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CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover. IDPRs lack a fixed secondary structure and possess emerging yet still equivocal roles in protein stability, interactions, and enzymatic activity. We find that the internal IDPR of CLEC16A is crucial for its degradation. CLEC16A turnover was promoted by RNF41, which binds and acts upon the internal IDPR to destabilize CLEC16A. Loss of this internal IDPR also destabilized the ubiquitin-dependent tripartite CLEC16A–RNF41–USP8 complex. Finally, the presence of an internal IDPR within CLEC16A was confirmed using NMR and CD spectroscopy. Together, our studies reveal that an IDPR is essential to control the reciprocal regulatory balance between CLEC16A and RNF41, which could be targeted to improve mitochondrial health in disease.

A central goal of physiological research is the understanding of cell-specific roles of disease-associated genes. Cre-mediated recombineering is the tool of choice for cell type–specific analysis of gene function in preclinical models. In the type 1 diabetes (T1D) research field, multiple lines of nonobese diabetic (NOD) mice have been engineered to express Cre recombinase in pancreatic β cells using insulin promoter fragments, but tissue promiscuity remains a concern. Constitutive Ins1tm1.1(cre)Thor (Ins1Cre) mice on the C57/bl6-J background have high β-cell specificity with no reported off-target effects. We explored whether NOD:Ins1Cre mice could be used to investigate β-cell gene deletion in T1D disease modeling. We studied wild-type (Ins1WT/WT), Ins1 heterozygous (Ins1Cre/WT or Ins1Neo/WT), and Ins1 null (Ins1Cre/Neo) littermates on a NOD background. Female Ins1Neo/WT mice exhibited significant protection from diabetes, with further near-complete protection in Ins1Cre/WT mice. The effects of combined neomycin and Cre knockin in Ins1Neo/Cre mice were not additive to the Cre knockin alone. In Ins1Neo/Cre mice, protection from diabetes was associated with reduced insulitis at age 12 weeks. Collectively, these data confirm previous reports that loss of Ins1 alleles protects NOD mice from diabetes development and demonstrates, for the first time, that Cre itself may have additional protective effects. This has important implications for the experimental design and interpretation of preclinical T1D studies using β-cell-selective Cre in NOD mice.

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.

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.

While exposure to inflammatory cytokines is thought to contribute to pancreatic β-cell damage during diabetes, primarily because cytokine-induced nitric oxide impairs β-cell function and causes cell death with prolonged exposure, we hypothesize that there is a physiological role for cytokine signaling that protects β-cells from a number of environmental stresses. This hypothesis is derived from the knowledge that β-cells are essential for survival even though they have a limited capacity to replicate, yet they are exposed to high cytokine levels during infection as most of the pancreatic blood flow is directed to islets. Here, mouse islets were subjected to single-cell RNA sequencing following 18-h cytokine exposure. Treatment with IL-1β and IFN-γ stimulates expression of inducible nitric oxide synthase (iNOS) mRNA and antiviral and immune-associated genes as well as repression of islet identity factors in a subset of β- and non-β-endocrine cells in a nitric oxide-independent manner. Nitric oxide-dependent expression of genes encoding heat shock proteins was observed in both β- and non-β-endocrine cells. Interestingly, cells with high expression of heat shock proteins failed to increase antiviral and immune-associated gene expression, suggesting that nitric oxide may be an internal “off switch” to prevent the negative effects of prolonged cytokine signaling in islet endocrine cells. We found no evidence for pro-apoptotic gene expression following 18-h cytokine exposure. Our findings suggest that the primary functions of cytokines and nitric oxide are to protect islet endocrine cells from damage, and only when regulation of cytokine signaling is lost does irreversible damage occur., Graphical Abstract Graphical Abstract

A central goal of physiological research is the understanding of cell-specific roles of disease-associated genes. Cre-mediated recombineering is the tool of choice for cell type-specific analysis of gene function in pre-clinical models. In the type 1 diabetes research field, multiple lines of NOD mice have been engineered to express Cre recombinase in pancreatic β-cells using insulin promoter fragments, but tissue promiscuity remains a concern. Constitutive Ins1tm1.1(cre)Thor (Ins1Cre) mice on the C57/bl6-J background has high β-cell specificity and with no reported off-target effects. We explored if NOD:Ins1Cre mice could be used to investigate β-cell gene deletion in type 1 diabetes disease modeling. We studied wildtype (Ins1WT/WT), Ins1 heterozygous (Ins1Cre/WT or Ins1Neo/WT), and Ins1 null (Ins1Cre/Neo) littermates on a NOD background. Female Ins1Neo/WT mice exhibited significant protection from diabetes, with further near-complete protection in Ins1Cre/WT mice. The effects of combined neomycin and Cre knock-in in Ins1Neo/Cre mice were not additive to the Cre knock-in alone. In Ins1Neo/Cre mice, protection from diabetes was associated with reduced insulitis at 12 weeks of age. Collectively, these data confirm previous reports that loss of Ins1 alleles protects NOD mice from diabetes development and demonstrates, for the first time, that Cre itself may have additional protective effects. This has significant implications for the experimental design and interpretation of pre-clinical type 1 diabetes studies using β-cell-specific Cre in NOD mice.

