Opposing effects on regulated insulin secretion of acute vs chronic stimulation of AMP-activated protein kinase.

AIMS/HYPOTHESIS: Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. METHODS: AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-ßH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for ß1- vs ß2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. RESULTS: Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (p<0.01, p<0.05 and p<0.001, respectively), despite a lowering of glucose-induced intracellular free Ca2+ dynamics in response to 991 (AUC, p<0.05) and to RA089 at the highest dose (25 µmol/l) at 5.59 min (p<0.05). Although abolished in the absence of AMPK, the effects of 991 were observed in the absence of the upstream kinase, liver kinase B1, further implicating 'amplifying' pathways. In marked contrast, chronic activation of AMPK, either globally or selectively in the beta cell, achieved using a gain-of-function mutant, impaired insulin release in vivo (p<0.05 at 15 min following i.p. injection of 3 mmol/l glucose) and in vitro (p<0.01 following incubation of islets with 17 mmol/l glucose), and lowered glucose tolerance (p<0.001). CONCLUSIONS/INTERPRETATION: AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators.

Harnessing the Endogenous Plasticity of Pancreatic Islets: A Feasible Regenerative Medicine Therapy for Diabetes?

Diabetes is a chronic metabolic disease caused by an absolute or relative deficiency in functional pancreatic ß-cells that leads to defective control of blood glucose. Current treatments for diabetes, despite their great beneficial effects on clinical symptoms, are not curative treatments, leading to a chronic dependence on insulin throughout life that does not prevent the secondary complications associated with diabetes. The overwhelming increase in DM incidence has led to a search for novel antidiabetic therapies aiming at the regeneration of the lost functional ß-cells to allow the re-establishment of the endogenous glucose homeostasis. Here we review several aspects that must be considered for the development of novel and successful regenerative therapies for diabetes: first, the need to maintain the heterogeneity of islet ß-cells with several subpopulations of ß-cells characterized by different transcriptomic profiles correlating with differences in functionality and in resistance/behavior under stress conditions; second, the existence of an intrinsic islet plasticity that allows stimulus-mediated transcriptome alterations that trigger the transdifferentiation of islet non-ß-cells into ß-cells; and finally, the possibility of using agents that promote a fully functional/mature ß-cell phenotype to reduce and reverse the process of dedifferentiation of ß-cells during diabetes.

The pore-forming subunit MCU of the mitochondrial Ca2+ uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice.

AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.

PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus.

AIMS/HYPOTHESIS: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. METHODS: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. RESULTS: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. CONCLUSIONS/INTERPRETATION: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.

The PTGS2/COX2-PGE2 signaling cascade in inflammation: Pro or anti? A case study with type 1 diabetes mellitus.

Prostaglandins are lipid mediators involved in physiological processes, such as constriction or dilation of blood vessels, but also pathophysiological processes, which include inflammation, pain and fever. They are produced by almost all cell types in the organism by activation of Prostaglandin endoperoxide synthases/Cyclooxygenases. The inducible Prostaglandin Endoperoxide Synthase 2/Cyclooxygenase 2 (PTGS2/COX2) plays an important role in pathologies associated with inflammatory signaling. The main product derived from PTGS2/COX2 expression and activation is Prostaglandin E2 (PGE2), which promotes a wide variety of tissue-specific effects, pending environmental inputs. One of the major sources of PGE2 are infiltrating inflammatory cells - the production of this molecule increases drastically in damaged tissues. Immune infiltration is a hallmark of type 1 diabetes mellitus, a multifactorial disease that leads to autoimmune-mediated pancreatic beta cell destruction. Controversial effects for the PTGS2/COX2-PGE2 signaling cascade in pancreatic islet cells subjected to diabetogenic conditions have been reported, allocating PGE2 as both, cause and consequence of inflammation. Herein, we review the main effects of this molecular pathway in a tissue-specific manner, with a special emphasis on beta cell mass protection/destruction and its potential role in the prevention or development of T1DM. We also discuss strategies to target this pathway for future therapies.

