Short- and long-term exposure to high glucose induces unique transcriptional changes in osteoblasts in vitro.

Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10 days, and prior to analysis, they were exposed to hyperglycemia (25 mM) for the short-term (1 or 3 days) or long-term (10 days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3 days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.

Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells.

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = -0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.

Skeletal muscle cystathionine γ-lyase deficiency promotes obesity and insulin resistance and results in hyperglycemia and skeletal muscle injury upon HFD in mice.

OBJECTIVES: The objective of this study was to investigate whether skeletal muscle cystathionine γ-lyase (CTH) contributes to high-fat diet (HFD)-induced metabolic disorders using skeletal muscle Cth knockout (CthΔskm) mice. METHODS: The CthΔskm mice and littermate Cth-floxed (Cthf/f) mice were fed with either HFD or chow diet for 13 weeks. Metabolomics and transcriptome analysis were used to assess the impact of CTH deficiency in skeletal muscle. RESULTS: Metabolomics coupled with transcriptome showed that CthΔskm mice displayed impaired energy metabolism and some signaling pathways linked to insulin resistance (IR) in skeletal muscle although the mice had normal insulin sensitivity. HFD led to reduced CTH expression and impaired energy metabolism in skeletal muscle in Cthf/f mice. CTH deficiency and HFD had some common pathways enriched in the aspects of amino acid metabolism, carbon metabolism, and fatty acid metabolism. CthΔskm+HFD mice exhibited increased body weight gain, fasting blood glucose, plasma insulin, and IR, and reduced glucose transporter 4 and CD36 expression in skeletal muscle compared to Cthf/f+HFD mice. Impaired mitochondria and irregular arrangement in myofilament occurred in CthΔskm+HFD mice. Omics analysis showed differential pathways enriched between CthΔskm mice and Cthf/f mice upon HFD. More severity in impaired energy metabolism, reduced AMPK signaling, and increased oxidative stress and ferroptosis occurred in CthΔskm+HFD mice compared to Cthf/f+HFD mice. DISCUSSION: Our results indicate that skeletal muscle CTH expression dysregulation contributes to metabolism disorders upon HFD.

Vitamin C alleviates hyperglycemic stress in retinal pigment epithelial cells.

BACKGROUND: Retinal pigment epithelial cells (RPECs) are a type of retinal cells that structurally and physiologically support photoreceptors. However, hyperglycemia has been shown to play a critical role in the progression of diabetic retinopathy (DR), which is one of the leading causes of vision impairment. In the diabetic eye, the high glucose environment damages RPECs via the induction of oxidative stress, leading to the release of excess reactive oxygen species (ROS) and triggering apoptosis. In this study, we aim to investigate the antioxidant mechanism of Vitamin C in reducing hyperglycemia-induced stress and whether this mechanism can preserve the function of RPECs. METHODS AND RESULTS: ARPE-19 cells were treated with high glucose in the presence or absence of Vitamin C. Cell viability was measured by MTT assay. Cleaved poly ADP-ribose polymerase (PARP) was used to identify apoptosis in the cells. ROS were detected by the DCFH-DA reaction. The accumulation of sorbitol in the aldose reductase (AR) polyol pathway was determined using the sorbitol detection assay. Primary mouse RPECs were isolated from adult mice and identified by Rpe65 expression. The mitochondrial damage was measured by mitochondrial membrane depolarization. Our results showed that high glucose conditions reduce cell viability in RPECs while Vitamin C can restore cell viability, compared to the vehicle treatment. We also demonstrated that Vitamin C reduces hyperglycemia-induced ROS production and prevents cell apoptosis in RPECs in an AR-independent pathway. CONCLUSIONS: These results suggest that Vitamin C is not only a nutritional necessity but also an adjuvant that can be combined with AR inhibitors for alleviating hyperglycemic stress in RPECs.

Pancreas Β-Cells in Type 1 and Type 2 Diabetes: Cell Death, Oxidative Stress and Immune Regulation. Recently Appearing Changes in Diabetes Consequences.