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.

More than a decade after the discovery of a novel type 1 diabetes risk locus on chromosome 16p13, there remains complexity and controversy over the specific gene(s) that regulate diabetes pathogenesis. A new study by Nieves-Bonilla et al. shows that one of these genes, DEXI, is unlikely to contribute to type 1 diabetes pathogenesis and positions the endolysosomal E3 ubiquitin ligase CLEC16A as the primary culprit by which this gene locus influences diabetes risk.

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.

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.

Inflammatory damage contributes to beta cell failure in all forms of diabetes, yet few protective responses to inflammation have been described. Mitochondria are a frequent target of inflammatory β-cell damage, thus impairing bioenergetics and initiating the pro-apoptotic machinery. Thus, we hypothesized that clearance of damaged mitochondria by mitophagy is a necessary protective response for inflammatory β-cell damage. Utilizing live-cell imaging, biochemical assays, and in vivo mitophagy biosensors, we observed that pro-inflammatory cytokines promote trafficking of damaged mitochondria through the mitophagy pathway in mouse and human islets. Cytokine treatment dissipated mitochondrial membrane potential, decreased cellular respiration, induced recruitment of the mitophagy initiator Parkin, activated turnover of mitochondrial proteins, and ultimately, targeted damaged mitochondria to lysosomes for clearance. To determine if mitophagy acts as a protective response, we assessed the role of pro-inflammatory agents in mice deficient for T1D gene and mitophagy regulator Clec16a in β-cells. Indeed, Clec16a-deficient β-cells had increased cytokine-mediated apoptosis and worsening hyperglycemia after streptozotocin treatment in vivo as well as an accumulation of fragmented, damaged mitochondria. To explore mechanisms by which mitophagy contributes to cytokine-mediated toxicity, we found that reactive O2 species and nitric oxide (NO) activate mitophagy and that mitophagy-deficient mouse and human islets treated with anti-oxidants or NO inhibitors ameliorated β-cell death. Finally, we observed that adenoviral overexpression of Clec16a rescued cytokine-induced apoptosis in human islets. Together, our studies position mitophagy as a crucial protective response to inflammatory β-cell demise activated by both oxidative and nitrosative stress, which could be targeted to prevent β-cell death in diabetes. Disclosure V. Sidarala: None. S. Soleimanpour: None.

Chronic hyperglycemia (HG) promotes pancreatic islet dysfunction which leads to the onset of T2DM. This study is aimed at defining regulatory roles of Rac1, a small G-protein, in the activation of p53 and ATM kinase in pancreatic β-cells, under the duress of HG conditions. We report significant stimulatory effects of HG (20 mM; 24 h) on p53 activation in INS-1 832/13 cells, normal rodent and human islets. Pharmacological inhibition of Rac1 (EHT1864 or NSC23766) significantly suppressed HG-induced p53 activation in INS-1 832/13 cells and rat islets, suggesting novel roles for this small G-protein in the activation of p53. Inhibition of Rac1 geranylgeranylation with simvastatin or GGTI-2147, significantly attenuated HG-induced p53 activation, suggesting requisite roles for this signaling step in HG-mediated effects on β-cells. HG-induced p53 activation was also suppressed by SB203580, a known inhibitor of p38MAPK. Additionally, we observed increased activation of ATM kinase under HG conditions, which was blocked in presence of EHT1864. Furthermore, pharmacological inhibition of ATM kinase (KU55933) reduced activation of ATM kinase, but not p53, suggesting that HG-mediated activation of p53 and ATM could represent independent pro-apoptotic events. In conclusion, these data indicate that sustained activation of Rac1-p38MAPK signaling axis leads to activation of p53 leading to β-cell dysfunction under the duress of chronic hyperglycemic conditions.