Metabolic reprogramming by Acly inhibition using SB-204990 alters glucoregulation and modulates molecular mechanisms associated with aging.

ATP-citrate lyase is a central integrator of cellular metabolism in the interface of protein, carbohydrate, and lipid metabolism. The physiological consequences as well as the molecular mechanisms orchestrating the response to long-term pharmacologically induced Acly inhibition are unknown. We report here that the Acly inhibitor SB-204990 improves metabolic health and physical strength in wild-type mice when fed with a high-fat diet, while in mice fed with healthy diet results in metabolic imbalance and moderated insulin resistance. By applying a multiomic approach using untargeted metabolomics, transcriptomics, and proteomics, we determined that, in vivo, SB-204990 plays a role in the regulation of molecular mechanisms associated with aging, such as energy metabolism, mitochondrial function, mTOR signaling, and folate cycle, while global alterations on histone acetylation are absent. Our findings indicate a mechanism for regulating molecular pathways of aging that prevents the development of metabolic abnormalities associated with unhealthy dieting. This strategy might be explored for devising therapeutic approaches to prevent metabolic diseases.

Identification of biomarkers for glycaemic deterioration in type 2 diabetes.

We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Roles of extracellular vesicles associated non-coding RNAs in Diabetes Mellitus.

Extracellular vesicles (EVs), especially exosomes (50 to 150 nm), have been shown to play important roles in a wide range of physiological and pathological processes, including metabolic diseases such as Diabetes Mellitus (DM). In the last decade, several studies have demonstrated how EVs are involved in cell-to-cell communication. EVs are enriched in proteins, mRNAs and non-coding RNAs (miRNAs, long non-coding RNAs and circRNAS, among others) which are transferred to recipient cells and may have a profound impact in either their survival or functionality. Several studies have pointed out the contribution of exosomal miRNAs, such as miR-l42-3p and miR-26, in the development of Type 1 and Type 2 DM (T1DM and T2DM), respectively. In addition, some miRNA families such as miR-let7 and miR-29 found in exosomes have been associated with both types of diabetes, suggesting that they share common etiological features. The knowledge about the role of exosomal long non-coding RNAs in this group of diseases is more immature, but the exosomal lncRNA MALAT1 has been found to be elevated in the plasma of individuals with T2DM, while more than 169 lncRNAs were reported to be differentially expressed between healthy donors and people with T1DM. Here, we review the current knowledge about exosomal non-coding RNAs in DM and discuss their potential as novel biomarkers and possible therapeutic targets.

Vertical Sleeve Gastrectomy Lowers SGLT2/Slc5a2 Expression in the Mouse Kidney.

Bariatric surgery improves glucose homeostasis, but the underlying mechanisms are not fully elucidated. Here, we show that the expression of sodium-glucose cotransporter 2 (SGLT2/Slc5a2) is reduced in the kidney of lean and obese mice following vertical sleeve gastrectomy (VSG). Indicating an important contribution of altered cotransporter expression to the impact of surgery, inactivation of the SGLT2/Slc5a2 gene by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 attenuated the effects of VSG, with glucose excursions following intraperitoneal injection lowered by ∼30% in wild-type mice but by ∼20% in SGLT2-null animals. The effects of the SGLT2 inhibitor dapaglifozin were similarly blunted by surgery. Unexpectedly, effects of dapaglifozin were still observed in SGLT2-null mice, consistent with the existence of metabolically beneficial off-target effects of SGLT2 inhibitors. Thus, we describe a new mechanism involved in mediating the glucose-lowering effects of bariatric surgery.

Vertical sleeve gastrectomy normalizes circulating glucocorticoid levels and lowers glucocorticoid action tissue-selectively in mice.

