OBJECTIVES:Skeletal muscle is a central regulator of metabolic health, serving as the primary site of postprandial glucose uptake and playing a critical role in whole-body insulin sensitivity. Despite its importance, the molecular mechanisms governing muscle differentiation (myogenesis) and their modulation by metabolic interventions remain poorly defined. This study identifies the clathrin adaptor protein Picalm (phosphatidylinositol-binding clathrin assembly protein) as a novel regulator of myogenesis and investigates its regulation in response to exercise training and intermittent fasting. METHODS:Functional characterization of Picalm was conducted in C2C12 myoblasts and primary myocytes using siRNA-mediated knockdown. Clathrin-mediated endocytosis was performed using dynamin inhibition (Dyngo-4a) and via an EGF internalization assay. Surface proteome alterations were analyzed by plasma membrane proteomics, and autophagy dynamics were assessed via immunoblotting and fluorescence imaging. Jasplakinolide was used to rescue differentiation defects by enhancing actin polymerization. RESULTS:Picalm-depleted C2C12 myoblasts exhibited impaired differentiation, presumably due to diminished intracellular trafficking dynamics of cell surface proteins. Inhibition of dynamin-dependent endocytosis phenocopied the differentiation defect and further aggravated myogenesis in Picalm-depleted cells, indicating that Picalm-dependent endocytic function is required for efficient differentiation. Consistent with this, Picalm knockdown significantly decreased clathrin-dependent uptake of EGF. Proteome analysis of a plasma membrane-enriched fraction revealed increased abundance of over 100 proteins after Picalm knockdown, particularly candidates involved in vesicular trafficking (Vamp3, Vamp5), actin remodeling (Actn1, Actn4, Rhog, Rock1, Rock2) and cell adhesion (integrin receptors). In line with this, Picalm knockdown resulted in impaired maturation and lysosomal degradation of autophagic vesicles. Remarkably, pharmacological stabilization of actin filaments with Jasplakinolide restored myogenic differentiation in Picalm-deficient cells, highlighting a functional link between actin remodeling and myogenesis. CONCLUSIONS:Picalm regulates skeletal muscle differentiation by supporting clathrin-mediated endocytosis and plasma membrane remodeling, thereby maintaining trafficking-dependent control of actin organization. Its expression is responsive to metabolic cues such as exercise and intermittent fasting. These findings reveal a novel molecular link between nutrient signaling and myogenesis, with implications for metabolic disease and muscle regeneration.

Golgi-associated membrane scaffolds, or tethers, have broad roles in membrane-bound protein and lipid trafficking and in maintaining Golgi architecture. Accordingly, they exert strong influence over cellular development, signalling, cargo modification and transport. An ever-expanding group of Golgins and multi-subunit tethering complexes assumes distinct functions in specific Golgi subcompartments in close partnership with Rab and ARL family GTPases. Their dysregulation or mutation impairs glycosylation, vesicle trafficking, and cytoskeletal dynamics, thereby contributing to a spectrum of human pathologies ranging from neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease) to cancers (e.g. lung, breast, colon) and metabolic defects (impaired insulin secretion and lipid droplet formation). Here, we review these diverse roles across molecular, cellular and organismal physiology.

Starch-binding domain-containing protein 1 (Stbd1) is a glycogen-binding protein which localizes to the endoplasmic reticulum (ER) membrane and ER-mitochondria contact sites (ERMCs). The protein undergoes N-myristoylation, which is a major determinant of its subcellular targeting. Stbd1 has been implicated in the control of glucose homeostasis, as evidenced by the finding that mice with targeted inactivation of Stbd1 display insulin resistance associated with increased ERMCs in the liver. In the present study, we addressed the effects of increased Stbd1 expression levels on insulin signaling. We show that Stbd1 overexpression enhances cellular sensitivity to insulin and improves insulin resistance in an in vitro hepatocyte cell model. We further demonstrate that increased Stbd1 expression levels are associated with enhanced activation of the AMP-activated protein kinase (AMPK), which is a central regulator of metabolism and an attractive therapeutic target for metabolic disorders related to insulin resistance, such as type 2 diabetes (T2D). The activation of AMPK signaling and the improved cellular response to insulin induced by Stbd1 overexpression occurred independently of N-myristoylation and associated changes in the number of ERMCs, glycogen levels, mitochondrial calcium, mitochondrial morphology, and respiratory function. Collectively, our findings uncover a new level of interaction between Stbd1 and AMPK, with Stbd1 acting as an upstream activator of AMPK signaling. Given that first-line drug treatments for insulin resistance and T2D are known activators of the AMPK pathway, these findings may provide a new perspective for the development of more effective therapeutic strategies.

