Regulatory Effects of Astragaloside IV on Hyperglycemia-Induced Mitophagy in Schwann Cells

Objective. This study aimed to observe the regulatory effects of astragaloside IV (AS-IV) on hyperglycemia-induced mitochondrial damage and mitophagy in Schwann cells and to provide references for clinical trials on AS-IV in the treatment of diabetic peripheral neuropathy. Methods. Schwann cells were grown in a high-glucose medium to construct an autophagy model; the cells were then treated with AS-IV and N-acetylcysteine (control) to observe the regulatory effects of AS-IV on oxidative stress and mitophagy. Results. AS-IV exhibited antioxidant activity and inhibited the overactivation of autophagy in Schwann cells, significantly reducing the level of reactive oxygen species and downregulating the expression of autophagy-related proteins (LC3, PINK, and Parkin) under hyperglycemic conditions, thereby exerting a protective effect on mitochondrial morphology and membrane potential. Conclusion. AS-IV can maintain the mitochondrial function of Schwann cells under hyperglycemic conditions by effectively alleviating oxidative stress and overactivation of mitophagy. The evidence from this study supports an AS-IV-based therapeutic strategy against diabetic peripheral neuropathy.

Cytokeratin 8 Inhibits Hepatic Glycogen Synthesis in Type 2 Diabetes Mellitus by Modulating Insulin-dependent IRS1/PI3K/Akt-GSK3β Pathway

Purpose: Cytokeratin 8(CK8) is a cytoskeletal protein mainly expressed in the liver. Recent studies have found that CK8 was closely related to glycogen synthesis. However, the role and the underlying mechanisms of CK8 in hepatic glycogen synthesis in type 2 diabetes mellitus (T2DM) have remained to be fully elucidated. Therefore, this study aimed to investigate the effects and the underlying mechanisms of CK8 on hepatic glycogen synthesis in T2DM.Methods The T2D mouse model was constructed by high energy feed of 8-10 weeks old C57BL/6J male mice. The model was validated by OGTT test and ITT test. The liver samples of T2DM patients were collected and the expression levels of CK8, IRS1, PI3K, Akt, GSK3β, p-PI3K p-Akt and p-GSK3β were determined by Western blotting. Then, at the cellular level the murine NCTC 1469 cells were used, and to up-regulate and down-regulate the CK8 gene the overexpression plasmid was constructed and transfected and RNA interference technology was applied, and CK8, IRS1, PI3K, Akt, GSK3β, p-IRS1, p-PI3K p-Akt and p-GSK3β were detected by Western blotting. Immunohistochemistry was used to detect the level of glycogen synthase (GS) and glycogen staining experiments was performed by PAS. At the animal level, the T2D mouse model was used to up-regulate and down-regulate the CK8 gene using an adenovirus vector. The levels of CK8, PI3K, Akt, GSK3β, p-PI3K, p-Akt and p-GSK3β in rat liver were detected by Western blotting, immunohistochemistry was used to detect the level of GS and glycogen staining experiments was performed by PAS.Results The expression levels of IRS1, p-PI3K, p-Akt and p-GSK3β were significantly higher, while the expression levels of CK8 was significantly lower in control group than in the liver of T2D mice or T2DM patients. Upregulation of CK8 in murine NCTC 1469 cells treated with high-glucose medium, we found IRS1, p-IRS1, p-PI3K, p-Akt and p-GSK3β were significantly decreased, the level of GS was significantly decreased, and glycogen synthesis was inhibited compared with NCTC1469 cells transfected with empty vector. Downregulation of CK8 in murine NCTC 1469 cells murine treated with high-glucose medium, we found IRS1, p-IRS1, p-PI3K, p-Akt and p-GSK3β were significantly higher, the level of GS was significantly increased, and glycogen synthesis was promoted compared with NCTC1469 cells transfected with sh-NC. Upregulation of CK8 in the T2D mouse model, we found p-PI3K, p-Akt and p-GSK3β were significantly lower compared with T2D mouse model transfected with empty vector. The level of GS was significantly decreased, and glycogen synthesis was inhibited. Downregulation of CK8 in the T2D mouse model, we found p-PI3K, p-Akt and p-GSK3β were significantly increased, and the level of GS was significantly increased, and glycogen synthesis was promoted compared with T2D mouse model transfected with sh-NC.Conclusions Overall, this experiment provides a new molecular target for the treatment of T2DM by revealing the role of CK8 inhibiting hepatic glycogen synthesis in T2DM via regulating insulin-dependent IRS1/PI3K-Akt-GSK3β pathway. CK8 may play an important role in the pathogenesis of glycogen synthesis in T2DM.