Objectives: Glucocorticoids produced by the adrenal cortex are essential for the maintenance of metabolic homeostasis. Glucocorticoid activation is catalysed by 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1). Excess glucocorticoids are associated with insulin resistance and hyperglycaemia. A small number of studies have demonstrated effects on glucocorticoid metabolism of bariatric surgery, a group of gastrointestinal procedures known to improve insulin sensitivity and secretion, which were assumed to result from weight loss. In this study, we hypothesize that a reduction in glucocorticoid action following bariatric surgery contributes to the widely observed euglycemic effects of the treatment. Methods: Glucose and insulin tolerance tests were performed at ten weeks post operatively and circulating corticosterone was measured. Liver and adipose tissues were harvested from fed mice and 11ß-HSD1 levels were measured by quantitative RT-PCR or Western (immuno-) blotting, respectively. 11ß-HSD1 null mice (Hsd11b1 -/-) were generated using CRISPR/Cas9 genome editing. Wild type and littermate Hsd11b1 -/- mice underwent Vertical Sleeve Gastrectomy (VSG) or sham surgery. Results: Under the conditions used, no differences in weight loss were observed between VSG treated and sham operated mice. However, both lean and obese WT VSG mice displayed significantly improved glucose clearance and insulin sensitivity. Remarkably, VSG restored physiological corticosterone production in HFD mice and reduced 11ß-HSD1 expression in liver and adipose tissue post-surgery. Elimination of the 11ß-HSD1/Hsd11b1 gene by CRISPR/Cas9 mimicked the effects of VSG on body weight and tolerance to 1g/kg glucose challenge. However, at higher glucose loads, the euglycemic effect of VSG was superior to Hsd11b1 elimination. Conclusions: Bariatric surgery improves insulin sensitivity and reduces glucocorticoid activation at the tissular level, under physiological and pathophysiological (obesity) conditions, irrespective of weight loss. These findings point towards a physiologically relevant gut-glucocorticoid axis, and suggest that lowered glucocorticoid exposure may represent an additional contribution to the health benefits of bariatric surgery.

Mitofusins Mfn1 and Mfn2 Are Required to Preserve Glucose- but Not Incretin-Stimulated ß-Cell Connectivity and Insulin Secretion.

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic ß-cells. Whether mitofusin gene expression, and hence, mitochondrial network integrity, is important for glucose or incretin signaling has not previously been explored. Here, we generated mice with ß-cell-selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (ßMfn1/2 dKO). ßMfn1/2-dKO mice displayed elevated fed and fasted glycemia and a more than fivefold decrease in plasma insulin. Mitochondrial length, glucose-induced polarization, ATP synthesis, and cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in ßMfn1/2-dKO mice, and glucagon-like peptide 1 or glucose-dependent insulinotropic peptide receptor agonists largely corrected defective glucose-stimulated insulin secretion through enhanced EPAC-dependent signaling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps-based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the ß-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in ß-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process.

Intravital imaging of islet Ca2+ dynamics reveals enhanced ß cell connectivity after bariatric surgery in mice.

Bariatric surgery improves both insulin sensitivity and secretion and can induce diabetes remission. However, the mechanisms and time courses of these changes, particularly the impact on ß cell function, are difficult to monitor directly. In this study, we investigated the effect of Vertical Sleeve Gastrectomy (VSG) on ß cell function in vivo by imaging Ca2+ dynamics in islets engrafted into the anterior eye chamber. Mirroring its clinical utility, VSG in mice results in significantly improved glucose tolerance, and enhanced insulin secretion. We reveal that these benefits are underpinned by augmented ß cell function and coordinated activity across the islet. These effects involve changes in circulating GLP-1 levels which may act both directly and indirectly on the ß cell, in the latter case through changes in body weight. Thus, bariatric surgery leads to time-dependent increases in ß cell function and intra-islet connectivity which are likely to contribute to diabetes remission.

The metabesity factor HMG20A potentiates astrocyte survival and reactive astrogliosis preserving neuronal integrity.

