexpress RGS16, respectively. 60 h post-infection, insulin secretion and cAMP levels were measured in static incubations in the presence of glucose and various secretagogues. ß-cell proliferation was measured in infected islets after 72 h in the presence of 16.7 mM glucose ± somatostatin and various inhibitors. RESULTS: RGS16 mRNA levels are strongly up-regulated in islets of Langerhans under hyperglycemic conditions in vivo and ex vivo. RGS16 overexpression stimulated glucose-induced insulin secretion in isolated mouse and human islets while, conversely, insulin secretion was impaired following RGS16 knock-down. Insulin secretion was no longer affected by RGS16 knock-down when islets were pre-treated with pertussis toxin to inactivate Gαi/o proteins, or in the presence of a somatostatin receptor antagonist. RGS16 overexpression increased intracellular cAMP levels, and its effects were blocked by an adenylyl cyclase inhibitor. Finally, RGS16 overexpression prevented the inhibitory effect of somatostatin on insulin secretion and ß-cell proliferation. CONCLUSIONS: Our results identify RGS16 as a novel regulator of ß-cell function that coordinately controls insulin secretion and proliferation by limiting the tonic inhibitory signal exerted by δ-cell-derived somatostatin in islets.

Overexpression of catalase prevents hypertension and tubulointerstitial fibrosis and normalization of renal angiotensin-converting enzyme-2 expression in Akita mice.

We investigated the relationship among oxidative stress, hypertension, renal injury, and angiotensin-converting enzyme-2 (ACE2) expression in type 1 diabetic Akita mice. Blood glucose, blood pressure, and albuminuria were monitored for up to 5 mo in adult male Akita and Akita catalase (Cat) transgenic (Tg) mice specifically overexpressing Cat, a key antioxidant enzyme in their renal proximal tubular cells (RPTCs). Same-age non-Akita littermates and Cat-Tg mice served as controls. In separate studies, adult male Akita mice (14 wk) were treated with ANG 1-7 (500 µg·kg⁻¹·day⁻¹ sc) ± A-779, an antagonist of the Mas receptor (10 mg·kg⁻¹·day⁻¹ sc), and euthanized at the age of 18 wk. The left kidneys were processed for histology and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess protein and gene expression. Urinary angiotensinogen (AGT), angiotensin II (ANG II), and ANG 1-7 were quantified by specific ELISAs. Overexpression of Cat attenuated renal oxidative stress; prevented hypertension; normalized RPTC ACE2 expression and urinary ANG 1-7 levels (both were low in Akita mice); ameliorated glomerular filtration rate, albuminuria, kidney hypertrophy, tubulointerstitial fibrosis, and tubular apoptosis; and suppressed profibrotic and proapoptotic gene expression in RPTCs of Akita Cat-Tg mice compared with Akita mice. Furthermore, daily administration of ANG 1-7 normalized systemic hypertension in Akita mice, which was reversed by A-779. These data demonstrate that Cat overexpression prevents hypertension and progression of nephropathy and highlight the importance of intrarenal oxidative stress and ACE2 expression contributing to hypertension and renal injury in diabetes.

Bcl-2-modifying factor induces renal proximal tubular cell apoptosis in diabetic mice.

This study investigated the mechanisms underlying tubular apoptosis in diabetes by identifying proapoptotic genes that are differentially upregulated by reactive oxygen species in renal proximal tubular cells (RPTCs) in models of diabetes. Total RNAs isolated from renal proximal tubules (RPTs) of 20-week-old heterozygous db/m+, db/db, and db/db catalase (CAT)-transgenic (Tg) mice were used for DNA chip microarray analysis. Real-time quantitative PCR assays, immunohistochemistry, and mice rendered diabetic with streptozotocin were used to validate the proapoptotic gene expression in RPTs. Cultured rat RPTCs were used to confirm the apoptotic activity and regulation of proapoptotic gene expression. Additionally, studies in kidney tissues from patients with and without diabetes were used to confirm enhanced proapoptotic gene expression in RPTs. Bcl-2-modifying factor (Bmf) was differentially upregulated (P<0.01) in RPTs of db/db mice compared with db/m+ and db/db CAT-Tg mice and in RPTs of streptozotocin-induced diabetic mice in which insulin reversed this finding. In vitro, Bmf cDNA overexpression in rat RPTCs coimmunoprecipated with Bcl-2, enhanced caspase-3 activity, and promoted apoptosis. High glucose (25 mmol/L) induced Bmf mRNA expression in RPTCs, whereas rotenone, catalase, diphenylene iodinium, and apocynin decreased it. Knockdown of Bmf with small interfering RNA reduced high glucose-induced apoptosis in RPTCs. More important, enhanced Bmf expression was detected in RPTs of kidneys from patients with diabetes. These data demonstrate differential upregulation of Bmf in diabetic RPTs and suggest a potential role for Bmf in regulating RPTC apoptosis and tubular atrophy in diabetes.

Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice.

We investigated the effects of dual renin-angiotensin system (RAS) blockade on angiotensin-converting enzyme-2 (Ace2) expression, hypertension, and renal proximal tubular cell (RPTC) apoptosis in type 1 diabetic Akita angiotensinogen (Agt)-transgenic (Tg) mice that specifically overexpress Agt in their RPTCs. Adult (11 wk old) male Akita and Akita Agt-Tg mice were treated with two RAS blockers (ANG II receptor type 1 blocker losartan, 30 mg·kg(-1)·day(-1)) and angiotensin-converting enzyme (ACE) inhibitor perindopril (4 mg·kg(-1)·day(-1)) in drinking water. Same-age non-Akita littermates and Agt-Tg mice served as controls. Blood pressure, blood glucose, and albuminuria were monitored weekly. The animals were euthanized at age 16 wk. The left kidneys were processed for immunohistochemistry and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess gene and protein expression. Urinary ANG II and ANG 1-7 were quantified by ELISA. RAS blockade normalized renal Ace2 expression and urinary ANG 1-7 levels (both of which were low in untreated Akita and Akita Agt-Tg), prevented hypertension, albuminuria, tubulointerstitial fibrosis and tubular apoptosis, and inhibited profibrotic and proapoptotic gene expression in RPTCs of Akita and Akita Agt-Tg mice compared with non-Akita controls. Our results demonstrate the effectiveness of RAS blockade in preventing intrarenal RAS activation, hypertension, and nephropathy progression in diabetes and support the important role of intrarenal Ace2 expression in modulating hypertension and renal injury in diabetes.