Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Sorcin Links Pancreatic ß-Cell Lipotoxicity to ER Ca2+ Stores.

Preserving ß-cell function during the development of obesity and insulin resistance would limit the worldwide epidemic of type 2 diabetes. Endoplasmic reticulum (ER) calcium (Ca(2+)) depletion induced by saturated free fatty acids and cytokines causes ß-cell ER stress and apoptosis, but the molecular mechanisms behind these phenomena are still poorly understood. Here, we demonstrate that palmitate-induced sorcin downregulation and subsequent increases in glucose-6-phosphatase catalytic subunit-2 (G6PC2) levels contribute to lipotoxicity. Sorcin is a calcium sensor protein involved in maintaining ER Ca(2+) by inhibiting ryanodine receptor activity and playing a role in terminating Ca(2+)-induced Ca(2+) release. G6PC2, a genome-wide association study gene associated with fasting blood glucose, is a negative regulator of glucose-stimulated insulin secretion (GSIS). High-fat feeding in mice and chronic exposure of human islets to palmitate decreases endogenous sorcin expression while levels of G6PC2 mRNA increase. Sorcin-null mice are glucose intolerant, with markedly impaired GSIS and increased expression of G6pc2 Under high-fat diet, mice overexpressing sorcin in the ß-cell display improved glucose tolerance, fasting blood glucose, and GSIS, whereas G6PC2 levels are decreased and cytosolic and ER Ca(2+) are increased in transgenic islets. Sorcin may thus provide a target for intervention in type 2 diabetes.

Mitochondrial exchanger NCLX plays a major role in the intracellular Ca2+ signaling, gliotransmission, and proliferation of astrocytes.

Mitochondria not only provide cells with energy, but are central to Ca(2+) signaling. Powered by the mitochondrial membrane potential, Ca(2+) enters the mitochondria and is released into the cytosol through a mitochondrial Na(+)/Ca(2+) exchanger. We established that NCLX, a newly discovered mitochondrial Na(+)/Ca(2+) exchanger, is expressed in astrocytes isolated from mice of either sex. Immunoblot analysis of organellar fractions showed that the location of NCLX is confined to mitochondria. Using pericam-based mitochondrial Ca(2+) imaging and NCLX inhibition either by siRNA or by the pharmacological blocker CGP37157, we demonstrated that NCLX is responsible for mitochondrial Ca(2+) extrusion. Suppression of NCLX function altered cytosolic Ca(2+) dynamics in astrocytes and this was mediated by a strong effect of NCLX activity on Ca(2+) influx via store-operated entry. Furthermore, Ca(2+) influx through the store-operated Ca(2+) entry triggered strong, whereas ER Ca(2+) release triggered only modest mitochondrial Ca(2+) transients, indicating that the functional cross talk between the plasma membrane and mitochondrial domains is particularly strong in astrocytes. Finally, silencing of NCLX expression significantly reduced Ca(2+)-dependent processes in astrocytes (i.e., exocytotic glutamate release, in vitro wound closure, and proliferation), whereas Ca(2+) wave propagation was not affected. Therefore, NCLX, by meditating astrocytic mitochondrial Na(+)/Ca(2+) exchange, links between mitochondria and plasma membrane Ca(2+) signaling, thereby modulating cytoplasmic Ca(2+) transients required to control a diverse array of astrocyte functions.

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange.

Mitochondrial Ca(2+) efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na(+)-dependent mechanism mediates mitochondrial Ca(2+) efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na(+)/Ca(2+) exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca(2+) and Na(+) fluorescent imaging, we demonstrate that mitochondrial Na(+)-dependent Ca(2+) efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca(2+) transport was inhibited, moreover, by CGP-37157 and exhibited Li(+) dependence, both hallmarks of mitochondrial Na(+)-dependent Ca(2+) efflux. Finally, NCLX-mediated mitochondrial Ca(2+) exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na(+)/Ca(2+) exchanger.