Present study explores the role of liver lipidome in driving T2D-associated metabolic changes. Elevated liver triacylglycerols, reduced PUFAs, and 86 differentially abundant lipid species were identified in diabetes-prone mice. Of these altered lipid species, 82 markedly overlap with human plasma lipids associated with T2D/CVD risk. Pathway enrichment highlighted sphingolipid metabolism, however, only five of all genes involved in the pathway were differentially expressed in the liver. Interestingly, overlap with adipose tissue transcriptome was much higher (57 genes), pointing toward an active adipose-liver interaction. Next, the integration of liver lipidome and transcriptome identified strongly correlated lipid-gene networks highlighting ceramide [Cer(22:0)], dihydroceramide(24:1), and triacylglycerol(58:6) playing a central role in transcriptional regulation. Putative molecular targets of Cer(22:0) were altered (Cyp3a44, Tgf-β1) in primary mouse hepatocytes treated with Cer(22:0). Early alteration of liver lipidome markedly depends on adipose tissue expression pattern and provides substantial evidence linking early liver lipidome alterations and risk of T2D.

Type 2 diabetes (T2D) is marked by progressive β-cell failure with concomitant impairment in insulin secretion. To expand our understanding of this process, we investigated the scaffold proteins intersectin 1 and 2 (Itsn1/2), which coordinate neurotransmitter release and endocytic recycling of synaptic vesicle components in neurons, and are also expressed in endocrine pancreatic islet cells.

Mediator, a co-regulator complex required for RNA Polymerase II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We observe reduced Mediator subunit MED15 expression in endocrine hormone-producing pancreatic islets isolated from people living with type 2 diabetes and sought to understand how MED15 and Mediator control gene expression programs important for the function of insulin-producing β-cells. Here we show that Med15 is expressed during mouse β-cell development and maturation. Knockout of Med15 in mouse β-cells causes defects in β-cell maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses found that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. In support of a conserved role during human development, human embryonic stem cell-derived β-like cells, genetically engineered to express high levels of MED15, express increased levels of maturation markers. We provide evidence of a conserved role for Mediator in β-cell maturation and demonstrate an additional layer of control that tunes β-cell transcription factor function.

The aim of this work was to understand the role of non-beta cells in pancreatic islets at early stages of type 2 diabetes pathogenesis. Specific clustering was employed to single-cell transcriptome data from islet cells of obese mouse strains differing in their diabetes susceptibility (diabetes-resistant B6.V.Lepob/ob [OB] and diabetes-susceptible New Zealand Obese [NZO] mice) on a diabetogenic diet. Refined clustering analysis revealed several heterogeneous subpopulations for alpha cells, delta cells and macrophages, of which 133 mapped to human diabetes genes identified by genome-wide association studies. Importantly, a similar non-beta cell heterogeneity was found in a dataset of human islets from donors at different stages of type 2 diabetes. The predominant alpha cell cluster in NZO mice displayed signs of cellular stress and lower mitochondrial capacity (97 differentially expressed genes [DEGs]), whereas delta cells from these mice exhibited higher expression levels of maturation marker genes (Hhex and Sst) but lower somatostatin secretion than OB mice (184 DEGs). Furthermore, a cluster of macrophages was almost twice as abundant in islets of OB mice, and displayed extensive cell–cell communication with beta cells of OB mice. Treatment of beta cells with IL-15, predicted to be released by macrophages, activated signal transducer and activator of transcription (STAT3), which may mediate anti-apoptotic effects. Similar to mice, humans without diabetes possess a greater number of macrophages than those with prediabetes (39 mmol/mol [5.7 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159211 ).