Excess Glucose Impedes the Proliferation of Skeletal Muscle Satellite Cells Under Adherent Culture Conditions

Glucose is a major energy source consumed by proliferating mammalian cells. Therefore, in general, proliferating cells have the preference of high glucose contents in extracellular environment. Here, we showed that high glucose concentrations impede the proliferation of satellite cells, which are muscle-specific stem cells, under adherent culture conditions. We found that the proliferation activity of satellite cells was higher in glucose-free DMEM growth medium (low-glucose medium with a glucose concentration of 2 mM) than in standard glucose DMEM (high-glucose medium with a glucose concentration of 19 mM). Satellite cells cultured in the high-glucose medium showed a decreased population of reserve cells, identified by staining for Pax7 expression, suggesting that glucose concentration affects cell fate determination. In conclusion, glucose is a factor that decides the cell fate of skeletal muscle-specific stem cells. Due to this unique feature of satellite cells, hyperglycemia may negatively affect the regenerative capability of skeletal muscle myofibers and thus facilitate sarcopenia.

CircRNA-DAPK1 promotes vascular cell pyroptosis in diabetes by regulating the circ-DAPK1/miR-4454/TXNIP axis.

Background Circular RNAs have been demonstrated to play an important role in the development of vascular diseases. However, little is known about the role of circ-021774, also named circ-DAPK1, in vascular cell pyroptosis. Methods Circ-DAPK1 was selected from circular RNA sequencing data of HUVECs treated with high glucose medium and normal medium. RT-qPCR was used to determine the expression of circ-DAPK1 in vivo and in vitro. Dual luciferase reporter assay, fluorescence in situ hybridization (FISH) and RNA immunoprecipitation (RIP) were performed to prove the interaction of circ-DAPK1, miRNA-4454 and thioredoxin-interactingprotein (TXNIP). Adeno-associated virus (AAV) was injected intravenously to establish mouse models. PI staining, western-blot and transmission electron microscopy (TEM) analyses were performed to identify the role of circ-DAPK1 in promoting pyroptosis. Results We found that circ-DAPK1 was highly expressed in high glucose medium cultured HUVECs and db/db mice. In vitro and in vivo experiments demonstrated that circ-DAPK1 knockdown decreased the number of PI+ cells, the expression of ASC, NLRP3, GSDMD-N, cleaved caspase-1, IL-18 and IL-1β. In a mechanistic study, the circ-DAPK1/miRNA-4454/TXNIP signaling axis was demonstrated to promote vascular cell pyroptosis in diabetes. Conclusions Circ-DAPK1 functions as a promoter of vascular cell pyroptosis in diabetes via the circ-DAPK1/miRNA-4454/TXNIP signaling axis.

Cinnamon Extract Changes the Expression of miRNAs26-b, 29a, 223 and320 in Insulin Resistant Adipocytes

Objective: Insulin resistance (IR) is the major cause in Type 2 diabetes mellitus (T2DM). Expression of some miRNAs can be changed in response to a drug treatment for IR, and used as the biomarker in IR. This study set out to determine the effect of cinnamon extract (cinnamaldehyde) on some miRNAs expression in IR adipocytes. Materials and Methods: In this In-vitro study the 3T3L1 cells were expanded in Dulbecco’s modified Eagle’s medium (DMEM), differentiated into adipocytes phenotype and insulin resistant with high glucose medium, then the cells were treated with cinnamaldehyde. To determine of the miRNAs profiling in 3T3L1 adipocytes, insulin-resistant adipocytes and treated insulin-resistant adipocytes quantitative real-time PCR method was performed. Results: IR adipocytes exhibited a significantly increase in miRs 29a, 223 and 320 expression, and decrease in miR26-b expression in compare to the normal adipocytes (P-value<0.001 and P-value= 0.024 respectively). However in response to cinnamaldehyde in IR adipocytes, expression of miRs 29a, 223 and 320 were down-regulated while expression of miR26-b was up-regulated neared it to the normal level (P-value= 0.003 and P-value= 0.002 respectively). Conclusion: IR changes expression of intended miRs, so that cinnamaldehyde treatment helps to improve and normalize the changes. Cinnamon as the herbal product can be helpful for IR particular in adipose tissue.

