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Mitochondrial dysfunction is a hallmark of metabolic diseases, including diabetes, yet the consequences of mitochondrial damage in metabolic tissues are often unclear. Here, we report that mitochondrial dysfunction engages a retrograde (mitonuclear) signaling program that impairs cellular identity and maturity across many metabolic tissues. Surprisingly, we demonstrate that impairments in the mitochondrial quality control machinery, which we observe in pancreatic β cells of humans with diabetes, cause reductions of β cell mass due to dedifferentiation, rather than apoptosis. Utilizing transcriptomic profiling, lineage tracing, and assessments of chromatin accessibility, we find that targeted defects anywhere in the mitochondrial quality control pathway (e.g., genome integrity, dynamics, or turnover) activate the mitochondrial integrated stress response and promote cellular immaturity in β cells, hepatocytes, and brown adipocytes. Intriguingly, pharmacologic blockade of mitochondrial retrograde signaling in vivo restores β cell mass and identity to ameliorate hyperglycemia following mitochondrial damage. Thus, we observe that a shared mitochondrial retrograde response controls cellular identity across metabolic tissues and may be a promising target to treat or prevent metabolic diseases.

Insulin-producing pancreatic β-cells are central to glucose homeostasis, and their failure is a principal driver of diabetes development. To preserve optimal health β-cells must withstand both intrinsic and extrinsic stressors, ranging from inflammation to increased peripheral insulin demand, in addition to maintaining insulin biosynthesis and secretory machinery. Autophagy is increasingly being appreciated as a critical β-cell quality control system vital for glycemic control. Here we focus on the underappreciated, yet crucial, roles for selective and organelle-specific forms of autophagy as mediators of β-cell health. We examine the unique molecular players underlying each distinct form of autophagy in β-cells, including selective autophagy of mitochondria, insulin granules, lipid, intracellular amyloid aggregates, endoplasmic reticulum, and peroxisomes. We also describe how defects in selective autophagy pathways contribute to the development of diabetes. As all forms of autophagy are not the same, a refined view of β-cell selective autophagy may inform new approaches to defend against the various insults leading to β-cell failure in diabetes.

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. However, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. IDPRs comprise nearly half of the human proteome, yet their mechanistic roles in human disease are poorly understood. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases CLEC16A ubiquitination and degradation, thus impairing assembly of the mitophagy regulatory machinery. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we elucidate how an IDPR in CLEC16A regulates mitophagy and implicate pathogenic human gene variants that disrupt IDPRs as novel contributors to diabetes and other CLEC16A-associated diseases. Abbreviations : CAS: carbon-detect amino-acid specific; IDPR: intrinsically disordered protein region; MEFs: mouse embryonic fibroblasts; NMR: nuclear magnetic resonance.

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. However, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. IDPRs comprise nearly half of the human proteome, yet their mechanistic roles in human disease are poorly understood. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases CLEC16A ubiquitination and degradation, thus impairing assembly of the mitophagy regulatory machinery. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we elucidate how an IDPR in CLEC16A regulates mitophagy and implicate pathogenic human gene variants that disrupt IDPRs as novel contributors to diabetes and other CLEC16A-associated diseases. Abbreviations : CAS: carbon-detect amino-acid specific; IDPR: intrinsically disordered protein region; MEFs: mouse embryonic fibroblasts; NMR: nuclear magnetic resonance.

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. While CLEC16A has ubiquitin ligase activity, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs pancreatic β-cell mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases its self-ubiquitination and destabilizes CLEC16A, thus impairing formation of a critical CLEC16A-dependent mitophagy complex. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we clarify how an IDPR in CLEC16A prevents diabetes, thus implicating the disruption of IDPRs as novel pathological contributors to diabetes and other CLEC16A-associated diseases.