Aim: Understand the role of non-beta-cells in islets of Langerhans at early stages of type 2 diabetes (T2D) pathogenesis.

MicroRNAs (miRNAs) recently emerged as means of communication between insulin-sensitive tissues to mediate diabetes development and progression, and as such they present a valuable proxy for epigenetic alterations associated with type 2 diabetes. In order to identify miRNA markers for the precursor of diabetes called prediabetes, we applied a translational approach encompassing analysis of human plasma samples, mouse tissues and an in vitro validation system. MiR-652-3p, miR-877-5p, miR-93-5p, miR-130a-3p, miR-152-3p and let-7i-5p were increased in plasma of women with impaired fasting glucose levels (IFG) compared to those with normal fasting glucose and normal glucose tolerance (NGT). Among these, let-7i-5p and miR-93-5p correlated with fasting blood glucose levels. Human data were then compared to miRNome data obtained from islets of Langerhans and adipose tissue of 10-week-old female New Zealand Obese mice, which differ in their degree of hyperglycemia and liver fat content. Similar to human plasma, let-7i-5p was increased in adipose tissue and islets of Langerhans of diabetes-prone mice. As predicted by the in silico analysis, overexpression of let-7i-5p in the rat β-cell line INS-1 832/12 resulted in downregulation of insulin signaling pathway components (Insr, Rictor, Prkcb, Clock, Sos1 and Kcnma1). Taken together, our integrated approach highlighted let-7i-5p as a potential regulator of whole-body insulin sensitivity and a novel marker of prediabetes in women.

OBJECTIVES:Better disease management can be achieved with earlier detection through robust, sensitive, and easily accessible biomarkers. The aim of the current study was to identify novel epigenetic biomarkers determining the risk of type 2 diabetes (T2D).METHODS:Livers of 10-week-old female New Zealand Obese (NZO) mice, slightly differing in their degree of hyperglycemia and liver fat content and thereby in their diabetes susceptibility were used for expression and methylation profiling. We screened for differences in hepatic expression and DNA methylation in diabetes-prone and -resistant mice, and verified a candidate (HAMP) in human livers and blood cells. Hamp expression was manipulated in primary hepatocytes and insulin-stimulated pAKT was detected. Luciferase reporter assays were conducted in a murine liver cell line to test the impact of DNA methylation on promoter activity.RESULTS:In livers of NZO mice, the overlap of methylome and transcriptome analyses revealed a potential transcriptional dysregulation of 12 hepatokines. The strongest effect with a 52% decreased expression in livers of diabetes-prone mice was detected for the Hamp gene, mediated by elevated DNA methylation of two CpG sites located in the promoter. Hamp encodes the iron-regulatory hormone hepcidin, which had a lower abundance in the livers of mice prone to developing diabetes. Suppression of Hamp reduces the levels of pAKT in insulin-treated hepatocytes. In liver biopsies of obese insulin-resistant women, HAMP expression was significantly downregulated along with increased DNA methylation of a homologous CpG site. In blood cells of incident T2D cases from the prospective EPIC-Potsdam cohort, higher DNA methylation of two CpG sites was related to increased risk of incident diabetes.CONCLUSIONS:We identified epigenetic changes in the HAMP gene which may be used as an early marker preceding T2D.