Diabetes mellitus type 1 (T1D) and type 2 (T2D) develop due to dysfunction of the Langerhans islet ß-cells in the pancreas, and this dysfunction is mediated by oxidative, endoplasmic reticulum (ER), and mitochondrial stresses. Although the two types of diabetes are significantly different, ß-cell failure and death play a key role in the pathogenesis of both diseases, resulting in hyperglycemia due to a reduced ability to produce insulin. In T1D, ß-cell apoptosis is the main event leading to hyperglycemia, while in T2D, insulin resistance results in an inability to meet insulin requirements. It has been suggested that autophagy promotes ß-cell survival by delaying apoptosis and providing adaptive responses to mitigate the detrimental effects of ER stress and DNA damage, which is directly related to oxidative stress. As people with diabetes are now living longer, they are more susceptible to a different set of complications. There has been a diversification in causes of death, whereby a larger proportion of deaths among individuals with diabetes is attributable to nonvascular conditions; on the other hand, the proportion of cancer-related deaths has remained stable or even increased in some countries. Due to the increasing cases of both T1D and T2D, these diseases become even more socially significant. Hence, we believe that search for any opportunities for control of this disease is an overwhelmingly important target for the modern science. We focus on two differences that are characteristic of the development of diabetes's last periods. One of them shows that all-cause death rates have declined in several diabetes populations, driven in part by large declines in vascular disease mortality but large increases in oncological diseases. Another hypothesis is that some T2D medications could be repurposed to control glycemia in patients with T1D.

Novel PLGA-encapsulated-nanopiperine promotes synergistic interaction of p53/PARP-1/Hsp90 axis to combat ALX-induced-hyperglycemia.

The present study predicts the molecular targets and druglike properties of the phyto-compound piperine (PIP) by in silico studies including molecular docking simulation, druglikeness prediction and ADME analysis for prospective therapeutic benefits against diabetic complications. PIP was encapsulated in biodegradable polymer poly-lactide-co-glycolide (PLGA) to form nanopiperine (NPIP) and their physico-chemical properties were characterized by AFM and DLS. ∼ 30 nm sized NPIP showed 86.68% encapsulation efficiency and - 6 mV zeta potential, demonstrated great interactive stability and binding with CT-DNA displaying upsurge in molar ellipticity during CD spectroscopy. NPIP lowered glucose levels in peripheral circulation by > 65 mg/dL compared to disease model and improved glucose influx in alloxan-induced in vivo and in vitro diabetes models concerted with 3-folds decrease in ROS production, ROS-induced DNA damage and 27.24% decrease in nuclear condensation. The 25% increase in % cell viability and inhibition in chromosome aberration justified the initiation of p53 and PARP DNA repairing protein expression and maintenance of Hsp90. Thus, the experimental study corroborated well with in silico predictions of modulating the p53/PARP-1/Hsp90 axis, with predicted dock score value of - 8.72, - 8.57, - 8.76 kcal/mol respectively, validated docking-based preventive approaches for unravelling the intricacies of molecular signalling and nano-drug efficacy as therapeutics for diabetics.

Effects of paradoxical sleep deprivation on oxidative parameters in the serum and brain of mice submitted to the animal model of hyperglycemia.

The present study aimed to investigate the effects of paradoxical sleep deprivation (PSD) on behavioral and oxidative stress parameters in the brain and serum of mice submitted to the animal model of hyperglycemia induced by alloxan, mimicking the main symptom of diabetes mellitus (DM). Adults C57BL/6 male and female mice received an injection of alloxan, and ten days later, the animals were submitted to the PSD for 36 h. The animals' behavioral parameters were evaluated in the open-field test. Oxidative stress parameters [Diacetyldichlorofluorescein (DCF), Thiobarbituric acid reactive substances (TBARS), Superoxide dismutase (SOD), and Glutathione] were assessed in the frontal cortex, hippocampus, striatum, and serum. The PSD increased the male and female mice locomotion, but the alloxan's pre-administration prevented the PSD-induced hyperactivity. In addition, the male mice receiving alloxan and submitted to the PSD had elevated latency time in the first quadrant and the number of fecal boli, demonstrating increased anxiety-like behavior. The HPA-axis was hyperactivating in male and female mice pre-administered alloxan and/or PSD-submitted animals. The oxidative stress parameters were also increased in the serum of the animals administered alloxan and/or sleep-deprived mice. Despite alloxan or PSD leading to behavioral or biochemical alterations, the one did not potentiate the other in mice. However, more studies are necessary to identify the link between sleep and hyperglycemia.

