Loss of ZnT8 function protects against diabetes by enhanced insulin secretion.
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ABSTRACT:
SUBMITTER: Dwivedi OP
PROVIDER: S-EPMC6858874 | biostudies-literature | 2019 Nov
REPOSITORIES: biostudies-literature
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Project description:In 12-month-old APOE targeted-replacement mice, we report that overall differences in gene expression were the most prominent when comparing the protective APOE2 to the other two alleles, with fewer differences found when comparing the risk-neutral APOE3 and disease-promoting APOE4 alleles. When compared with either APOE3 or APOE4, differential expression of genes within the endosomal pathways is a prominent feature of APOE2 expression in the brain. We hypothesized that the protective effects of APOE2 are mediated through the endosomal pathway during aging. In contrast to Alzheimer’s disease and APOE4 models, we detected normal morphology and abundance of early endosomes within cortical neurons of APOE2 targeted-replacement mice during aging despite decreased rab5b recruitment to early endosomes. Similarly, the morphology and abundance of retromer-associated vesicles was normal in APOE2 mice, despite reduced recruitment of vesicle-associated VPS35. Significantly, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, indicative of an enhanced endosomal cargo clearance to the extracellular space that contributes to a homeostatic balance of endosomal functions. Our findings thus demonstrate that APOE2 effectively offsets endosomal pathway changes during aging to preserve its integrity by enhancing exosome biogenesis, mitigating age-driven endosomal dysfunction that contributes to Alzheimer’s disease risk.
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Project description:Recent genome-wide association studies demonstrated that common variants of solute carrier family 30 member 8 gene (SLC30A8) increase susceptibility to type 2 diabetes. SLC30A8 encodes zinc transporter-8 (ZnT8), which delivers zinc ion from the cytoplasm into insulin granules. Although it is well known that insulin granules contain high amounts of zinc, the physiological role of secreted zinc remains elusive. In this study, we generated mice with β cell-specific Slc30a8 deficiency (ZnT8KO mice) and demonstrated an unexpected functional linkage between Slc30a8 deletion and hepatic insulin clearance. The ZnT8KO mice had low peripheral blood insulin levels, despite insulin hypersecretion from pancreatic β cells. We also demonstrated that a substantial amount of the hypersecreted insulin was degraded during its first passage through the liver. Consistent with these findings, ZnT8KO mice and human individuals carrying rs13266634, a major risk allele of SLC30A8, exhibited increased insulin clearance, as assessed by c-peptide/insulin ratio. Furthermore, we demonstrated that zinc secreted in concert with insulin suppressed hepatic insulin clearance by inhibiting clathrin-dependent insulin endocytosis. Our results indicate that SLC30A8 regulates hepatic insulin clearance and that genetic dysregulation of this system may play a role in the pathogenesis of type 2 diabetes.
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Project description:Background: Although several approaches have revealed much about individual factors that regulate pancreatic development, we have yet to fully understand their complicated interplay during pancreas morphogenesis. Gfi1 is transcription factor specifically expressed in pancreatic acinar cells, whose role in pancreas cells fate identity and specification is still elusive. Methods: In order to gain further insight into the function of this factor in the pancreas, we generated animals deficient for Gfi1 specifically in the pancreas. Gfi1 conditional knockout animals were phenotypically characterized by immunohistochemistry, RT-qPCR, and RNA scope. To assess the role of Gfi1 in the pathogenesis of diabetes, we challenged Gfi1-deficient mice with two models of induced hyperglycemia: long-term high-fat/high-sugar feeding and streptozotocin injections. Results: Interestingly, mutant mice did not show any obvious deleterious phenotype. However, in depth analyses demonstrated a significant decrease in pancreatic amylase expression, leading to a diminution in intestinal carbohydrates processing and thus glucose absorption. In fact, Gfi1-deficient mice were found resistant to diet-induced hyperglycemia, appearing normoglycemic even after long-term high-fat/high-sugar diet. Another feature observed in mutant acinar cells was the misexpression of ghrelin, a hormone previously suggested to exhibit anti-apoptotic effects on β-cells in vitro. Impressively, Gfi1 mutant mice were found to be resistant to the cytotoxic and diabetogenic effects of high-dose streptozotocin administrations, displaying a negligible loss of β-cells and an imperturbable normoglycemia. Conclusions: Together, these results demonstrate that Gfi1 could turn to be extremely valuable for the development of new therapies and could thus open new research avenues in the context of diabetes research.
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Project description:ObjectiveInsulin resistance develops in tandem with obesity. Ablating myostatin (Mstn) prevents obesity, so we investigated if Mstn deficiency could improve insulin sensitivity. A loss-of-function mutation (Mstn(Ln)) in either one or both alleles of the Mstn gene shows how Mstn deficiency protects whole-body insulin sensitivity.Research design and methodsMstn(Ln/Ln) mice were weaned onto a high-fat diet (HFD) or standard diet. HFD-fed Mstn(Ln/Ln) mice exhibited high lean, low-fat body compositions compared with wild types. Wild-type and heterozygous and homozygous mutant mice were bled to determine basal levels of insulin, glucose, and homeostasis model assessment of insulin resistance. To evaluate postprandial insulin sensitivity between animals of a similar size, glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamp studies were performed with heterozygous and homozygous mutant mice. Quantitative RT-PCR quantified TNF proportional, variant, IL-6, IL-1beta, F4/80, GPR43, and CD36 expression in muscle, fat, and liver. Histological analysis measured hepatosteatosis.ResultsHomozygous mutants were glucose tolerant and protected against overall insulin resistance compared with heterozygous mice. Hyperinsulinemic-euglycemic clamp studies revealed a dramatically improved glucose infusion rate, glucose disposal rate, and hepatic glucose production in 11-month-old Mstn(Ln/Ln) mice on an HFD. Improvements to muscle and liver insulin sensitivity (approximately 200-400%) correlated with 50-75% decreased tumor necrosis factor (TNF)alpha production and coincided with severe Mstn deficiency. Hepatosteatosis appeared to be ameliorated. Short-term treatment of Mstn(Ln/Ln) mice with recombinant Mstn led to increased plasma TNFalpha and insulin resistance.ConclusionsWe find that severe Mstn deficiency caused by Ln (lean) mutations in HFD-fed mice protects muscle and liver against obesity-induced insulin resistance.