Progressive dysfunction and failure of insulin-releasing β-cells are a hallmark of type 2 diabetes (T2D). To study mechanisms of β-cell loss in T2D, we performed islet single-cell RNA sequencing of two obese mouse strains differing in their diabetes susceptibility. With mice on a control diet, we identified six β-cell clusters with similar abundance in both strains. However, after feeding of a diabetogenic diet for 2 days, β-cell cluster composition markedly differed between strains. Islets of diabetes-resistant mice developed into a protective β-cell cluster (Beta4), whereas those of diabetes-prone mice progressed toward stress-related clusters with a strikingly different expression pattern. Interestingly, the protective cluster showed indications of reduced β-cell identity, such as downregulation of GLUT2, GLP1R, and MafA, and in vitro knockdown of GLUT2 in β-cells-mimicking its phenotype-decreased stress response and apoptosis. This might explain enhanced β-cell survival of diabetes-resistant islets. In contrast, β-cells of diabetes-prone mice responded with expression changes indicating metabolic pressure and endoplasmic reticulum stress, presumably leading to later β-cell loss. In conclusion, failure of diabetes-prone mice to adapt gene expression toward a more dedifferentiated state in response to rising blood glucose levels leads to β-cell failure and diabetes development.

Aims/ hypothesis Sorting-related receptor with type A repeats (SORLA) is a neuronal sorting receptor that prevents accumulation of amyloid-beta peptides, the main constituent of senile plaques in Alzheimer disease. Recent transcriptomic studies show that SORLA transcripts are also found in pancreatic islet beta cells, yet the role of SORLA in islets is unclear so far. Based on its protective role in reducing amyloid burden in the brain, we hypothesized that SORLA may have a similar function in the pancreas, regulating islet amyloid plaque formation from islet amyloid polypeptide (IAPP). Methods We generated human IAPP transgenic mice lacking SORLA (hIAPP:SORLA KO) to assess the consequences of receptor deficiency for islet histopathology and function in vivo. Using both primary islet cells and established cell lines, we further investigated the molecular mechanisms whereby SORLA controls the cellular metabolism and accumulation of IAPP. Results Loss of SORLA activity in hIAPP:SORLA KO resulted in a significant increase in islet amyloid deposits and associated islet cell death as compared to hIAPP:SORLA WT animals expressing the receptor. Aggravated islet amyloid deposition was observed in mice fed a normal chow diet, not requiring high-fat diet feeding typically needed to induce islet amyloidosis in mouse models. Further in vitro studies showed that SORLA binds to and mediates the endocytic uptake of proIAPP, but not mature IAPP, delivering the propeptide to an endolysosomal fate. Conclusions/interpretation SORLA functions as a clearance receptor specific for proIAPP, protecting against islet amyloid deposition and associated cell death caused by IAPP.

Current attempts to prevent and manage type 2 diabetes have been moderately effective, and a better understanding of the molecular roots of this complex disease is important to develop more successful and precise treatment options. Recently, we initiated the collective diabetes cross, where four mouse inbred strains differing in their diabetes susceptibility were crossed with the obese and diabetes-prone NZO strain and identified the quantitative trait loci (QTL) Nidd13/NZO, a genomic region on chromosome 13 that correlates with hyperglycemia in NZO allele carriers compared to B6 controls. Subsequent analysis of the critical region, harboring 644 genes, included expression studies in pancreatic islets of congenic Nidd13/NZO mice, integration of single-cell data from parental NZO and B6 islets as well as haplotype analysis. Finally, of the five genes (Acot12, S100z, Ankrd55, Rnf180, and Iqgap2) within the polymorphic haplotype block that are differently expressed in islets of B6 compared to NZO mice, we identified the calcium-binding protein S100z gene to affect islet cell proliferation as well as apoptosis when overexpressed in MIN6 cells. In summary, we define S100z as the most striking gene to be causal for the diabetes QTL Nidd13/NZO by affecting β-cell proliferation and apoptosis. Thus, S100z is an entirely novel diabetes gene regulating islet cell function.