Background/Aims: Type 1 diabetes (T1D) is characterized by absolute insulin deficiency due to destruction of pancreatic β-cells by cytokines (e.g., interleukin-1β; IL-1β) released by invading immune cells. The mechanisms by which these cytokines induce β-cell dysfunction remain poorly understood. Recent evidence suggests that excessive generation of reactive oxygen species (ROS) by the phagocyte-like NADPH oxidase2 (Nox2), along with significantly low levels of antioxidants in β-cells, drive them toward oxidative damage. Rac1, a small G-protein, is one of the members of Nox2 holoenzyme. We recently reported that NSC23766, a known inhibitor of Rac1, significantly attenuated cytokine-induced Nox2 activation and ROS generation in pancreatic islet β-cells in vitro. Herein, we determined the effects of NSC23766 (2.5 mg/kg/day, i.p/daily) on the development of diabetes in the NOD mouse, a model for T1D. Methods: Two groups of experimental animals (Balb/c and NOD mice) received NSC23766, while the two control groups received equal volume of saline. Body weights and blood glucose were measured every week for 34 weeks. Rac1 activation in pancreatic islets was measured by GLISA activation assay. Rac1 and CHOP expression was determined by Western Blotting. Results: Our findings indicate that administration of NSC23766 significantly prevented the development of spontaneous diabetes in the NOD mice. Furthermore, NSC23766 markedly suppressed Rac1 expression and activity and the endoplasmic reticulum stress (CHOP expression) in NOD islets. Conclusions: Our findings provide the first evidence implicating the role of Tiam1-Rac1-Nox2 signaling pathway in the onset of spontaneous diabetes in the NOD mouse model.

It is well established that glucotoxicity (caused by high glucose concentrations; HG) underlies pathogenesis of islet dysfunction in diabetes. We have recently demonstrated that Nox2 plays a requisite role in the generation of reactive oxygen species (ROS) under HG conditions, resulting in mitochondrial dysregulation and loss of islet β-cell function. Herein, we investigated roles of Nox2 in the regulation of downstream stress kinase (p38MAPK) activation under HG conditions (20 mM; 24 h) in normal rodent islets and INS-1 832/13 cells. We observed that gp91-ds-tat, a specific inhibitor of Nox2, but not its inactive analog, significantly attenuated HG-induced Nox2 activation, ROS generation and p38MAPK activation, thus suggesting that Nox2 activation couples with p38MAPK activation. Since Rac1, is an integral member of the Nox2 holoenzyme, we also assessed the effects of Rac1 inhibitors (EHT 1864, NSC23766 and Ehop-016) on HG-induced p38MAPK activation in isolated β-cells. We report a significant inhibition of p38MAPK phosphorylation by Rac1 inhibitors, implying a regulatory role for Rac1 in promoting the Nox2-p38MAPK signaling axis in the β-cell under the duress of HG. 2-Bromopalmitate, a known inhibitor of protein (Rac1) palmitoylation, significantly reduced HG-induced p38MAPK phosphorylation. However, GGTI-2147, a specific inhibitor of geranylgeranylation of Rac1, failed to exert any significant effects on HG-induced p38MAPK activation. In conclusion, we present the first evidence that the Rac1-Nox2 signaling module plays novel regulatory roles in HG-induced p38MAPK activation and loss in glucose-stimulated insulin secretion (GSIS) culminating in metabolic dysfunction and the onset of diabetes.

Rho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells.Co-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA).Western blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016.Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

Glucose-stimulated insulin secretion (GSIS) in the pancreatic β-cells entails a variety of signaling mechanisms including activation of small GTP-binding proteins (G-proteins). Previous studies from our laboratory in human islets, rodent islets and clonal β-cells have demonstrated that G-proteins (e.g., Arf6, Cdc42 and Rac1) play novel roles in cytoskeletal remodeling, which is a critical step in the trafficking of insulin-laden secretory granules for fusion with plasma membrane and release of insulin. To further understand regulatory roles of Rac1 in GSIS, we utilized, herein, EHT 1864, a small molecule inhibitor, which attenuates Rac1 activation by retaining the G-protein in an inert/inactive state, thereby preventing activation of its downstream effector proteins. We demonstrate that EHT 1864 markedly attenuated GSIS in INS-1 832/13 cells. In addition, EHT 1864 significantly reduced glucose-induced activation and membrane targeting of Rac1 in INS-1 832/13 cells. This Rac1 inhibitor also suppressed glucose-induced activation of ERK1/2 and p53, but not Akt. Lastly, unlike the inhibitors of protein prenylation (simvastatin), EHT 1864 did not exert any significant effects on cell morphology (cell rounding) under the conditions it attenuated Rac1-sensitive signaling steps leading to GSIS. Based on these findings, we conclude that EHT 1864 specifically inhibits glucose-induced Rac1 activation and membrane association and associated downstream signaling events culminating in inhibition of GSIS.