The synergism of Lactobacillaceae, inulin, polyglucose, and aerobic exercise ameliorates hyperglycemia by modulating the gut microbiota community and the metabolic profiles in db/db mice.

This study aimed to assess the impact of Lactobacillaceae (L or H represents a low or high dose), inulin (I), and polydextrose (P) combined with aerobic exercise (A) on the composition of the gut microbiota and metabolic profiles in db/db mice. After a 12-week intervention, LIP, LIPA, and HIPA groups exhibited significant improvements in hyperglycemia, glucose tolerance, insulin resistance, inflammatory response, and short-chain fatty acid (SCFA) and blood lipid levels compared to type 2 diabetes mice (MC). After treatment, the gut microbiota composition shifted favorably in the treatment groups which significantly increased the abundance of beneficial bacteria, such as Bacteroides, Blautia, Akkermansia, and Faecalibaculum, and significantly decreased the abundance of Proteus. Metabolomics analysis showed that compared to the MC group, the contents of 5-hydroxyindoleacetic acid, 3-hydroxysebacic acid, adenosine monophosphate (AMP), xanthine and hypoxanthine were significantly decreased, while 3-ketosphinganine, sphinganine, and sphingosine were significantly increased in the LIP and LIPA groups, respectively. Additionally, LIP and LIPA not only improved sphingolipid metabolism and purine metabolism pathways but also activated AMP-activated protein kinase to promote ß-oxidation by increasing the levels of SCFAs. Faecalibaculum, Blautia, Bacteroides, and Akkermansia exhibited positive correlations with sphingosine, 3-ketosphinganine, and sphinganine, and exhibited negative correlations with hypoxanthine, xanthine and AMP. Faecalibaculum, Blautia, Bacteroides, and Akkermansia may have the potential to improve sphingolipid metabolism and purine metabolism pathways. These findings suggest that the synergism of Lactobacillaceae, inulin, polydextrose, and aerobic exercise provides a promising strategy for the prevention and management of type 2 diabetes.

Sirt7/HIC1 complex participates in hyperglycaemia-mediated EndMT via modulation of SDC1 expression in diabetic kidney disease and metabolic memory.

Diabetic kidney disease (DKD), a primary microvascular complication arising from diabetes, may result in end-stage renal disease. Epigenetic regulation of endothelial mesenchymal transition (EndMT) has been recently reported to exert function in metabolic memory and DKD. Here, we investigated the mechanism which Sirt7 modulated EndMT in human glomerular endothelial cells (HGECs) in the occurrence of metabolic memory in DKD. Lower levels of SDC1 and Sirt7 were noted in the glomeruli of both DKD patients and diabetes-induced renal injury rats, as well as in human glomerular endothelial cells (HGECs) with high blood sugar. Endothelial-to-mesenchymal transition (EndMT) was sustained despite the normalization of glycaemic control. We also found that Sirt7 overexpression associated with glucose normalization promoted the SDC1 expression and reversed EndMT in HGECs. Furthermore, the sh-Sirt7-mediated EndMT could be reversed by SDC1 overexpression. The ChIP assay revealed enrichment of Sirt7 and H3K18ac in the SDC1 promoter region. Furthermore, hypermethylated in cancer 1 (HIC1) was found to be associated with Sirt7. Overexpression of HIC1 with normoglycaemia reversed high glucose-mediated EndMT in HGECs. The knockdown of HIC1-mediated EndMT was reversed by SDC1 upregulation. In addition, the enrichment of HIC1 and Sirt7 was observed in the same promoter region of SDC1. The overexpressed Sirt7 reversed EndMT and improved renal function in insulin-treated diabetic models. This study demonstrated that the hyperglycaemia-mediated interaction between Sirt7 and HIC1 exerts a role in the metabolic memory in DKD by inactivating SDC1 transcription and mediating EndMT despite glucose normalization in HGECs.

Betaine postpones hyperglycemia-related senescence in ovarian and testicular cells: Involvement of RAGE and ß-galactosidase.