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Project description:Xerostomia as a result of salivary gland damage is a permanent and debilitating side effect of radiotherapy for head and neck cancers. Effective treatments for protecting, or restoring, salivary gland function are not available. Here we report that irradiation treatment leads to activation of the calcium-permeable channel, transient potential melastatin-like 2 (TRPM2), via stimulation of poly-ADP-ribose polymerase. Importantly, irradiation induced an irreversible loss of salivary gland fluid secretion in TRPM2+/+ mice while a transient loss was seen in TRPM2-/- mice with >60% recovery by 30 days after irradiation. Treatment of TRPM2+/+ mice with the free radical scavenger Tempol or the PARP1 inhibitor 3-aminobenzamide attenuated irradiation-induced activation of TRPM2 and induced significant recovery of salivary fluid secretion. Furthermore, TPL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) induced complete recovery of function in irradiated TRPM2-/- mice. These novel data demonstrate that TRPM2 is activated by irradiation, via PARP1 activation, and contributes to irreversible loss of salivary gland function.
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Project description:AIMS/HYPOTHESIS:Zinc is highly concentrated in pancreatic beta cells, is critical for normal insulin storage and may regulate glucagon secretion from alpha cells. Zinc transport family member 8 (ZnT8) is a zinc efflux transporter that is highly abundant in beta cells. Polymorphisms of ZnT8 (also known as SLC30A8) gene in man are associated with increased risk of type 2 diabetes. While global Znt8 knockout (Znt8KO) mice have been characterised, ZnT8 is also present in other islet cell types and extra-pancreatic tissues. Therefore, it is important to find ways of understanding the role of ZnT8 in beta and alpha cells without the difficulties caused by the confounding effects of ZnT8 in these other tissues. METHODS:We generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8, and performed in vivo and in vitro characterisation of the phenotypes to determine the functional and anatomical impact of ZnT8 in these cells. Thus we assessed zinc accumulation, insulin granule morphology, insulin biosynthesis and secretion, and glucose homeostasis. RESULTS:Znt8BKO mice are glucose-intolerant, have reduced beta cell zinc accumulation and atypical insulin granules. They also display reduced first-phase glucose-stimulated insulin secretion, reduced insulin processing enzyme transcripts and increased proinsulin levels. In contrast, Znt8AKO mice show no evident abnormalities in plasma glucagon and glucose homeostasis. CONCLUSIONS/INTERPRETATION:This is the first report of specific beta and alpha cell deletion of Znt8. Our data indicate that while, under the conditions studied, ZnT8 is absolutely essential for proper beta cell function, it is largely dispensable for alpha cell function.
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Project description:The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo.
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Project description:Objective: The overall objective of this study was to investigate the effects of catalpol on bone remodeling of diabetic osteoporosis by regulating osteoblast differentiation and migration. Method: Using a murine model of diabetic osteoporosis, to detect the protective effects of catalpol on bone loss, architectural deterioration of trabecular bone and bone metabolism biomarkers were tested. A model of MC3T3-E1 cells was established by treatment with high glucose; the regulatory role of catalpol in the differentiation and migration was tested by Western blot, ALP staining, and Alizarin Red staining. Results: Catalpol treatment markedly ameliorated trabecular bone deterioration by reducing degenerative changes of the trabecular structure by improving the bone formation marker levels of ALP, osteopontin, type I collagen, and osteocalcin, as well as the level of OPG/RANKL. Catalpol enhanced cell motility and scattering following gap formation of MC3T3-E1 cells. Conclusion: The results indicated that catalpol exhibits a protective effect against diabetic osteoporosis by regulating the differentiation and migration of osteoblast.
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Project description:Polymorphisms in the SLC30A8 gene, which encodes the ZnT8 zinc transporter, are associated with altered susceptibility to type 2 diabetes (T2D), and SLC30A8 haploinsufficiency is protective against the development of T2D in obese humans. SLC30A8 is predominantly expressed in pancreatic islet ?-cells, but surprisingly, multiple knockout mouse studies have shown little effect of Slc30a8 deletion on glucose tolerance or glucose-stimulated insulin secretion (GSIS). Multiple other Slc30a isoforms are expressed at low levels in pancreatic islets. We hypothesized that functional compensation by the Slc30a7 isoform, which encodes ZnT7, limits the impact of Slc30a8 deletion on islet function. We therefore analyzed the effect of Slc30a7 deletion alone or in combination with Slc30a8 on in vivo glucose metabolism and GSIS in isolated islets. Deletion of Slc30a7 alone had complex effects in vivo, impairing glucose tolerance and reducing the glucose-stimulated increase in plasma insulin levels, hepatic glycogen levels, and pancreatic insulin content. Slc30a7 deletion also affected islet morphology and increased the ratio of islet ?- to ?-cells. However, deletion of Slc30a7 alone had no effect on GSIS in isolated islets, whereas combined deletion of Slc30a7 and Slc30a8 abolished GSIS. These data demonstrate that the function of ZnT8 in islets can be unmasked by removal of ZnT7 and imply that ZnT8 may affect T2D susceptibility through actions in other tissues where it is expressed at low levels rather than through effects on pancreatic islet function.
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