Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis.

Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and ß-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged ß-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

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.

A Novel Receivership Model for Transition of Young Adults With Diabetes: Experience From a Single-center Academic Transition Program.

OBJECTIVE: The transition from pediatric to adult care for young adults with diabetes represents an important but often challenging time characterized by a shift from a family-centered care model of pediatrics to a patient-centered care model of adult medicine. We developed a structured transition program based on an adult receivership model at a large academic medical center to improve care coordination and patient satisfaction with the transition process. METHODS: From 2016 to 2020, we implemented a series of quality improvement efforts for young adults aged 18 to 23 years with diabetes by incorporating best practices from the American Diabetes Association guidelines on care for emerging adults. We measured transition orientation attendance, patient satisfaction, hemoglobin A1c (HbA1c) pre- and post-transfer, and care gaps to determine the impact of the program. RESULTS: In this study, 307 individuals with type 1 diabetes and 16 individuals with type 2 diabetes were taken care of by the adult endocrinology department at the University of Michigan between January 1, 2016 and October 31, 2020. We observed high attendance rates (86% among internal transfers) and favorable patient satisfaction scores for the transition orientation session. Despite the glycemic challenges posed during the transition, HbA1c modestly yet significantly improved 1-year after transfer (-0.4%, P < .01). CONCLUSION: We successfully established and maintained a young adult diabetes transition program using a quality improvement approach. Future work will focus on reducing care gaps at the time of transfer, assessing long-term retention rates, and enhancing care coordination for patients referred from outside the health network.

A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic ß-Cells.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional ß-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in ß-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify ß-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for ß-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in ß-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous ß-cell models.

Protein disorder in the regulatory control of mitophagy.

Mitophagy is a central component of the mitochondrial quality control machinery, which is necessary for cellular viability and bioenergetics. The E3 ubiquitin ligase CLEC16A (C-type lectin domain containing 16A) forms a tripartite mitophagy regulatory complex together with the E3 ligase RNF41 (ring finger protein 41) and the ubiquitin-specific peptidase USP8 (ubiquitin specific peptidase 8), yet CLEC16A structural/functional domains relevant for mitophagy are unknown. We identify that CLEC16A contains an internal intrinsically disordered region (IDR), which is important for CLEC16A function and stability. IDRs are flexible domains lacking fixed secondary structure and regulate an emerging number of diverse processes, yet they have been largely unstudied in mitophagy. We observe that the internal CLEC16A IDR is essential for CLEC16A degradation and is bound by RNF41 to promote CLEC16A turnover. This IDR also promotes assembly of the CLEC16A-RNF41-USP8 mitophagy regulatory complex. Thus, our study revealed the importance of IDRs in mitophagy via the regulation of CLEC16A abundance by RNF41, opening new structural insights into mitochondrial quality control.

Mitochondrial metabolism and dynamics in pancreatic beta cell glucose sensing.

Glucose-regulated insulin secretion becomes defective in all forms of diabetes. The signaling mechanisms through which the sugar acts on the ensemble of beta cells within the islet remain a vigorous area of research after more than 60 years. Here, we focus firstly on the role that the privileged oxidative metabolism of glucose plays in glucose detection, discussing the importance of 'disallowing' in the beta cell the expression of genes including Lactate dehydrogenase (Ldha) and the lactate transporter Mct1/Slc16a1 to restrict other metabolic fates for glucose. We next explore the regulation of mitochondrial metabolism by Ca2+ and its possible role in sustaining glucose signaling towards insulin secretion. Finally, we discuss in depth the importance of mitochondrial structure and dynamics in the beta cell, and their potential for therapeutic targeting by incretin hormones or direct regulators of mitochondrial fusion. This review, and the 2023 Sir Philip Randle Lecture which GAR will give at the Islet Study Group meeting in Vancouver, Canada in June 2023, honor the foundational, and sometimes under-appreciated, contributions made by Professor Randle and his colleagues towards our understanding of the regulation of insulin secretion.

Evaluation of polygenic risk scores to differentiate between type 1 and type 2 diabetes.

