Age is a risk factor for type 2 diabetes (T2D). We aimed to elucidate whether β-cell glucose metabolism is altered with aging and contributes to T2D.
We used senescence-accelerated mice (SAM), ...C57BL/6J (B6) mice, and ob/ob mice as aging models. As a diabetes model, we used db/db mice. The glucose responsiveness of insulin secretion and the U-13C-glucose metabolic flux were examined in isolated islets. We analyzed the expression of β-cell-specific genes in isolated islets and pancreatic sections as molecular signatures of β-cell identity. β cells defective in the malate-aspartate (MA) shuttle were previously generated from MIN6-K8 cells by the knockout of Got1, a component of the shuttle. We analyzed Got1 KO β cells as a model of increased glycolysis.
We identified hyperresponsiveness to glucose and compromised cellular identity as dysfunctional phenotypes shared in common between aged and diabetic mouse β cells. We also observed a metabolic commonality between aged and diabetic β cells: hyperactive glycolysis through the increased expression of nicotinamide mononucleotide adenylyl transferase 2 (Nmnat2), a cytosolic nicotinamide adenine dinucleotide (NAD)-synthesizing enzyme. Got1 KO β cells showed increased glycolysis, β-cell dysfunction, and impaired cellular identity, phenocopying aging and diabetes. Using Got1 KO β cells, we show that attenuation of glycolysis or Nmnat2 activity can restore β-cell function and identity.
Our study demonstrates that hyperactive glycolysis is a metabolic signature of aged and diabetic β cells, which may underlie age-related β-cell dysfunction and loss of cellular identity. We suggest Nmnat2 suppression as an approach to counteract age-related T2D.
Display omitted
•Glucose hypersensitivity and impaired identity are common features of aged and diabetic β cells.•Metabolic tracing reveals increased glycolysis and altered NAD production in aged and diabetic β cells.•Increased glycolysis induces β-cell dysfunction and loss of identity.•NAD production by Nmnat2 can be targeted to restore β-cell phenotypes.
Diabetic peripheral neuropathy (DPN) includes symptoms of thermosensory impairment, which are reported to involve changes in the expression or function, or both, of nociceptive TRPV1 and TRPA1 ...channels in rodents. In the present study, we did not find changes in the expression or function of TRPV1 or TRPA1 in DPN mice caused by STZ, although thermal hypoalgesia was observed in a murine model of DPN or TRPV1
mice with a Plantar test, which specifically detects temperature avoidance. With a Thermal Gradient Ring in which mice can move freely in a temperature gradient, temperature preference can be analyzed, and we clearly discriminated the temperature-dependent phenotype between DPN and TRPV1
mice. Accordingly, we propose approaches with multiple behavioral methods to analyze the progression of DPN by response to thermal stimuli. Attention to both thermal avoidance and preference may provide insight into the symptoms of DPN.
Incretins (GLP-1 and GIP) potentiate insulin secretion through cAMP signaling in pancreatic β-cells in a glucose-dependent manner. We recently proposed a mechanistic model of incretin-induced insulin ...secretion (IIIS) that requires two critical processes: 1) generation of cytosolic glutamate through the malate-aspartate (MA) shuttle in glucose metabolism and 2) glutamate transport into insulin granules by cAMP signaling to promote insulin granule exocytosis. To directly prove the model, we have established and characterized CRISPR/Cas9-engineered clonal mouse β-cell lines deficient for the genes critical in these two processes: aspartate aminotransferase 1 (AST1, gene symbol Got1), a key enzyme in the MA shuttle, which generates cytosolic glutamate, and the vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3, gene symbol Slc17a7, Slc17a6, and Slc17a8, respectively), which participate in glutamate transport into secretory vesicles. Got1 knockout (KO) β-cell lines were defective in cytosolic glutamate production from glucose and showed impaired IIIS. Unexpectedly, different from the previous finding that global Slc17a7 KO mice exhibited impaired IIIS from pancreatic islets, β-cell specific Slc17a7 KO mice showed no significant impairment in IIIS, as assessed by pancreas perfusion experiment. Single Slc17a7 KO β-cell lines also retained IIIS, probably due to compensatory upregulation of Slc17a6. Interestingly, triple KO of Slc17a7, Slc17a6, and Slc17a8 diminished IIIS, which was rescued by exogenously introduced wild-type Slc17a7 or Slc17a6 genes. The present study provides direct evidence for the essential roles of AST1 and VGLUTs in β-cell glutamate signaling for IIIS and also shows the usefulness of the CRISPR/Cas9 system for studying β-cells by simultaneous disruption of multiple genes.
Aims
Muscle atrophy is a diabetic complication, which results in a deterioration in glycemic control in type 2 diabetes mellitus (T2DM) individuals. The psoas muscle mass index (PMI) is a reliable ...indicator for estimating whole-body muscle mass. We aimed to examine the relationship between clinical parameters and the PMI to clarify the mechanism underlying muscle atrophy in diabetes.