The structural and functional disorders of the testis and ovary are one of the main complications of hyperglycemia. Betaine is a trimethyl glycine with antioxidant, antidiabetic, and anti-inflammatory potential. The aim of this study is to investigate the potential of betaine on the expression of aging and oxidative stress markers in ovarian and testicular cells under hyperglycemic conditions. Testicular and ovarian cells were subjected to four different conditions, including normal glucose and hyperglycemia, with or without betaine (5 mM). The cells with hyperglycemia saw an increase in malondialdehyde (MDA), methylglyoxal (MGO), expression of a receptor for AGE, and aging-related genes (ß-GAL), and a decrease in the activity of antioxidant enzymes including catalase, glutathione peroxidase, and superoxide dismutase. The treatment with betaine, in contrast, decreased the amount of MGO and MDA, and also downregulated aging-related signaling. Although hyperglycemia induces senescence in testicular and ovarian cells, the use of betaine may have a protective effect against the cell senescence, which may be useful in the management of infertility.

Roles of heat shock protein A12A in the development of diabetic cardiomyopathy.

Long-term hyperglycemia can lead to diabetic cardiomyopathy (DCM), a main lethal complication of diabetes. However, the mechanisms underlying DCM development have not been fully elucidated. Heat shock protein A12A (HSPA12A) is the atypic member of the Heat shock 70kDa protein family. In the present study, we found that the expression of HSPA12A was upregulated in the hearts of mice with streptozotocin-induced diabetes, while ablation of HSPA12A improved cardiac systolic and diastolic dysfunction and increased cumulative survival of diabetic mice. An increased expression of HSPA12A was also found in H9c2 cardiac cells following treatment with high glucose (HG), while overexpression of HSPA12A-enhanced the HG-induced cardiac cell death, as reflected by higher levels of propidium iodide cells, lactate dehydrogenase leakage, and caspase 3 cleavage. Moreover, the HG-induced increase of oxidative stress, as indicated by dihydroethidium staining, was exaggerated by HSPA12A overexpression. Further studies demonstrated that the HG-induced increases of protein kinase B and forkhead box transcription factors 1 phosphorylation were diminished by HSPA12A overexpression, while pharmacologically inhibition of protein kinase B further enhanced the HG-induced lactate dehydrogenase leakage in HSPA12A overexpressed cardiac cells. Together, the results suggest that hyperglycemia upregulated HSPA12A expression in cardiac cells, by which induced cell death to promote DCM development. Targeting HSPA12A may serve as a potential approach for DCM management.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Investigating the Role of 17-Beta Estradiol in the Regulation of the Unfolded Protein Response (UPR) in Pancreatic Beta Cells.

Diabetes mellitus is clinically defined by chronic hyperglycemia. Sex differences in the presentation and outcome of diabetes exist with premenopausal women having a reduced risk of developing diabetes, relative to men, or women after menopause. Accumulating evidence shows a protective role of estrogens, specifically 17-beta estradiol, in the maintenance of pancreatic beta cell health; however, the mechanisms underlying this protection are still unknown. To elucidate these potential mechanisms, we used a pancreatic beta cell line (BTC6) and a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse, exhibiting sexual dimorphism in glucose regulation. In this study we hypothesize that 17-beta estradiol protects pancreatic beta cells by modulating the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress. We observed that ovariectomized female and male ApoE-/-:Ins2+/Akita mice show significantly increased expression of apoptotic UPR markers. Sham operated female and ovariectomized female ApoE-/-:Ins2+/Akita mice supplemented with exogenous 17-beta estradiol increased the expression of adaptive UPR markers compared to non-supplemented ovariectomized female ApoE-/-:Ins2+/Akita mice. These findings were consistent to what was observed in cultured BTC6 cells, suggesting that 17-beta estradiol may protect pancreatic beta cells by repressing the apoptotic UPR and enhancing the adaptive UPR activation in response to pancreatic ER stress.

Canagliflozin protects against hyperglycemia-induced cerebrovascular injury by preventing blood-brain barrier (BBB) disruption via AMPK/Sp1/adenosine A2A receptor.