High Glucose Induces the Loss of Retinal Pericytes Partly via NLRP3-Caspase-1-GSDMD-Mediated Pyroptosis

Diabetic retinopathy (DR) is one of the hallmark complications of diabetes and a leading cause of vision loss in adults. Retinal pericyte death seems to be a prominent feature in the onset of DR. Pyroptosis is an inflammatory form of programmed cell death, defined as being caspase-gasdermin-D (GSDMD)-dependent. The NOD-like receptor pyrin 3 (NLRP3) inflammasome plays an important role in mediating GSDMD activation. However, the role and mechanism of pyroptosis in the loss of retinal pericytes during the pathogenesis of DR are still unclear. In the present study, we cultured primary human retinal pericytes (HRPs) in high glucose medium; caspase-3 inhibitor DEVD, caspase-1 inhibitor YVAD, or NLRP3 inhibitor glyburide was used as intervention reagents; GSDMD was overexpressed or suppressed by transfection with an expressing vector or retroviral silencing of GSDMD, respectively. Our data showed that high glucose induced NLRP3-caspase-1-GSDMD activation and pore formation in a dose- and time-dependent manner (p<0.05) and resulted in the inflammatory cytokines IL-1β and IL-18 and lactate dehydrogenase (LDH) release from HRPs (p<0.05), which are all signs of HRP pyroptosis. Overexpression of GSDMD facilitated high glucose-induced pyroptosis (all p<0.05). However, these effects were blunted by synergistically treating DEVD, YVAD, and silencing GSDMD (p<0.05). Taken together, our results firstly revealed that high glucose induced the loss of retinal pericytes partly via NLRP3-caspase-1-GSDMD-mediated pyroptosis.

Glucose regulates tissue-specific chondro-osteogenic differentiation of human cartilage endplate stem cells via O-GlcNAcylation of Sox9 and Runx2

Background The degenerative disc disease (DDD) is a major cause of low back pain. The physiological low-glucose microenvironment of the cartilage endplate (CEP) is disrupted in DDD. Glucose influences protein O-GlcNAcylation via the hexosamine biosynthetic pathway (HBP), which is the key to stem cell fate. Thiamet-G is an inhibitor of O-GlcNAcase for accumulating O-GlcNAcylated proteins while 6-diazo-5-oxo-l-norleucine (DON) inhibits HBP. Mechanisms of DDD are incompletely understood but include CEP degeneration and calcification. We aimed to identify the molecular mechanisms of glucose in CEP calcification in DDD. Methods We assessed normal and degenerated CEP tissues from patients, and the effects of chondrogenesis and osteogenesis of the CEP were determined by western blot and immunohistochemical staining. Cartilage endplate stem cells (CESCs) were induced with low-, normal-, and high-glucose medium for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. CESCs were induced with low-glucose and high-glucose medium with or without Thiamet-G or DON for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. Sox9 and Runx2 O-GlcNAcylation were measured by immunofluorescence. The effects of O-GlcNAcylation on the downstream genes of Sox9 and Runx2 were determined by Q-PCR and western blot. Results Degenerated CEPs from DDD patients lost chondrogenesis, acquired osteogenesis, and had higher protein O-GlcNAcylation level compared to normal CEPs from LVF patients. CESC chondrogenic differentiation gradually decreased while osteogenic differentiation gradually increased from low- to high-glucose differentiation medium. Furthermore, Thiamet-G promoted CESC osteogenic differentiation and inhibited chondrogenic differentiation in low-glucose differentiation medium; however, DON acted opposite role in high-glucose differentiation medium. Interestingly, we found that Sox9 and Runx2 were O-GlcNAcylated in differentiated CESCs. Finally, O-GlcNAcylation of Sox9 and Runx2 decreased chondrogenesis and increased osteogenesis in CESCs. Conclusions Our findings demonstrate the effect of glucose concentration on regulating the chondrogenic and osteogenic differentiation potential of CESCs and provide insight into the mechanism of how glucose concentration regulates Sox9 and Runx2 O-GlcNAcylation to affect the differentiation of CESCs, which may represent a target for CEP degeneration therapy.