Polygenic risk scores (PRS) quantify the genetic liability to disease and are calculated using an individual's genotype profile and disease-specific genome-wide association study (GWAS) summary statistics. Type 1 (T1D) and type 2 (T2D) diabetes both are determined in part by genetic loci. Correctly differentiating between types of diabetes is crucial for accurate diagnosis and treatment. PRS have the potential to address possible misclassification of T1D and T2D. Here we evaluated PRS models for T1D and T2D in European genetic ancestry participants from the UK Biobank (UKB) and then in the Michigan Genomics Initiative (MGI). Specifically, we investigated the utility of T1D and T2D PRS to discriminate between T1D, T2D, and controls in unrelated UKB individuals of European ancestry. We derived PRS models using external non-UKB GWAS. The T1D PRS model with the best discrimination between T1D cases and controls (area under the receiver operator curve [AUC] = 0.805) also yielded the best discrimination of T1D from T2D cases in the UKB (AUC = 0.792) and separation in MGI (AUC = 0.686). In contrast, the best T2D model did not discriminate between T1D and T2D cases (AUC = 0.527). Our analysis suggests that a T1D PRS model based on independent single nucleotide polymorphisms may help differentiate between T1D, T2D, and controls in individuals of European genetic ancestry.

Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.

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.

An intrinsically disordered protein region encoded by the human disease gene CLEC16A regulates mitophagy.

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.

ß-Cell Cre Expression and Reduced Ins1 Gene Dosage Protect Mice From Type 1 Diabetes.

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 ß 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 ß-cell specificity with no reported off-target effects. We explored whether NOD:Ins1Cre mice could be used to investigate ß-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 ß-cell-selective Cre in NOD mice.

Mitofusins Mfn1 and Mfn2 Are Required to Preserve Glucose- but Not Incretin-Stimulated ß-Cell Connectivity and Insulin Secretion.

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic ß-cells. Whether mitofusin gene expression, and hence, mitochondrial network integrity, is important for glucose or incretin signaling has not previously been explored. Here, we generated mice with ß-cell-selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (ßMfn1/2 dKO). ßMfn1/2-dKO mice displayed elevated fed and fasted glycemia and a more than fivefold decrease in plasma insulin. Mitochondrial length, glucose-induced polarization, ATP synthesis, and cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in ßMfn1/2-dKO mice, and glucagon-like peptide 1 or glucose-dependent insulinotropic peptide receptor agonists largely corrected defective glucose-stimulated insulin secretion through enhanced EPAC-dependent signaling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps-based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the ß-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in ß-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process.

Mitofusin 1 and 2 regulation of mitochondrial DNA content is a critical determinant of glucose homeostasis.

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 ß-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 ß-cells. Thus, the primary physiologic role of Mfn1 and 2 in ß-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.

New Aspects of Diabetes Research and Therapeutic Development.

Both type 1 and type 2 diabetes mellitus are advancing at exponential rates, placing significant burdens on health care networks worldwide. Although traditional pharmacologic therapies such as insulin and oral antidiabetic stalwarts like metformin and the sulfonylureas continue to be used, newer drugs are now on the market targeting novel blood glucose-lowering pathways. Furthermore, exciting new developments in the understanding of beta cell and islet biology are driving the potential for treatments targeting incretin action, islet transplantation with new methods for immunologic protection, and the generation of functional beta cells from stem cells. Here we discuss the mechanistic details underlying past, present, and future diabetes therapies and evaluate their potential to treat and possibly reverse type 1 and 2 diabetes in humans. SIGNIFICANCE STATEMENT: Diabetes mellitus has reached epidemic proportions in the developed and developing world alike. As the last several years have seen many new developments in the field, a new and up to date review of these advances and their careful evaluation will help both clinical and research diabetologists to better understand where the field is currently heading.

Colorectal cancer cells utilize autophagy to maintain mitochondrial metabolism for cell proliferation under nutrient stress.

Cancer cells reprogram cellular metabolism to maintain adequate nutrient pools to sustain proliferation. Moreover, autophagy is a regulated mechanism to break down dysfunctional cellular components and recycle cellular nutrients. However, the requirement for autophagy and the integration in cancer cell metabolism is not clear in colon cancer. Here, we show a cell-autonomous dependency of autophagy for cell growth in colorectal cancer. Loss of epithelial autophagy inhibits tumor growth in both sporadic and colitis-associated cancer models. Genetic and pharmacological inhibition of autophagy inhibits cell growth in colon cancer-derived cell lines and patient-derived enteroid models. Importantly, normal colon epithelium and patient-derived normal enteroid growth were not decreased following autophagy inhibition. To couple the role of autophagy to cellular metabolism, a cell culture screen in conjunction with metabolomic analysis was performed. We identified a critical role of autophagy to maintain mitochondrial metabolites for growth. Loss of mitochondrial recycling through inhibition of mitophagy hinders colon cancer cell growth. These findings have revealed a cell-autonomous role of autophagy that plays a critical role in regulating nutrient pools in vivo and in cell models, and it provides therapeutic targets for colon cancer.