Diabetes mellitus causes brain microvascular endothelial cell (MEC) damage, inducing dysfunctional angiogenic response and disruption of the blood-brain barrier (BBB). Canagliflozin is a revolutionary hypoglycemic drug that exerts neurologic and/or vascular-protective effects beyond glycemic control; however, its underlying mechanism remains unclear. In the present study, we hypothesize that canagliflozin ameliorates BBB permeability by preventing diabetes-induced brain MEC damage. Mice with high-fat diet/streptozotocin-induced diabetes received canagliflozin for 8 weeks. We assessed vascular integrity by measuring cerebrovascular neovascularization indices. The expression of specificity protein 1 (Sp1), as well as tight junction proteins (TJs), phosphorylated AMP-activated protein kinase (p-AMPK), and adenosine A2A receptors was examined. Mouse brain MECs were grown in high glucose (30 mM) to mimic diabetic conditions. They were treated with/without canagliflozin and assessed for migration and angiogenic ability. We also performed validation studies using AMPK activator (AICAR), inhibitor (Compound C), Sp1 small interfering RNA (siRNA), and adenosine A2A receptor siRNA. We observed that cerebral pathological neovascularization indices were significantly normalized in mice treated with canagliflozin. Increased Sp1 and adenosine A2A receptor expression and decreased p-AMPK and TJ expression were observed under diabetic conditions. Canagliflozin or AICAR treatment alleviated these changes. However, this alleviation effect of canagliflozin was diminished again after Compound C treatment. Either Sp1 siRNA or adenosine A2A receptor siRNA could increase the expression of TJs. Luciferase reporter assay confirmed that Sp1 could bind to the adenosine A2A receptor gene promoter. Our study identifies the AMPK/Sp1/adenosine A2A receptor pathway as a treatment target for diabetes-induced cerebrovascular injury.

Artesunate improves glucose and lipid metabolism in db/db mice by regulating the metabolic profile and the MAPK/PI3K/Akt signalling pathway.

BACKGROUND: Diabetes is a metabolic disorder characterized by chronic hyperglycaemia. Chronic metabolic abnormalities and long-term hyperglycaemia may result in a wide range of acute and chronic consequences. Previous studies have demonstrated that artesunate(ART) has antidiabetic, anti-inflammatory, antiatherosclerotic, and other beneficial effects, but the specific regulatory mechanism is not completely clear. AIM: This study investigated the effects of ART on metabolic disorders in type 2 diabetes mellitus (T2DM) model db/db mice and explored the underlying mechanisms involved. METHODS: C57BL/KsJ-db/db mice were used to identify the targets and molecular mechanism of ART. Metabolomic methods were used to evaluate the efficacy of ART in improving T2DM-related metabolic disorders. Network pharmacology and transcriptomic sequencing were used to analyse the targets and pathways of ART in T2DM. Finally, molecular biology experiments were performed to verify the key targets and pathways selected by network pharmacology and transcriptomic analyses. RESULTS: After a 7-week ART intervention (160 mg/kg), the glucose and lipid metabolism levels of the db/db mice improved. Additionally, the oxidative stress indices, namely, the MDA and SOD levels, significantly improved (p<0.01). Linoleic acid and glycerophospholipid metabolism, amino acid metabolism, bile acid synthesis, and purine metabolism disorders in db/db mice were partially corrected after ART treatment. Network pharmacology analysis identified important targets of ART for the treatment of metabolic disorders in T2DM . These targets are involved in key signalling pathways, including the highest scores observed for the PI3K/Akt signalling pathway. Transcriptomic analysis revealed that ART could activate the MAPK signalling pathway and two key gene targets, HGK and GADD45. Immunoblotting revealed that ART increases p-PI3K, p-AKT, Glut2, and IRS1 protein expression and suppresses the phosphorylation of p38, ERK1/2, and JNK, returning HGK and GADD45 to their preartesunate levels. CONCLUSION: Treatment of db/db mice with 160 mg/kg ART for 7 weeks significantly reduced fasting blood glucose and lipid levels. It also improved metabolic imbalances in amino acids, lipids, purines, and bile acids, thereby improving metabolic disorders. These effects are achieved by activating the PI3K/AKT pathway and inhibiting the MAPK pathway, thus demonstrating the efficacy of the drug.

The impact of glucagon to support postabsorptive glucose flux and glycemia in healthy rats and its attenuation in male Zucker diabetic fatty rats.