Rationale: We recently demonstrated that the 'Metabesity' factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1-CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. Methods: HMG20A expression profile was assessed by quantitative PCR (QT-PCR), Western blotting and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to either a high-fat diet or a high-fat high-sucrose regimen, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals and 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes. The impact of ORY1001, an inhibitor of the LSD1-CoREST complex, on HMG20A expression, reactive astrogliosis and glucose metabolism was evaluated in vitro and in vivo in high-fat high-sucrose fed mice. Results: We show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. HMG20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and reactive astrogliosis. Hmg20a transcript levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways which are hallmarks of reactive astrogliosis. Accordingly, HMG20A depleted astrocytes exhibited reduced mitochondrial bioenergetics and increased susceptibility to apoptosis. Neuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. ORY1001 treatment rescued expression of reactive astrogliosis-linked genes in HMG20A ablated astrocytes while enhancing cell surface area, GFAP intensity and STAT3 expression in healthy astrocytes, mimicking the effect of HMG20A. Furthermore, ORY1001 treatment protected against obesity-associated glucose intolerance in mice correlating with a regression of hypothalamic HMG20A expression, indicative of reactive astrogliosis attenuation with improved health status. Conclusion: HMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce reactive astrogliosis in an attempt to preserve the neuronal network and re-establish glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote reactive astrogliosis and increase susceptibility of neurons to stress-induced apoptosis. Such effects could be reversed by ORY1001 treatment both in vitro and in vivo, paving the way for a new therapeutic approach for Type 2 Diabetes Mellitus.

Long Non-Coding RNAs as Key Modulators of Pancreatic ß-Cell Mass and Function.

Numerous studies have sought to decipher the genetic and other mechanisms contributing to ß-cell loss and dysfunction in diabetes mellitus. However, we have yet to fully understand the etiology of the disease or to develop satisfactory treatments. Since the majority of diabetes susceptibility loci are mapped to non-coding regions within the genome, understanding the functions of non-coding RNAs in ß-cell biology might provide crucial insights into the pathogenesis of type 1 (T1D) and type 2 (T2D) diabetes. During the past decade, numerous studies have indicated that long non-coding RNAs play important roles in the maintenance of ß-cell mass and function. Indeed, lncRNAs have been shown to be involved in controlling ß-cell proliferation during development and/or ß-cell compensation in response to hyperglycaemia. LncRNAs such as TUG-1 and MEG3 play a role in both ß-cell apoptosis and function, while others sensitize ß-cells to apoptosis in response to stress signals. In addition, several long non-coding RNAs have been shown to regulate the expression of ß-cell-enriched transcription factors in cis or in trans. In this review, we provide an overview of the roles of lncRNAs in maintaining ß-function and mass, and discuss their relevance in the development of diabetes.

Inadequate control of thyroid hormones sensitizes to hepatocarcinogenesis and unhealthy aging.

An inverse correlation between thyroid hormone levels and longevity has been reported in several species and reduced thyroid hormone levels have been proposed as a biomarker for healthy aging and metabolic fitness. However, hypothyroidism is a medical condition associated with compromised health and reduced life expectancy. Herein, we show, using wild-type and the Pax8 ablated model of hypothyroidism in mice, that hyperthyroidism and severe hypothyroidism are associated with an overall unhealthy status and shorter lifespan. Mild hypothyroid Pax8 +/- mice were heavier and displayed insulin resistance, hepatic steatosis and increased prevalence of liver cancer yet had normal lifespan. These pathophysiological conditions were precipitated by hepatic mitochondrial dysfunction and oxidative damage accumulation. These findings indicate that individuals carrying mutations on PAX8 may be susceptible to develop liver cancer and/or diabetes and raise concerns regarding the development of interventions aiming to modulate thyroid hormones to promote healthy aging or lifespan in mammals.

Transient PAX8 Expression in Islets During Pregnancy Correlates With ß-Cell Survival, Revealing a Novel Candidate Gene in Gestational Diabetes Mellitus.

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8 A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 affects islet viability and that PAX8 could be considered as a candidate gene for the study of GDM.

The type 2 diabetes-associated HMG20A gene is mandatory for islet beta cell functional maturity.

