Two Weeks High Glucose is Enough to Induce Liver and Gut Lesion

Background:High glucose is critical for diabetes.But in which way it induces diabetes, and which organ trigger the formation of diabetes are not clear.The goal of this study is to see if there is a risk of acquiring diabetic symptoms following a 2 weeks short infusion of 2g/kg/day and dietary 2.5g/kg/day in SD rats on various body organs.Methods：Twelve weeks old SD rats were randomly divided into control group,high glucose infusion group(IHG,infusion 2g/kg/day) and oral high glucose group OHG,dietary 2.5g/kg/day).Fasten blood sugar,TNF-a and IL-6 were measured. Intestine and liver samples were collected for pathological examination.Feces of rats were collected for gut microbiota tests.Results：The results indicated that short time high glucose induced hyperglycemia lasted for at least 2 weeks after ceasing of high glucose.It increased serum levels of IL-6 and TNF-a obviously.It led to jejunum mucosa injury, obvious steatosis of hepatocytes, and disturbed the balance of gut microbiota.OHG led to swelling and necrosis of individual intestinal villi.IHG led to necrosis and disappearence of cells in the upper layer of intestinal mucosa.The lesion was confined to the mucosa.Conclusions:Short time high glucose induced lesion in liver and intestine,disturbed the balance of gut microbiota and consequently induced inflammation and triggered diabetes.

Effect of Liver Kinase B1 on Osteogenic/Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in High Glucose Environment

Bone marrow mesenchymal stem cells (BMSCs) present reduced proliferation under high glucose condition. Liver kinase B1 (LKB1) can maintain the homeostasis of hematopoietic stem cells. However, whether LKB1 regulates BMSCs osteogenic/adipogenic differentiation under high glucose is unclear. Rat BMSCs were isolated and separated into control group, high glucose group, and LKB1 group (BMSCs were transfected with pc-DNA 3.1-LKB1 plasmid under high glucose condition) followed by analysis of LKB1 expression by Real time PCR and Western blot, osteocalcin, type I collagen, RUNX2 and OPN mRNA level by real-time PCR, FABP4 and PPARγ2 level by western blot. In high glucose group, LKB1 expression was significantly decreased, with reduced expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA and elevated FABP4 and PPARγ2 level compared to control group (P < 0.05). Transfection of LKB1 plasmid reduced LKB1 expression, upregulated osteocalcin, type I collagen, RUNX2 and OPN mRNA and downregulated FABP4 and PPARγ2. Compared with the high glucose group, there was a statistical difference (P <0.05). High glucose can inhibit LKB1 expression and BMSCs osteogenic differentiation, and promote adipogenic differentiation. Upregulating LKB1 expression can promote BMSCs osteogenic differentiation.

High glucose promotes apoptosis and autophagy of MC3T3-E1 osteoblasts

IntroductionDiabetes and osteoporosis are common metabolic diseases. Abnormal high glucose can lead to the apoptosis of osteoblasts. Autophagy is a highly conserved cellular process that degrades proteins or organelles. In the present study, we comparatively analyzed the effects of high glucose and glucose fluctuation on apoptosis and autophagy of MC3T3-E1 osteoblasts.Material and methodsMC3T3-E1 cells were respectively treated with different concentrations of D-glucose: 5.5 mM for the control group, 25 mM for the high glucose group and 5.5/25 mM for the glucose fluctuation group.ResultsHigh glucose and glucose fluctuation decreased MC3T3-E1 proliferation and activated autophagy. Also, high glucose and glucose fluctuation might induce the production of reactive oxygen species, decline the mitochondrial membrane potential and trigger apoptosis. The differences in the glucose fluctuation treatment group were more significant. Moreover, N-acetylcysteine, an antioxidant reagent, dramatically eliminated the intracellular reactive oxygen species induced by high glucose and glucose fluctuation, and significantly inhibited the autophagy and apoptosis in MC3T3-E1 osteoblasts. Furthermore, treatment with chloroquine, an inhibitor of autophagy, significantly increased the apoptosis of MC3T3-E1 osteoblasts.ConclusionsHigh glucose, especially high glucose fluctuation, inhibits proliferation and promotes apoptosis and autophagy of MC3T3-E1 osteoblasts. This may occur through inducing oxidative stress and mitochondrial damage in the osteoblasts.

