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The dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are essential for mitochondrial function, which has been principally attributed to their regulation of fission/fusion dynamics. Here, we report that Mfn1 and 2 are critical for glucose-stimulated insulin secretion (GSIS) primarily through control of mitochondrial DNA (mtDNA) content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in β-cells reduced mtDNA content, impaired mitochondrial morphology and networking, and decreased respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies unexpectedly revealed that Mfn1/2 control of glucose homeostasis was dependent on maintenance of mtDNA content, rather than mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient β-cells. Thus, the primary physiologic role of Mfn1 and 2 in β-cells is coupled to the preservation of mtDNA content rather than mitochondrial architecture, and Mfn1 and 2 may be promising targets to overcome mitochondrial dysfunction and restore glucose control in diabetes. Sidarala et al. examine the importance of the mitochondrial structural proteins, Mitofusins 1 and 2 (Mfn1/2), in diabetes. They find that Mfn1/2 control blood glucose by preserving mitochondrial DNA content, rather than mitochondrial structure.

Inflammatory damage contributes to β cell failure in type 1 and 2 diabetes (T1D and T2D, respectively). Mitochondria are damaged by inflammatory signaling in β cells, resulting in impaired bioenergetics and initiation of proapoptotic machinery. Hence, the identification of protective responses to inflammation could lead to new therapeutic targets. Here, we report that mitophagy serves as a protective response to inflammatory stress in both human and rodent β cells. Utilizing in vivo mitophagy reporters, we observed that diabetogenic proinflammatory cytokines induced mitophagy in response to nitrosative/oxidative mitochondrial damage. Mitophagy-deficient β cells were sensitized to inflammatory stress, leading to the accumulation of fragmented dysfunctional mitochondria, increased β cell death, and hyperglycemia. Overexpression of CLEC16A, a T1D gene and mitophagy regulator whose expression in islets is protective against T1D, ameliorated cytokine-induced human β cell apoptosis. Thus, mitophagy promotes β cell survival and prevents diabetes by countering inflammatory injury. Targeting this pathway has the potential to prevent β cell failure in diabetes and may be beneficial in other inflammatory conditions.

Pluripotent stem cell (SC)-derived islets offer hope as a renewable source for β cell replacement for type 1 diabetes (T1D), yet functional and metabolic immaturity may limit their long-term therapeutic potential. Here, we show that limitations in mitochondrial transcriptional programming impede the formation of SC-derived β (SC-β) cells. Utilizing transcriptomic profiling, assessments of chromatin accessibility, mitochondrial phenotyping, and lipidomics analyses, we observe that SC-β cells exhibit reduced oxidative and mitochondrial fatty acid metabolism compared to primary human islets that are related to limitations in key mitochondrial transcriptional networks. Surprisingly, we find that reductions in glucose-stimulated mitochondrial respiration in SC-islets were not associated with alterations in mitochondrial mass, structure, or genome integrity. In contrast, SC-islets show limited expression of targets of PPARα, which regulate mitochondrial programming, yet whose functions in β cell differentiation are unknown. Importantly, treatment with WY14643, a potent PPARα agonist, induces expression of mitochondrial targets, improves insulin secretion, and increases the formation of SC-β cells both in vitro and following transplantation. Thus, PPARα-dependent mitochondrial programming promotes the differentiation of SC-β cells and may be a promising target to improve β cell replacement efforts for T1D. Here they show that PPARα-dependent mitochondrial programming promotes the differentiation of pluripotent stem cell-derived β cells. Targeting mitochondria has the potential to improve β cell replacement efforts for the treatment of type 1 diabetes.

ER stress and proinsulin misfolding are heralded as contributing factors to β cell dysfunction in type 2 diabetes, yet how ER function becomes compromised is not well understood. Recent data identify altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple β cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that β cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas thioredoxin-interacting protein suppression restored ER redox and proinsulin trafficking. Taken together, we propose that β cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.

Aims/hypothesis Lipolytic breakdown of endogenous lipid pools in pancreatic beta cells contributes to glucose-stimulated insulin secretion (GSIS) and is thought to be mediated by acute activation of neutral lipases in the amplification pathway. Recently it has been shown in other cell types that endogenous lipid can be metabolised by autophagy, and this lipophagy is catalysed by lysosomal acid lipase (LAL). This study aimed to elucidate a role for LAL and lipophagy in pancreatic beta cells. Methods We employed pharmacological and/or genetic inhibition of autophagy and LAL in MIN6 cells and primary islets. Insulin secretion following inhibition was measured using RIA. Lipid accumulation was assessed by MS and confocal microscopy (to visualise lipid droplets) and autophagic flux was analysed by western blot. Results Insulin secretion was increased following chronic (≥8 h) inhibition of LAL. This was more pronounced with glucose than with non-nutrient stimuli and was accompanied by augmentation of neutral lipid species. Similarly, following inhibition of autophagy in MIN6 cells, the number of lipid droplets was increased and GSIS was potentiated. Inhibition of LAL or autophagy in primary islets also increased insulin secretion. This augmentation of GSIS following LAL or autophagy inhibition was dependent on the acute activation of neutral lipases. Conclusions/interpretation Our data suggest that lysosomal lipid degradation, using LAL and potentially lipophagy, contributes to neutral lipid turnover in beta cells. It also serves as a constitutive negative regulator of GSIS by depletion of substrate for the non-lysosomal neutral lipases that are activated acutely by glucose.