Mitochondrial function is pivotal to β-cell competence. The mitochondrial life cycle balances mitochondrial biogenesis and turnover (mitophagy) to ensure optimal metabolic function. Human type 2 diabetic (T2D) β-cells are known to develop mitochondrial structural/functional defects, suggestive of a defective mitochondrial life cycle. However, it is unclear if these defects are a cause or consequence of T2D. Here, we observed that human T2D β-cells had reduced mitophagic flux, mtDNA content, and expression of mitochondrially encoded genes. To test the importance of the mitochondrial life cycle to drive β-cell failure in T2D, we developed 2 distinct β-cell specific mouse models: βTfamKO (to deplete mtDNA) and βClec16aΚΟ (to impair mitophagy). We observed age dependent loss of glucose tolerance, glucose stimulated insulin secretion and β-cell mass in both models, which was exacerbated by obesity in βClec16aΚΟ mice. Loss of β-cell mass was largely independent of changes in proliferation or apoptosis but rather, due to an induction of β-cell immaturity. Using lineage tracing approaches, we confirmed that βClec16aKO and βTfamKO mice induce formation of both insulin-negative immature β-cells and β-to-α cell transdifferentiation. Further, high-throughput gene expression profiling, biochemical, and metabolic assays highlighted that either Clec16a or Tfam deficiency induces an aberrant retrograde signaling program, manifested by reductions in cellular ATP and activation of the integrated stress response (ISR). Importantly, inhibition of the ISR in vivo ameliorated glucose intolerance, defective insulin secretion, β-cell immaturity, and loss of β-cell mass, suggesting that aberrant retrograde signaling may directly lead to loss of β-cell identity. Taken together, our studies illustrate that a unified mitochondrial lifecycle is necessary to maintain β-cell mass and identity and may be targeted to prevent β-cell failure in T2D. Disclosure G. Pearson: None. N. Lawlor: None. J. Zhu: None. E. M. Walker: None. E. C. Reck: None. M. L. Stitzel: None. S. Soleimanpour: None. Funding American Diabetes Association (1-19-PDF-063 to G.P.)

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 mtDNA content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in β-cells reduced mtDNA content, induced mitochondrial fragmentation, and impaired 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. Indeed, pharmacologic mitofusin agonists rescued islet mtDNA depletion due to mitofusin deficiency independent of changes on 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 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.

Relative deficiency of TOLLIP expression in monocytes is associated with increased tuberculosis (TB) susceptibility in genetic studies, despite antagonizing host innate immune pathways that control Mycobacterium tuberculosis (Mtb) infection. In this study, we investigated the mechanisms by which TOLLIP influences Mtb immunity. Tollip-/- mice developed worsened disease, consistent with prior genetic observations, and developed large numbers of foam cells. Selective TOLLIP deletion in alveolar macrophages (AM) was sufficient to induce lipid accumulation and increased Mtb persistence 28 days after infection, despite increased antimicrobial responses. We analyzed sorted, Mtb-infected Tollip-/- AM from mixed bone marrow chimeric mice to measure global gene expression 28 days post-infection. We found transcriptional profiles consistent with increased EIF2 signaling. Selective lipid administration to Tollip-/- macrophages induced lipid accumulation, and Mtb infection of lipid laden, Tollip-/- macrophages induced cellular stress and impaired Mtb control. EIF2 activation induced increased Mtb replication within macrophages, irrespective of TOLLIP expression, and EIF2 kinases were enriched in human caseous granulomas. Our findings define a critical checkpoint for TOLLIP to prevent lipid-induced EIF2 activation and demonstrate an important mechanism for EIF2 signaling to permit Mtb replication within macrophages.

Inflammatory damage contributes to β-cell failure in type 1 and 2 diabetes (T1D and T2D). Mitochondria are damaged by inflammatory signaling in β-cells, resulting in impaired bioenergetics and initiation of pro-apoptotic 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. Utilizingin vivomitophagy reporters, we observed that diabetogenic pro-inflammatory 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 ofCLEC16A, 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.

Mitophagy is a cellular quality-control pathway, which is essential for elimination of unhealthy mitochondria. While mitophagy is critical to pancreatic β-cell function, the posttranslational signals governing β-cell mitochondrial turnover are unknown. Here, we report that ubiquitination is essential for the assembly of a mitophagy regulatory complex, comprised of the E3 ligase Nrdp1, the deubiquitinase enzyme USP8, and Clec16a, a mediator of β-cell mitophagy with unclear function. We discover that the diabetes gene Clec16a encodes an E3 ligase, which promotes nondegradative ubiquitin conjugates to direct its mitophagy effectors and stabilize the Clec16a-Nrdp1-USP8 complex. Inhibition of the Clec16a pathway by the chemotherapeutic lenalidomide, a selective ubiquitin ligase inhibitor associated with new-onset diabetes, impairs β-cell mitophagy, oxygen consumption, and insulin secretion. Indeed, patients treated with lenalidomide develop compromised β-cell function. Moreover, the β-cell Clec16a-Nrdp1-USP8 mitophagy complex is destabilized and dysfunctional after lenalidomide treatment as well as after glucolipotoxic stress. Thus, the Clec16a-Nrdp1-USP8 complex relies on ubiquitin signals to promote mitophagy and maintain mitochondrial quality control necessary for optimal β-cell function.