Effects of icariin and quercetin on high glucose-induced neuronal cell apoptosis

Purpose: To study the effects of icariin and quercetin on cell apoptotic changes in neurons induced by elevated glucose condition, and the mechanism involved. Methods: Neonatal male Sprague Dawley rats (n = 48) weighing 5 – 7 g were used. Neuronal cells were isolated from rat hippocampus and cultured after purification. The cells were randomly assigned to six groups: control, high glucose, icariin, quercetin, serine/threonine-specific protein kinase (Akt) inhibitor, and Akt agonist groups. The Akt inhibitor and agonist used in this study were MK-22062hci and SC79, respectively. The influence of icariin and quercetin on neuronal apoptotic changes were determined flow cytometrically, while their effects on levels of expression of Akt, p-Akt, bax and bcl-2 were determined with Western blotting. Results: Treatment with icariin or quercetin significantly inhibited apoptosis induced by high glucose. The concentrations of Akt, p-Akt, and bcl-2 proteins were markedly upregulated in high glucose group, relative to control (p < 0.05). The corresponding expression of bax was significantly down-regulated in high glucose group, relative to control (p < 0.05). Treatment with icariin or quercetin, or their agonists reversed high glucose-mediated alterations in these protein levels (p < 0.05). Conclusion: Icariin and quercetin inhibit neuronal cell apoptosis induced by high glucose through upregulation of bcl-2 expression and down- regulations of bax expression and Akt-induced protein phosphorylation. Thus, Icariin and quercetin possess potential benefits for the treatment of neurological diseases. Keywords: Apoptosis, High glucose condition, Hippocampal neurons, Icariin, Quercetin

Effect of Insulin-Like Growth Factor-1 on Bone Formation of Bone Marrow Mesenchymal Stem Cells Under High Glucose Environment by Regulating Insulin Like Growth Factor Binding Protein 2(IGFBP-2)/p38 Pathway

Diabetes easily affects the biological properties of bone marrow mesenchymal stem cells (BMSCs). Insulin-like growth factor-1 (IGF-1) promotes bone healing during osteoporotic fractures. However, IGF-1's effect on BMSCs high glucose is unclear. Rat BMSCs were assigned into control group, high glucose group, and IGF-1 group in which BMSCs were transfected with pc-DNA 3.1-IGF-1 plasmid on the basis of high glucose followed by analysis of IGF-1, RUNX2 and OPN mRNA level by real time PCR, cell proliferation by MTT assay, alkaline phosphatase (ALP) activity, IGFBP-2 level by ELISA, and p38 phosphorylation by Western blot. High glucose group showed significantly decreased IGF-1, RUNX2 and OPN mRNA level, reduced ALP activity, IGFBP-2 expression and p38 phosphorylation compared to control group (P < 0 05). Transfection of IGF-1 plasmid under high-glucose environment significantly upregulated IGF-1, RUNX2 and OPN mRNA, increased ALP activity, IGFBP-2 expression and p38 phosphorylation compared to high-glucose group (P < 0 05). IGF-1 expression is reduced in high glucose environment. Up-regulation of IGF-1 can promote BMSCs osteogenic differentiation through the IGFBP-2/p38 pathway.

Effect of adiponectin on oxidative stress-apoptotic pathway in podocytes under high glucose conditions

To investigate the effect of adiponectin (APN) on the oxidative stress-apoptotic pathway of podocytes under high glucose conditions, podocytes were categorized into a control group (5.5 mmol/L, normal glucose, NG), high glucose group (30 mmol/L, high glucose, HG), and an APN intervention group (HG+APN). The expression of podocyte cytoskeleton proteins (nephrin/podocin/synaptopodin), p-AMPK activity, and the NADPH oxidase family (NOX1/NOX4) and apoptosis-related proteins p53 and PUMA (p53 up-regulated apoptotic regulator) were detected by RT-PCR and Western blotting. The total RNA extracted by nano-magnetic beads was retrieved into DNA by the MagBeads Total RNA Extraction Kit, and cDNA was synthesized through reverse transcription. Podocyte apoptosis was detected by flow cytometry. In comparison with the control group, the high glucose group exhibited the reduced expression of podocyte cytoskeleton proteins, decreased p-AMPK activity, increased expression of NOX1, NOX4, P53, and PUMA, and increased podocyte apoptosis (28.15%±1.38%). APN intervention could significantly restore the expression of cytoskeleton proteins, increase the activity of p-AMPK, reduce the expression of NOX1, NOX4, P53, and PUMA, and reduce the apoptosis of podocytes (9.15%±1.98%). The protective effect of APN disappeared when AMPK was inhibited. APN may inhibit oxidative stress-apoptosis of podocytes under high glucose conditions through the activation of AMPK.