HMG20A (also known as iBRAF) is a chromatin factor involved in neuronal differentiation and maturation. Recently small nucleotide polymorphisms (SNPs) in the HMG20A gene have been linked to type 2 diabetes mellitus (T2DM) yet neither expression nor function of this T2DM candidate gene in islets is known. Herein we demonstrate that HMG20A is expressed in both human and mouse islets and that levels are decreased in islets of T2DM donors as compared to islets from non-diabetic donors. In vitro studies in mouse and human islets demonstrated that glucose transiently increased HMG20A transcript levels, a result also observed in islets of gestating mice. In contrast, HMG20A expression was not altered in islets from diet-induced obese and pre-diabetic mice. The T2DM-associated rs7119 SNP, located in the 3' UTR of the HMG20A transcript reduced the luciferase activity of a reporter construct in the human beta 1.1E7 cell line. Depletion of Hmg20a in the rat INS-1E cell line resulted in decreased expression levels of its neuronal target gene NeuroD whereas Rest and Pax4 were increased. Chromatin immunoprecipitation confirmed the interaction of HMG20A with the Pax4 gene promoter. Expression levels of Mafa, Glucokinase, and Insulin were also inhibited. Furthermore, glucose-induced insulin secretion was blunted in HMG20A-depleted islets. In summary, our data demonstrate that HMG20A expression in islet is essential for metabolism-insulin secretion coupling via the coordinated regulation of key islet-enriched genes such as NeuroD and Mafa and that depletion induces expression of genes such as Pax4 and Rest implicated in beta cell de-differentiation. More importantly we assign to the T2DM-linked rs7119 SNP the functional consequence of reducing HMG20A expression likely translating to impaired beta cell mature function.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

Targeting pancreatic expressed PAX genes for the treatment of diabetes mellitus and pancreatic neuroendocrine tumors.

INTRODUCTION: Four members of the PAX family, PAX2, PAX4, PAX6 and PAX8 are known to be expressed in the pancreas. Accumulated evidences indicate that several pancreatic expressed PAX genes play a significant role in pancreatic development/functionality and alterations in these genes are involved in the pathogenesis of pancreatic diseases. Areas covered: In this review, we summarize the ongoing research related to pancreatic PAX genes in diabetes mellitus and pancreatic neuroendocrine tumors. We dissect the current knowledge at different levels; from mechanistic studies in cell lines performed to understand the molecular processes controlled by pancreatic PAX genes, to in vivo studies using rodent models that over-express or lack specific PAX genes. Finally, we describe human studies associating variants on pancreatic-expressed PAX genes with pancreatic diseases. Expert opinion: Based on the current literature, we propose that future interventions to treat pancreatic neuroendocrine tumors and diabetes mellitus could be developed via the modulation of PAX4 and/or PAX6 regulated pathways.

Levothyroxine enhances glucose clearance and blunts the onset of experimental type 1 diabetes mellitus in mice.

BACKGROUND AND PURPOSE: Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis. EXPERIMENTAL APPROACH: C57BL/6 mice were treated with levothyroxine from birth to 24 weeks of age, when mice were killed. The effects of levothyroxine supplementation on metabolic health were determined. C57BL/6 mice treated with levothyroxine for 2 weeks and then challenged with streptozotocin to monitor survival. Mechanistic experiments were conducted in the pancreas, liver and skeletal muscle. RIP-B7.1 mice were treated with levothyroxine for 2 weeks and were subsequently immunized to trigger experimental autoimmune diabetes (EAD). Metabolic tests were performed. Mice were killed and metabolic tissues were extracted for immunohistological analyses. KEY RESULTS: Long-term levothyroxine supplementation enhanced glucose clearance and reduced circulating glucose in C57BL/6 mice. Levothyroxine increased simultaneously the proliferation and apoptosis of pancreatic beta cells, promoting the maintenance of a highly insulin-expressing beta cell population. Levothyroxine increased circulating insulin levels, inducing sustained activation of IRS1-AKT signalling in insulin-target tissues. Levothyroxine-treated C57BL/6 mice challenged with streptozotocin exhibited extended survival. Levothyroxine blunted the onset of EAD in RIP-B7.1 mice by inducing beta cell proliferation and preservation of insulin-expressing cells. CONCLUSIONS AND IMPLICATIONS: Interventions based on the use of thyroid hormones or thyromimetics could be explored to provide therapeutic benefit in patients with type 1 diabetes mellitus.