PTPN2 improves implant osseointegration in T2DM via inducing the dephosphorylation of ERK

Type 2 diabetes mellitus (T2DM) is considered to compromise implant osseointegration. Protein tyrosine phosphatase non-receptor type 2 (PTPN2) regulates glucose metabolism, systemic inflammation, and bone regeneration. This study aimed to investigate the role of PTPN2 in implant osseointegration in T2DM and explore the potential mechanisms. Streptozotocin-induced diabetic rats received implant surgery, with or without local overexpression of PTPN2 for three months, and implant osseointegration was examined by histological evaluation, micro-CT analysis, pull-out test, and scanning electron microscope. Rat bone marrow stem cells (RBMSCs) were isolated and exposed to high glucose, and osteogenic differentiation was evaluated by alizarin red staining, ALP assay, and Western blot analysis. Overexpression of PTPN2 could improve impaired implant osseointegration in T2DM rats and promote osteogenic differentiation of RBMSCs in high glucose. In addition, p-ERK level in RBMSCs was increased in high glucose and decreased after PTPN2 overexpression. These results suggest that PTPN2 promotes implant osseointegration in T2DM rats and enhances osteogenesis of RBMSCs in high glucose medium via inducing the dephosphorylation of ERK. PTPN2 may be a novel target for the therapy of impaired implant osseointegration in T2DM patients. Impact statement Using both in vivo and in vitro approaches, we made important findings that PTPN2 promoted implant osseointegration in T2DM rats and enhanced osteogenesis of RBMSCs in high glucose medium. The positive effects of PTPN2 on osteogenesis are related to the dephosphorylation of ERK and the inhibition of MAPK/ERK pathway. PTPN2 may be a novel target for the therapy of impaired implant osseointegration in T2DM patients.

Production, Characterization, and Function of Pseudoislets from Perinatal Canine Pancreas

We evaluated the cell composition and function of canine pancreatic pseudoislets (PIs) produced from 42- to 55-day-old fetuses, 1- to 21-day-old pups, and an adult dog pancreas. After mild collagenase treatment, partially digested tissues were cultured for 2–3 weeks. PI production started on culture day 3, was marked for 6 to 9 days, and then stopped. PI production was greatest with the neonatal specimens, reaching about 12 million aggregates per litter (55-day-old fetus) or per pancreas (1-day-old pup). Cell composition at all stages was similar to that in adult pancreatic islets, with predominant β cells, scant α cells and, most importantly, presence of δ cells. Among pancreatic markers assessed by quantitative real-time PCR (qRT-PCR) mRNA assay, insulin showed the highest expression levels in PIs from newborn and adult pancreas, although these were more than 1000 times lower than in adult islets. Pdx1 mRNA expression was high in PIs from 55-day-old pancreases and was lower at later stages. Consistent with the qRT-PCR results, the insulin content was far lower than reported in adult dog pancreatic islets. However, insulin release by PIs from 1-day-old pups was demonstrated and was stimulated by a high-glucose medium. PIs were transplanted into euglycemic and diabetic SCID mice. In euglycemic animals, the transplant cell composition underwent maturation and transplants were still viable after 6 months. In diabetic mice, the PI transplants produced insulin and partially controlled the hyperglycemia. These data indicate that PIs can be produced ex vivo from canine fetal or postnatal pancreases. Although functional PIs can be obtained, the production yield is most likely insufficient to meet the requirements for diabetic dog transplantation without further innovation in cell culture amplification.