A Selective Look at Autophagy in Pancreatic ß-Cells.

Insulin-producing pancreatic ß-cells are central to glucose homeostasis, and their failure is a principal driver of diabetes development. To preserve optimal health ß-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 ß-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 ß-cell health. We examine the unique molecular players underlying each distinct form of autophagy in ß-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 ß-cell selective autophagy may inform new approaches to defend against the various insults leading to ß-cell failure in diabetes.

Cell Cycle Regulation of the Pdx1 Transcription Factor in Developing Pancreas and Insulin-Producing ß-Cells.

Current evidence indicates that proliferating ß-cells express lower levels of some functional cell identity genes, suggesting that proliferating cells are not optimally functional. Pdx1 is important for ß-cell specification, function, and proliferation and is mutated in monogenic forms of diabetes. However, its regulation during the cell cycle is unknown. Here we examined Pdx1 protein expression in immortalized ß-cells, maternal mouse islets during pregnancy, and mouse embryonic pancreas. We demonstrate that Pdx1 localization and protein levels are highly dynamic. In nonmitotic cells, Pdx1 is not observed in constitutive heterochromatin, nucleoli, or most areas containing repressive epigenetic marks. At prophase, Pdx1 is enriched around the chromosomes before Ki67 coating of the chromosome surface. Pdx1 uniformly localizes in the cytoplasm at prometaphase and becomes enriched around the chromosomes again at the end of cell division, before nuclear envelope formation. Cells in S phase have lower Pdx1 levels than cells at earlier cell cycle stages, and overexpression of Pdx1 in INS-1 cells prevents progression toward G2, suggesting that cell cycle-dependent regulation of Pdx1 is required for completion of mitosis. Together, we find that Pdx1 localization and protein levels are tightly regulated throughout the cell cycle. This dynamic regulation has implications for the dichotomous role of Pdx1 in ß-cell function and proliferation.

Adrenal metastasis as the initial diagnosis of synchronous papillary and follicular thyroid cancer.

BACKGROUND: Differentiated thyroid cancer uncommonly presents with distant metastases. Adrenal metastasis from differentiated thyroid cancer presenting as the initial finding is even less common. CASE PRESENTATION: A 71-year-old male was incidentally found on chest CT to have bilateral thyroid nodules, which were confirmed on ultrasound. Fine needle aspiration of the dominant right 3.3 cm nodule contained histologic features most consistent with Bethesda classification III, and repeat fine needle aspiration revealed pathology consistent with Bethesda classification II. Follow-up thyroid ultrasound showed 1% increase and 14% increase in nodule volume at one and two years, respectively, compared to baseline. Prior to the second annual thyroid ultrasound, the patient was incidentally found to have a 4.1 cm heterogeneously enhancing mass in the right adrenal gland on CT of the abdomen and pelvis. Biochemical evaluation was unremarkable with the exception of morning cortisol of 3.2 µg/dL after dexamethasone suppression. The patient then underwent laparoscopic right adrenal gland excision, which revealed metastatic follicular thyroid carcinoma. Total thyroidectomy was then performed, with pathology showing a 4.8 cm well-differentiated follicular thyroid carcinoma of the right lobe, a 0.5 cm noninvasive follicular thyroid neoplasm with papillary-like nuclear features of the left lobe, and a 0.1 cm papillary microcarcinoma of the left lobe. Thyrotropin-stimulated whole body scan showed normal physiologic uptake of the remnant thyroid tissue without evidence of other iodine avid disease. The patient then received radioactive iodine. At follow-up 14 months after total thyroidectomy, he remains free of recurrent disease. CONCLUSION: Despite following the recommended protocol for evaluation and surveillance of thyroid nodules, thyroid cancer can be challenging to diagnose, and may not be diagnosed until distant metastases are identified.