Identification of MicroRNAs Associated with Prediabetic Status in Obese Women.

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 ß-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.

Differences in DNA methylation of HAMP in blood cells predicts the development of type 2 diabetes.

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.

SORLA mediates endocytic uptake of proIAPP and protects against islet amyloid deposition.

OBJECTIVE: 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 beta cells of pancreatic islets, yet the role of SORLA in islets is unknown. Based on its protective role in reducing the amyloid burden in the brain, we hypothesized that SORLA has a similar function in the pancreas via regulation of amyloid 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 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 compared to hIAPP:SORLA WT animals. 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. 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: SORLA functions as a proIAPP-specific clearance receptor, protecting against islet amyloid deposition and associated cell death caused by IAPP.

Heterogeneous Development of ß-Cell Populations in Diabetes-Resistant and -Susceptible Mice.

Progressive dysfunction and failure of insulin-releasing ß-cells are a hallmark of type 2 diabetes (T2D). To study mechanisms of ß-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 ß-cell clusters with similar abundance in both strains. However, after feeding of a diabetogenic diet for 2 days, ß-cell cluster composition markedly differed between strains. Islets of diabetes-resistant mice developed into a protective ß-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 ß-cell identity, such as downregulation of GLUT2, GLP1R, and MafA, and in vitro knockdown of GLUT2 in ß-cells-mimicking its phenotype-decreased stress response and apoptosis. This might explain enhanced ß-cell survival of diabetes-resistant islets. In contrast, ß-cells of diabetes-prone mice responded with expression changes indicating metabolic pressure and endoplasmic reticulum stress, presumably leading to later ß-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 ß-cell failure and diabetes development.

Identification of Novel Genes Involved in Hyperglycemia in Mice.

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 ß-cell proliferation and apoptosis. Thus, S100z is an entirely novel diabetes gene regulating islet cell function.

Friend and foe: ß-cell Ca2+ signaling and the development of diabetes.

BACKGROUND: The divalent cation Calcium (Ca2+) regulates a wide range of processes in disparate cell types. Within insulin-producing ß-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 ß-cell. Additionally, there is evidence that prolonged activation of these same pathways is detrimental to ß-cell health and may contribute to Type 2 Diabetes pathogenesis. SCOPE OF REVIEW: This review aims to briefly highlight canonical Ca2+ signaling pathways in ß-cells and how ß-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 ß-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 ß-cell function, including increased viability, replication, and insulin production and secretion. Conversely, chronic stimulation of Ca2+ signaling pathways increases ß-cell ER stress and results in the loss of ß-cell differentiation status. Together, decades of study demonstrate that Ca2+ movement is tightly regulated within the ß-cell, which is at least partially due to its dual roles as a potent signaling molecule.

Neuronal PAS Domain Protein 4 Suppression of Oxygen Sensing Optimizes Metabolism during Excitation of Neuroendocrine Cells.

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 ß cells results in reduced OXPHOS, loss of insulin secretion, ß 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.

SOX4 Allows Facultative ß-Cell Proliferation Through Repression of Cdkn1a.

The high-mobility group box transcription factor SOX4 is the most highly expressed SOX family protein in pancreatic islets, and mutations in Sox4 are associated with an increased risk of developing type 2 diabetes. We used an inducible ß-cell knockout mouse model to test the hypothesis that Sox4 is essential for the maintenance of ß-cell number during the development of type 2 diabetes. Knockout of Sox4 at 6 weeks of age resulted in time-dependent worsening of glucose tolerance, impairment of insulin secretion, and diabetes by 30 weeks of age. Immunostaining revealed a decrease in ß-cell mass in knockout mice that was caused by a 39% reduction in ß-cell proliferation. Gene expression studies revealed that induction of the cell cycle inhibitor Cdkn1a was responsible for the decreased proliferation in the knockout animals. Altogether, this study demonstrates that SOX4 is necessary for adult ß-cell replication through direct regulation of the ß-cell cycle.

Npas4 Transcription Factor Expression Is Regulated by Calcium Signaling Pathways and Prevents Tacrolimus-induced Cytotoxicity in Pancreatic Beta Cells.

Cytosolic calcium influx activates signaling pathways known to support pancreatic beta cell function and survival by modulating gene expression. Impaired calcium signaling leads to decreased beta cell mass and diabetes. To appreciate the causes of these cytotoxic perturbations, a more detailed understanding of the relevant signaling pathways and their respective gene targets is required. In this study, we examined the calcium-induced expression of the cytoprotective beta cell transcription factor Npas4. Pharmacological inhibition implicated the calcineurin, Akt/protein kinase B, and Ca(2+)/calmodulin-dependent protein kinase signaling pathways in the regulation of Npas4 transcription and translation. Both Npas4 mRNA and protein had high turnover rates, and, at the protein level, degradation was mediated via the ubiquitin-proteasome pathway. Finally, beta cell cytotoxicity of the calcineurin inhibitor and immunosuppressant tacrolimus (FK-506) was prevented by Npas4 overexpression. These results delineate the pathways regulating Npas4 expression and stability and demonstrate its importance in clinical settings such as islet transplantation.