Hyperglucagonemia is a hallmark of type 2 diabetes (T2DM), yet the role of elevated plasma glucagon (P-GCG) to promote excessive postabsorptive glucose production and contribute to hyperglycemia in patients with this disease remains debatable. We investigated the acute action of P-GCG to safeguard/support postabsorptive endogenous glucose production (EGP) and euglycemia in healthy Zucker control lean (ZCL) rats. Using male Zucker diabetic fatty (ZDF) rats that exhibit the typical metabolic disorders of human T2DM, such as excessive EGP, hyperglycemia, hyperinsulinemia, and hyperglucagonemia, we examined the ability of hyperglucagonemia to promote greater rates of postabsorptive EGP and hyperglycemia. Euglycemic or hyperglycemic basal insulin (INS-BC) and glucagon (GCG-BC) clamps were performed in the absence or during an acute setting of glucagon deficiency (GCG-DF, ∼10% of basal), either alone or in combination with insulin deficiency (INS-DF, ∼10% of basal). Glucose appearance, disappearance, and cycling rates were measured using [2-3H] and [3-3H]-glucose. In ZCL rats, GCG-DF reduced the levels of hepatic cyclic AMP, EGP, and plasma glucose (PG) by 50%, 32%, and 50%, respectively. EGP fell in the presence GCG-DF and INS-BC, but under GCG-DF and INS-DF, EGP and PG increased two- and threefold, respectively. GCG-DF revealed the hyperglucagonemia present in ZDF rats lacked the ability to regulate hepatic intracellular cyclic AMP levels and glucose flux, since EGP and PG levels fell by only 10%. We conclude that the liver in T2DM suffers from resistance to all three major regulatory factors, glucagon, insulin, and glucose, thus leading to a loss of metabolic flexibility.NEW & NOTEWORTHY In postabsorptive state, basal plasma insulin (P-INS) and plasma glucose (PG) act dominantly to increase hepatic glucose cycling and reduce endogenous glucose production (EGP) and PG in healthy rats, which is only counteracted by the acute action of basal plasma glucagon (P-GCG) to support EGP and euglycemia. Hyperglucagonemia, a hallmark of type 2 diabetes (T2DM) present in Zucker diabetic fatty (ZDF) rats, is not the primary mediator of hyperglycemia and high EGP as commonly thought; instead, the liver is resistant to glucagon as well as insulin and glucose.

S-nitrosylation of AMPKγ impairs coronary collateral circulation and disrupts VSMC reprogramming.

Collateral circulation is essential for blood resupply to the ischemic heart, which is dictated by the contractile phenotypic restoration of vascular smooth muscle cells (VSMC). Here we investigate whether S-nitrosylation of AMP-activated protein kinase (AMPK), a key regulator of the VSMC phenotype, impairs collateral circulation. In rats with collateral growth and development, nitroglycerin decreases coronary collateral blood flow (CCBF), inhibits vascular contractile phenotypic restoration, and increases myocardial infarct size, accompanied by reduced AMPK activity in the collateral zone. Nitric oxide (NO) S-nitrosylates human recombinant AMPKγ1 at cysteine 131 and decreases AMP sensitivity of AMPK. In VSMCs, exogenous expression of S-nitrosylation-resistant AMPKγ1 or deficient NO synthase (iNOS) prevents the disruption of VSMC reprogramming. Finally, hyperhomocysteinemia or hyperglycemia increases AMPKγ1 S-nitrosylation, prevents vascular contractile phenotypic restoration, reduces CCBF, and increases the infarct size of the heart in Apoe-/- mice, all of which is rescued in Apoe-/-/iNOSsm-/- mice or Apoe-/- mice with enforced expression of the AMPKγ1-C130A mutant following RI/MI. We conclude that nitrosative stress disrupts coronary collateral circulation during hyperhomocysteinemia or hyperglycemia through AMPK S-nitrosylation.

The integrated analysis and underlying mechanisms of FNDC5 on diabetic induced cognitive deficits.