Methods
This retrospective, cross-sectional study examined 51 patients (31 men and 20 women) with T2DM and a mean HbA1c value of 9.9 ± 1.7%. These patients were admitted to Aichi Medical University Hospital and underwent abdominal computed tomography imaging from July 2020 to April 2021. Multiple clinical parameters were assessed with the PMI.
Results
In a multiple regression analysis adjusted for age and sex, the PMI was correlated with body weight, body mass index, serum concentrations of corrected calcium, aspartate aminotransferase, alanine aminotransferase, creatine kinase, thyroid-stimulating hormone (TSH), urinary C-peptide concentrations, the free triiodothyronine/free thyroxine (FT3/FT4) ratio, and the young adult mean score at the femur neck. Receiver operating characteristic curves were created using TSH concentrations and the FT3/FT4 ratio for diagnosing a low PMI. The area under the curve was 0.593 and 0.699, respectively. The cut-off value with maximum accuracy for TSH concentrations was 1.491 μIU/mL, sensitivity was 56.1%, and specificity was 80.0%. Corresponding values for the FT3/FT4 ratio were 1.723, 78.0, and 66.7%, respectively.
Conclusion
TSH concentrations and the FT3/FT4 ratio are correlated with the PMI, and their thresholds may help prevent muscle mass loss in Japanese individuals with T2DM.
We present a case of a 32‐year‐old diabetic woman with Prader–Willi syndrome who developed severe ketoacidosis caused by a sodium‐glucose cotransporter 2 (SGLT2) inhibitor, a novel class of ...antihyperglycemic agents, during a strict low‐carbohydrate diet. At admission, a serum glucose level of 191 mg/dL was relatively low, though laboratory evaluations showed severe ketoacidosis. This is the first report of ketoacidosis caused by a SGLT2 inhibitor. It is necessary to not only pay attention when using a SGLT2 inhibitor in patients following a low‐carbohydrate diet, but also to start a low‐carbohydrate diet in patients treated with a SGLT2 inhibitor because of a high risk for developing ketoacidosis.
This is the first report of ketoacidosis caused by a SGLT2 inhibitor. It is necessary to pay attention not only to use a SGLT2 inhibitor in the patients during a low‐carbohydrate diet but also to start a low‐carbohydrate diet in the patients treated with a SGLT2 inhibitor because of a high risk for developing ketoacidosis.
Aims/Introduction
Pancreatic islets are heterogenous. To clarify the relationship between islet heterogeneity and incretin action in the islets, we studied gene expression and metabolic profiles of ...non‐large and enlarged islets of the Zucker fatty diabetes mellitus rat, an obese diabetes model, as well as incretin‐induced insulin secretion (IIIS) in these islets.
Materials and Methods
Pancreatic islets of control (fa/+) and fatty (fa/fa) rats at 8 and 12 weeks‐of‐age were isolated. The islets of fa/fa rats at 12 weeks‐of‐age were separated into non‐large islets (≤200 μm in diameter) and enlarged islets (>300 μm in diameter). Morphological analyses, insulin secretion experiments, transcriptome analysis, metabolome analysis and oxygen consumption analysis were carried out on these islets.
Results
The number of enlarged islets was increased with age in fatty rats, and IIIS was significantly reduced in the enlarged islets. Markers for β‐cell differentiation were markedly decreased in the enlarged islets, but those for cell proliferation were increased. Glycolysis was enhanced in the enlarged islets, whereas the tricarboxylic acid cycle was suppressed. The oxygen consumption rate under glucose stimulation was reduced in the enlarged islets. Production of glutamate, a key signal for IIIS, was decreased in the enlarged islets.
Conclusions
The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for IIIS, show tumor cell‐like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age‐dependent increase in such islets could contribute to the pathophysiology of obese diabetes.
The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for incretin‐induced insulin secretion, show tumor cell‐like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age‐dependent increase in such islets could contribute to the pathophysiology of obese diabetes.
Aims/Introduction
A high‐carbohydrate diet is known to increase insulin secretion and induce obesity. However, whether or not a high‐carbohydrate diet affects β‐cell mass (BCM) has been little ...investigated.
Materials and Methods
Both wild‐type (WT) mice and adenosine triphosphate‐sensitive potassium channel‐deficient (Kir6.2KO) mice were fed normal chow or high‐starch (ST) diets for 22 weeks. BCM and the numbers of islets were analyzed by immunohistochemistry, and gene expression levels in islets were investigated by quantitative real‐time reverse transcription polymerase chain reaction. MIN6‐K8 β‐cells were stimulated in solution containing various concentrations of glucose combined with nifedipine and glimepiride, and gene expression was analyzed.