Deletion of PKCε Selectively Enhances the Amplifying Pathways of Glucose-Stimulated Insulin Secretion via Increased Lipolysis in Mouse β-Cells James Cantley 1 , James G. Burchfield 1 , 2 , Gemma L. Pearson 1 , Carsten Schmitz-Peiffer 1 , 2 , Michael Leitges 3 and Trevor J. Biden 1 , 2 1 Diabetes and Obesity Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia; 2 St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Darlinghurst, New South Wales, Australia; 3 Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway. Corresponding author: Trevor Biden, t.biden{at}garvan.org.au . Abstract OBJECTIVE Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of β-cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase C ε (PKCε) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCε. RESEARCH DESIGN AND METHODS We cultured islets isolated from PKCε knockout (PKCεKO) mice in palmitate prior to measuring GSIS, Ca 2+ responses, palmitate esterification products, lipolysis, lipase activity, and gene expression. RESULTS The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated Ca 2+ influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCεKO β-cells was revealed under conditions in which functional ATP-sensitive K + channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCεKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCεKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCε deletion. CONCLUSIONS Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine β-cells, and they suggest an interaction between PKCε and lipolysis. These results further highlight the therapeutic potential of PKCε inhibition to enhance GSIS from the β-cell under conditions of lipid excess. Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received January 29, 2009. Accepted April 24, 2009. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. © 2009 by the American Diabetes Association.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip − / − mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip − / − alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip − / − AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip − / − AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology. Toll-interacting protein (TOLLIP) prevents inflammation and lipid accumulation in alveolar macrophages to limit integrated stress response activation, macrophage necrosis and promote control of Mycobacterium tuberculosis .

OBJECTIVE--Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of G-cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase Cζ (PKCζ) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCζ. RESEARCH DESIGN AND METHODS--We cultured islets isolated from PKCζ knockout (PKCcKO) mice in palmitate prior to measuring GSIS, [Ca.sup.2+] responses, palmitate esterification products, lipolysis, lipase activity, and gene expression. RESULTS--The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated [Ca.sup.2+] influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCζKO β-cells was revealed under conditions in which functional ATP-sensitive [K.sup.+] channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCζKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCζKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCs deletion. CONCLUSIONS--Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine β-cells, and they suggest an interaction between PKCζ and lipolysis. These results further highlight the therapeutic potential of PKCζ inhibition to enhance GSIS from the β-cell under conditions of lipid excess. Diabetes 58:1826-1834, 2009

Pluripotent stem cell (SC)-derived islets offer hope as a renewable source for β cell replacement for type 1 diabetes (T1D), yet functional and metabolic immaturity may limit their long-term therapeutic potential. Here, we show that limitations in mitochondrial transcriptional programming impede the formation of SC-derived β (SC-β) cells. Utilizing transcriptomic profiling, assessments of chromatin accessibility, mitochondrial phenotyping, and lipidomics analyses, we observed that SC-β cells exhibit reduced oxidative and mitochondrial fatty acid metabolism compared to primary human islets that are related to limitations in key mitochondrial transcriptional networks. Surprisingly, we found that reductions in glucose-stimulated mitochondrial respiration in SC-islets were not associated with alterations in mitochondrial mass, structure, or genome integrity. In contrast, SC-islets show limited expression of targets of PPAR⍺, which regulate mitochondrial programming, yet whose functions in β cell differentiation are unknown. Importantly, treatment with WY14643, a potent PPAR⍺ agonist, induced expression of mitochondrial targets, improved insulin secretion, and increased the formation of SC-β cells both and following transplantation. Thus, PPAR⍺-dependent mitochondrial programming promotes the differentiation of SC-β cells and may be a promising target to improve β cell replacement efforts for T1D.

The dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are essential for mitochondrial function, which has been principally attributed to their regulation of fission/fusion dynamics. Here, we report that Mfn1 and 2 are critical for glucose-stimulated insulin secretion (GSIS) primarily through control of mitochondrial DNA (mtDNA) content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in β-cells reduced mtDNA content, impaired mitochondrial morphology and networking, and decreased respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies unexpectedly revealed that Mfn1/2 control of glucose homeostasis was dependent on maintenance of mtDNA content, rather than mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient β-cells. Thus, the primary physiologic role of Mfn1 and 2 in β-cells is coupled to the preservation of mtDNA content rather than mitochondrial architecture, and Mfn1 and 2 may be promising targets to overcome mitochondrial dysfunction and restore glucose control in diabetes. Competing Interest Statement The authors have declared no competing interest.