OBJECTIVES: Chronic hyperglycemia is considered as an important factor to promote the neurodegenerative process of brain, and the synaptic plasticity as well as heterogeneity of hippocampal cells are thought to be associated with cognitive dysfunction in the early process of neurodegeneration. To date, fibronectin type III domain-containing protein 5 (FNDC5) has been highlighted its protective role in multiple neurodegenerative diseases. However, the potential molecular and cellular mechanisms of FNDC5 on synaptic plasticity regulation in cognitive impairment (CI) induced by diabetics are still need to known. METHODS/DESIGN: To investigate the heterogeneity and synaptic plasticity of hippocampus in animals with CI state induced by hyperglycemia, and explore the potential role of FNDC5 involved in this process. Firstly, the single cell sequencing was performed based on the hippocampal tissue from db diabetic mice induced CI and normal health control mice by ex vivo experiments; and then the integrated analysis and observations validation using Quantitative Real-time PCR, western blot as well as other in vitro studies. RESULTS: We observed and clarified the sub-cluster of type IC spiral ganglion neurons expressed marker genes as Trmp3 and sub-cluster of astrocytes with marker gene as Atp1a2 in hippocampal cells from diabetic animals induced CI and the effect of those on neuron-glial communication. We also found that FNDC5\BDNF-Trk axis was involved in the synaptic plasticity regulation of hippocampus. In high glucose induced brain injury model in vitro, we investigated that FNDC5 significantly regulates BDNF expression and that over-expression of FNDC5 up-regulated BDNF expression (p < 0.05) and can also significantly increase the expression of synapsin-1 (p < 0.05), which is related to synaptic plasticity, In addition, the unbalanced methylation level between H3K4 and H3K9 in Fndc5 gene promoter correlated with significantly down-regulated expression of FNDC5 (p < 0.05) in the hyperglycemia state. CONCLUSION: The current study revealed that the synaptic plasticity of hippocampal cells in hyperglycemia might be regulated by FNDC5\BDNF-Trk axis, playing the protective role in the process of CI induced by hyperglycemia and providing a target for the early treatment of hyperglycemia induced cognitive dysfunction in clinic.

Two Prenylated Chalcones, 4-Hydroxyderricin, and Xanthoangelol Prevent Postprandial Hyperglycemia by Promoting GLUT4 Translocation via the LKB1/AMPK Signaling Pathway in Skeletal Muscle Cells.

SCOPE: Stimulation of glucose uptake in the skeletal muscle is crucial for the prevention of postprandial hyperglycemia. Insulin and certain polyphenols enhance glucose uptake through the translocation of glucose transporter 4 (GLUT4) in the skeletal muscle. The previous study reports that prenylated chalcones, 4-hydroxyderricin (4-HD), and xanthoangelol (XAG) promote glucose uptake and GLUT4 translocation in L6 myotubes, but their underlying molecular mechanism remains unclear. This study investigates the mechanism in L6 myotubes and confirms antihyperglycemia by 4-HD and XAG. METHODS AND RESULTS: In L6 myotubes, 4-HD and XAG promote glucose uptake and GLUT4 translocation through the activation of adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B1 (LKB1) signaling pathway without activating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and Janus kinases (JAKs)/signal transducers and activators of transcriptions (STATs) pathways. Moreover, Compound C, an AMPK-specific inhibitor, as well as siRNA targeting AMPK and LKB1 completely canceled 4-HD and XAG-increased glucose uptake. Consistently, oral administration of 4-HD and XAG to male ICR mice suppresses acute hyperglycemia in an oral glucose tolerance test. CONCLUSION: In conclusion, LKB1/AMPK pathway and subsequent GLUT4 translocation in skeletal muscle cells are involved in Ashitaba chalcone-suppressed acute hyperglycemia.

Increasing HbA1c is associated with reduced CD8+ T cell functionality in response to influenza virus in a TCR-dependent manner in individuals with diabetes mellitus.

Diabetes mellitus is on the rise globally and is a known susceptibility factor for severe influenza virus infections. However, the mechanisms by which diabetes increases the severity of an influenza virus infection are yet to be fully defined. Diabetes mellitus is hallmarked by high glucose concentrations in the blood. We hypothesized that these high glucose concentrations affect the functionality of CD8+ T cells, which play a key role eliminating virus-infected cells and have been shown to decrease influenza disease severity. To study the effect of hyperglycemia on CD8+ T cell function, we stimulated peripheral blood mononuclear cells (PBMCs) from donors with and without diabetes with influenza A virus, anti-CD3/anti-CD28-coated beads, PMA and ionomycin (PMA/I), or an influenza viral peptide pool. After stimulation, cells were assessed for functionality [as defined by expression of IFN-γ, TNF-α, macrophage inflammatory protein (MIP)-1ß, and lysosomal-associated membrane protein-1 (CD107a)] using flow cytometry. Our results showed that increasing HbA1c correlated with a reduction in TNF-α production by CD8+ T cells in response to influenza stimulation in a TCR-specific manner. This was not associated with any changes to CD8+ T cell subsets. We conclude that hyperglycemia impairs CD8+ T cell function to influenza virus infection, which may be linked with the increased risk of severe influenza in patients with diabetes.