The E3 ligase parkin is a critical regulator of mitophagy and has been identified as a susceptibility gene for type 2 diabetes (T2D), but its role in metabolically active tissues to modulate the development of T2D is unknown. Pancreatic β-cells and adipocytes both rely heavily on mitochondrial function to regulate optimal glycemic control to prevent T2D, but the role of parkin to modulate β-cell or adipocyte mitochondrial quality control is unclear. Despite the reported importance of parkin to control mitophagy, here we report that parkin surprisingly is dispensable in both β-cells and adipocytes for glucose homeostasis during diet-induced insulin resistance. We observe that insulin secretion, β-cell formation, and islet architecture were preserved in parkin deficient β-cells and islets, suggesting parkin is not necessary for control of β-cell function and islet compensation for diet-induced obesity. While transient parkin deficiency mildly impaired mitochondrial turnover in β-cell lines, parkin deletion in primary β-cells yielded no deficits in mitochondrial clearance. In adipocyte-specific deletion models, lipid uptake and β-oxidation were increased in cultured cells, while adipose tissue morphology, glucose homeostasis, or the beige-to-white adipocyte transition were unaffected in vivo. In key metabolic tissues where mitochondrial dysfunction is implicated in the development of T2D, our studies unexpectedly demonstrate that parkin is not an essential regulator of glucose tolerance, whole-body energy metabolism, or mitochondrial quality control. These findings highlight a novel role for parkin-independent processes to maintain β-cell and adipocyte mitochondrial quality control with diet-induced obesity. Disclosure G. Pearson: None. C. Corsa: None. S. Soleimanpour: None. O.A. MacDougald: None. Funding American Diabetes Association (1-18-PDF-064 to C.C.); National Institutes of Health; JDRF

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose- stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

The E3 ubiquitin ligase parkin is a critical regulator of mitophagy and has been identified as a susceptibility gene for type 2 diabetes (T2D). However, its role in metabolically active tissues that precipitate T2D development is unknown. Specifically, pancreatic β cells and adipocytes both rely heavily on mitochondrial function in the regulation of optimal glycemic control to prevent T2D, but parkin's role in preserving quality control of β cell or adipocyte mitochondria is unclear. Although parkin has been reported previously to control mitophagy, here we show that, surprisingly, parkin is dispensable for glucose homeostasis in both β cells and adipocytes during diet-induced insulin resistance in mice. We observed that insulin secretion, β cell formation, and islet architecture were preserved in parkin-deficient β cells and islets, suggesting that parkin is not necessary for control of β cell function and islet compensation for diet-induced obesity. Although transient parkin deficiency mildly impaired mitochondrial turnover in β cell lines, parkin deletion in primary β cells yielded no deficits in mitochondrial clearance. In adipocyte-specific deletion models, lipid uptake and β-oxidation were increased in cultured cells, whereas adipose tissue morphology, glucose homeostasis, and beige-to-white adipocyte transition were unaffected in vivo. In key metabolic tissues where mitochondrial dysfunction has been implicated in T2D development, our experiments unexpectedly revealed that parkin is not an essential regulator of glucose tolerance, whole-body energy metabolism, or mitochondrial quality control. These findings highlight that parkin-independent processes maintain β cell and adipocyte mitochondrial quality control in diet-induced obesity.

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.

AIMS/HYPOTHESIS: Proinflammatory cytokines contribute to beta cell destruction in type 1 diabetes, but the mechanisms are incompletely understood. The aim of the current study was to address the role of the protein kinase C (PKC) isoform PKCδ, a diverse regulator of cell death, in cytokine-stimulated apoptosis in primary beta cells. METHODS: Islets isolated from wild-type or Prkcd(-/-) mice were treated with IL-1β, TNF-α and IFNγ and assayed for apoptosis, nitric oxide (NO) generation and insulin secretion. Activation of signalling pathways, apoptosis and endoplasmic reticulum (ER) stress were determined by immunoblotting. Stabilisation of mRNA transcripts was measured by RT-PCR following transcriptional arrest. Mice were injected with multiple low doses of streptozotocin (MLD-STZ) and fasting blood glucose monitored. RESULTS: Deletion of Prkcd inhibited apoptosis and NO generation in islets stimulated ex vivo with cytokines. It also delayed the onset of hyperglycaemia in MLD-STZ-treated mice. Activation of ERK, p38, JNK, AKT1, the ER stress markers DDIT3 and phospho-EIF2α and the intrinsic apoptotic markers BCL2 and MCL1 was not different between genotypes. However, deletion of Prkcd destabilised mRNA transcripts for Nos2, and for multiple components of the toll-like receptor 2 (TLR2) signalling complex, which resulted in disrupted TLR2 signalling. CONCLUSIONS/INTERPRETATION: Loss of PKCδ partially protects against hyperglycaemia in the MLD-STZ model in vivo, and against cytokine-mediated apoptosis in vitro. This is accompanied by reduced NO generation and destabilisation of Nos2 and components of the TLR2 signalling pathway. The results highlight a mechanism for regulating proinflammatory gene expression in beta cells independently of transcription.

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, Ca2+ 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 Ca2+ 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.