Background Insulin-secreting β-cells are important regulators of blood glucose homeostasis. In individuals susceptible to develop type 2 diabetes (T2D), chronically elevated levels of glucose and fatty acids can cause β-cell stress, exhaustion, and death. We previously described β-cell expression of the transcription factor NPAS4 in response to glucose exposure or cellular stressors. NPAS4 promotes β-cell survival by dampening insulin production and secretion. Our goal was to identify target genes mediating these effects. The GTPase-accelerating protein RGS2 of the Regulator of G-protein Signalling (RGS) family was a promising candidate, since other RGS proteins regulate β-cell function and survival. Consequently, we hypothesized that RGS2 inhibits β-cell insulin secretion. Methods: We employed both gain-of-function (adenoviral overexpression in MIN6 cells or mouse islets) and loss-of-function (MIN6-derived Rgs2 knockout cell lines) models. Glucose-stimulated β-cell function was assessed via insulin ELISA, calcium imaging, and measurement of oxygen consumption rate (OCR). Results: Rgs2 overexpression diminished glucose-stimulated insulin secretion (GSIS) in islets and MIN6 cells by 30 % and 55-70 %, respectively, while KCl-driven insulin release and insulin content were unchanged. Additionally, Rgs2-overexpressing MIN6 cells showed 17 % lower glucose-stimulated calcium influx. Changes in these parameters coincided with a 30 % reduction in OCR during high glucose exposure of Rgs2-overexpressing islets. In contrast, Rgs2 knockout cells exhibited slightly elevated GSIS. Finally, RGS2 expression was decreased in islets from T2 D donors (results presented as: SEM; Student’s t-test; p < 0.05). Conclusion: Our findings suggest that RGS2 reduces oxidative phosphorylation, thus limiting calcium influx and insulin secretion. The exact mechanism remains to be discovered.

Background: The divalent cation Calcium (Ca2+) regulates a wide range of processes in disparate cell types. Within insulin-producing beta-cells, increases in cytosolic Ca2+ directly stimulate insulin vesicle exocytosis, but also initiate multiple signaling pathways. Mediated through activation of downstream kinases and transcription factors, Ca2+-regulated signaling pathways leverage substantial influence on a number of critical cellular processes within the beta-cell. Additionally, there is evidence that prolonged activation of these same pathways is detrimental to beta-cell health and may contribute to Type 2 Diabetes pathogenesis. Scope of review: This review aims to briefly highlight canonical Ca2+ signaling pathways in beta-cells and how beta-cells regulate the movement of Ca2+ across numerous organelles and microdomains. As a main focus, this review synthesizes experimental data from in vitro and in vivo models on both the beneficial and detrimental effects of Ca2+ signaling pathways for beta-cell function and health. Major conclusions: Acute increases in intracellular Ca2+ stimulate a number of signaling cascades, resulting in (de-) phosphorylation events and activation of downstream transcription factors. The short-term stimulation of these Ca2+ signaling pathways promotes numerous cellular processes critical to beta-cell function, including increased viability, replication, and insulin production and secretion. Conversely, chronic stimulation of Ca2+ signaling pathways increases beta-cell ER stress and results in the loss of beta-cell differentiation status. Together, decades of study demonstrate that Ca2+ movement is tightly regulated within the beta-cell, which is at least partially due to its dual roles as a potent signaling molecule. (C) 2018 The Authors. Published by Elsevier GmbH.

Depolarization of neuroendocrine cells results in calcium influx, which induces vesicle exocytosis and alters gene expression. These processes, along with the restoration of resting membrane potential, are energy intensive. We hypothesized that cellular mechanisms exist to maximize energy production during excitation. Here, we demonstrate that NPAS4, an immediate early basic helix-loop-helix (bHLH)-PAS transcription factor, acts to maximize energy production by suppressing hypoxia-inducible factor 1α (HIF1α). As such, knockout of Npas4 from insulin-producing β cells results in reduced OXPHOS, loss of insulin secretion, β cell dedifferentiation, and type 2 diabetes. NPAS4 plays a similar role in the nutrient-sensing cells of the hypothalamus. Its knockout here results in increased food intake, reduced locomotor activity, and elevated peripheral glucose production. In conclusion, NPAS4 is critical for the coordination of metabolism during the stimulation of electrically excitable cells; its loss leads to the defects in cellular metabolism that underlie the cellular dysfunction that occurs in metabolic disease.