Results
Both WT and Kir6.2KO mice fed ST showed hyperinsulinemia and body weight gain. BCM, the number of islets and the expression levels of cyclinD2 messenger ribonucleic acid were increased in WT mice fed ST compared with those in WT mice fed normal chow. In contrast, no significant difference in BCM, the number of islets or the expression levels of cyclinD2 messenger ribonucleic acid were observed between Kir6.2KO mice fed normal chow and those fed ST. Incubation of MIN6‐K8 β‐cells in high‐glucose media or with glimepiride increased cyclinD2 expression, whereas nifedipine attenuated a high‐glucose‐induced increase in cyclinD2 expression.
Conclusions
These results show that a high‐starch diet increases BCM in an adenosine triphosphate‐sensitive potassium channel‐dependent manner, which is mediated through upregulation of cyclinD2 expression.
High starch diet increases β‐cell mass due to increasing the number of islets in a KATP channel‐dependent manner.
Aims
We aimed to identify patients who would benefit from basal insulin-supported oral therapy (BOT) with a glinide and an α-glucosidase inhibitor (a fixed-dose combination tablet of mitiglinide ...10 mg and voglibose 0.2 mg) in Japanese type 2 diabetic patients.
Methods
Patients who were hospitalized to improve hyperglycemia received basal–bolus insulin therapy. After the reduction of glucose toxicity, a 75 g oral glucose tolerance test and a glucagon test were performed. Thereafter, the basal–bolus insulin therapy was switched to BOT with mitiglinide, followed by further addition of voglibose. Interstitial glucose levels were continuously monitored throughout the study period. Diurnal glucose profile was recorded and analyzed. Patients were divided into two groups according to whether their percentage of time in range (TIR, 70–180 mg/dL) under BOT with mitiglinide/voglibose was higher than 70% or not, and the differences in clinical characteristics between the groups were analyzed.
Results
Twenty patients were enrolled, and 19 of them completed the study. BOT with mitiglinide/voglibose achieved ≥ 70% of TIR in thirteen patients. The area under the curve of serum C-peptide levels during the oral glucose tolerance test was significantly higher in the patients with ≥ 70% of TIR. The daily insulin dosages and blood glucose profiles were comparable between the two groups.
Conclusions
The efficacy of BOT with mitiglinide/voglibose depended on residual insulin secretory abilities. This therapy would be a useful therapeutic option for patients with type 2 diabetes.
Diabetes is a major risk factor for atherosclerosis and ischemic vascular diseases. Recently, regenerative medicine is expected to be a novel therapy for ischemic diseases. Our previous studies have ...reported that transplantation of stem cells promoted therapeutic angiogenesis for diabetic neuropathy and ischemic vascular disease in a paracrine manner, but the precise mechanism is unclear. Therefore, we examined whether secreted factors from stem cells had direct beneficial effects on endothelial cells to promote angiogenesis. The soluble factors were collected as conditioned medium (CM) 48 h after culturing stem cells from human exfoliated deciduous teeth (SHED) in serum-free DMEM. SHED-CM significantly increased cell viability of human umbilical vein endothelial cells (HUVECs) in MTT assays and accelerated HUVECs migration in wound healing and Boyden chamber assays. In a Matrigel plug assay of mice, the migrated number of primary endothelial cells was markedly increased in the plug containing SHED-CM or SHED suspension. SHED-CM induced complex tubular structures of HUVECs in a tube formation assay. Furthermore, SHED-CM significantly increased neovascularization from the primary rat aorta, indicating that SHED-CM stimulated primary endothelial cells to promote comprehensive angiogenesis processes. The angiogenic effects of SHED-CM were the same or greater than the effective concentration of VEGF. In conclusion, SHED-CM directly stimulates vascular endothelial cells to promote angiogenesis and is promising for future clinical application.
Morphological analysis of peripheral nerves in mouse models can be used to characterize the pathophysiology of peripheral nerve disease, but obtaining high-quality electron micrographs can be ...challenging. Here, we present a protocol to obtain electron micrographs of mouse peripheral nerves. We detail the procedures of sampling, fixation, and embedding of peripheral nerves. We then outline the steps for ultrathin sectioning and transmission electron microscopy imaging. Finally, we describe morphological evaluation of nerve fibers in these images using ImageJ and AxonSeg.
For complete details on the use and execution of this protocol, please refer to Nakai-Shimoda et al. (2021).
Display omitted
•Morphological analysis of mouse peripheral nerves using transmission electron microscopy•Step-by-step guide for sampling, fixation, and embedding of peripheral nerves•Ultrathin sectioning by glass knife and imaging using transmission electron microscopy•Image analysis of unmyelinated and myelinated nerve fibers using ImageJ and AxonSeg
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Morphological analysis of peripheral nerves in mouse models can be used to characterize the pathophysiology of peripheral nerve disease, but obtaining high-quality electron micrographs can be challenging. Here, we present a protocol to obtain electron micrographs of mouse peripheral nerves. We detail the procedures of sampling, fixation, and embedding of peripheral nerves. We then outline the steps for ultrathin sectioning and transmission electron microscopy imaging. Finally, we describe morphological evaluation of nerve fibers in these images using ImageJ and AxonSeg.
PDF
