The role of mitochondrial fission protein Drp1 in pancreatic islets and beta cells.

The development of Type 2 Diabetes (T2D) is often associated with impaired mitochondrial dynamics and bioenergetics, particularly in pancreatic beta (β) cells. In this thesis, I show that alteration in mitochondrial dynamics affects mitochondrial bioenergetics and subsequently glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and islets. The transient phosphorylation of Drp1, a mitochondrial fission regulator, at its serine residues 616 and 637 in response to glucose indicates involvement of Drp1 during GSIS. Genetic silencing of Drp1 altered mitochondrial morphology, increased mitochondrial proton leak and decreased GSIS in mouse insulinoma MIN6 cells, consistent with another study in INS-1E cell line. Acute pharmacological inhibition of Drp1 by mitochondrial division inhibitor (mdivi-1) mimicked genetic knockdown effects not only in MIN6 cells but also in mouse and human pancreatic islets. Surprisingly, comprehensive analysis of bioenergetics in MIN6 cells and islets revealed that Drp1 deficiency attenuated GSIS by lowering glucose-fuelled respiration linked to ATP production, instead of its effect on proton leak as suggested previously. Strikingly, the impaired ATP output and insulin secretion was rescued by supplying fission-deficient cells and islets with pyruvate. It thus transpires that Drp1-dependent mitochondrial dynamics influences mitochondrial function and GSIS, both by controlling mitochondrial substrate delivery upstream of oxidative phosphorylation. Furthermore, transient Drp1 overexpression rescued the impaired insulin secretion triggering in Drp1 knockdown MIN6 cells. In the last part of my thesis, I explore bioenergetics of islets from diet-induced obese (DIO) and chow fed mice using respirometry as a tool to predict pancreatic β-cell function. Although there were no apparent pathologies in absolute GSIS values, I found high-fat diet-induced increase in insulin content and marked decrease in glucose-stimulated respiration, resulting in reduced ATP-linked respiration. Normalizing GSIS to insulin content uncovered compromised insulin secretion triggering in DIO islets. Additionally, I also disclose that plotting mitochondrial respiratory parameters vs. GSIS classifies dysfunctional properties of pancreatic insulin secretion. Using this prediction model, the data of DIO islets suggested defects in or upstream of oxidative phosphorylation. Moreover, internally standardizing mitochondrial respiration as coupling efficiency (CE) reveals a bioenergetic threshold for insulin triggering that can be used to address mitochondrial failure across independent studies

<em>MiR-184</em> regulates pancreatic &beta;-cell function according to glucose metabolism.

In response to fasting or hyperglycemia, the pancreatic &beta;-cell alters its output of secreted insulin; however the pathways governing this adaptive response are not entirely established. While the precise role of microRNAs (miRNAs) is also unclear, a recurring theme emphasizes their function in cellular stress responses. We recently showed that miR-184, an abundant miRNA in the &beta;-cell, regulates compensatory proliferation and secretion during insulin resistance. Consistent with previous studies showing miR-184 suppresses insulin release, expression of this miRNA was increased in islets after fasting, demonstrating an active role in the &beta;-cell as glucose levels lower and the insulin demand ceases. Additionally, miR-184 was negatively regulated upon administration of a sucrose-rich diet in Drosophila demonstrating strong conservation of this pathway through evolution. Furthermore, miR-184 and its target Argonaute2 (Ago2) remained inversely correlated as concentrations of extracellular glucose increased, underlining a functional relationship between this miRNA and its targets. Lastly, restoration of Ago2 in the presence of miR-184 rescued suppression of miR-375-targeted genes suggesting these genes act in a coordinated manner during changes in the metabolic context. Together, these results highlight the adaptive role of miR-184 according to glucose metabolism and suggest the regulatory role of this miRNA in energy homeostasis is highly conserved

Hypothalamic leptin action is mediated by histone deacetylase 5.

Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and greater diet-induced obesity when fed high-fat diet. Pharmacological and genetic inhibition of HDAC5 activity in the mediobasal hypothalamus increases food intake and modulates pathways implicated in leptin signalling. We show HDAC5 directly regulates STAT3 localization and transcriptional activity via reciprocal STAT3 deacetylation at Lys685 and phosphorylation at Tyr705. In vivo, leptin sensitivity is substantially impaired in HDAC5 loss-of-function mice. Hypothalamic HDAC5 overexpression improves leptin action and partially protects against HFD-induced leptin resistance and obesity. Overall, our data suggest that hypothalamic HDAC5 activity is a regulator of leptin signalling that adapts food intake and body weight to our dietary environment