Down-Regulation of Notch-2 Promotes the Osteogenic Differentiation of Bone Marrow Stromal Cells in High Glucose Environment by Targeting Fat Mass and Obesity-Associated Protein

BMSCs Differentiation into osteoblasts is beneficial for treating osteoporosis. Notch signaling pathway regulates the proliferation of BMSCs. However, Notch-2's effect on osteogenic differentiation of BMSCs in high glucose environment remains unclear. Rat BMSCs were isolated and divided into control group, high glucose group, Notch-2 siRNA group, in which BMSCs cells were transfected with Notch-2 siRNA under high glucose environment followed by analysis of Notch-2 mRNA expression by Real time PCR, cell proliferation by MTT assay, Caspase 3 activity, ALP activity, expression of osteogenic genes Runx2 and BMP-2 by Real time PCR, TNF-α and IL-2 secretion by ELISA, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity and FTO protein expression by Western blot. In high glucose group, Notch-2 expression was increased with inhibited cell proliferation, increased Caspase 3 activity and decreased ALP activity. Meanwhile, high glucose significantly increased MPO content, decreased SOD activity, increased TNF-α and IL-2 secretion and FTO expression, and reduced Runx2 and BMP-2 expression (P < 0.05); Notch-2 siRNA transfection significantly down-regulated Notch-2 expression, promoted BMSCs cell proliferation, decreased Caspase 3 activity and increased ALP activity. In addition, it also significantly decreased MPO content, increased SOD activity, decreased TNF-α and IL-2 and FTO expression as well as increased Runx2 and BMP-2 expression (P < 0.05). Notch-2 is upregulated in BMSCs under high glucose. Down-regulation of Notch-2 in BMSCs under high glucose environment could promote BMSCs proliferation and osteogenic differentiation.

Effect of SIRT1 on Bone Marrow Stromal Stem Cells in High Glucose Environment and Related Mechanisms

Silencing information regulator (SIRT1) involves in endocrine diseases. However, whether SIRT1 participates in BMSCs under high glucose environment remains unclear. Rat BMSCs were isolated and divided into control group; high glucose group; and SIRT1 group, in which SIRT1 agonist (Resveratrol) was added to high glucose BMSCs followed by analysis of SIRT1, Bax and Bcl-2 expression by real time PCR and ELISA, cell proliferation by MTT assay, Caspase3 activity, ALP activity, calcification nodule formation by alizarin red staining, changes of ROS and SOD activities, PI3K signaling pathway by Western blot. SIRT1 expression was significantly decreased in BMSCs in high glucose group, with inhibited cell proliferation, increased Caspase 3 activity, Bax expression, and ROS content, decreased Bcl-2 expression, ALP activity, SOD activity, as well as reduced formation of calcified nodules and phosphorylation of PI3K compared to control (P < 0.05). Addition of Resveratrol significantly promoted SIRT1 expression and cell proliferation of BMSCs in high glucose group, decreased Caspase 3 activity, Bax expression, and ROS content, increased Bcl-2 expression, ALP activity, SOD activity, as well as increased formation of calcified nodules and phosphorylation of PI3K (P < 0.05). SIRT1 expression is decreased in BMSCs in high glucose group. Increasing SIRT1 expression in BMSCs under high glucose environment promoted proliferation of BMSCs and the formation of calcified nodules by regulating oxidative stress and activating PI3K signaling pathway.

RIPK2-Mediated Autophagy and Negatively Regulated ROS-NLRP3 Inflammasome Signaling in GMCs Stimulated with High Glucose

Background. Hyperglycemia plays a vital role in diabetic nephropathy (DN); autophagy and its potential upregulator receptor-interacting protein kinase 2 (RIPK2) are associated with ROS, which play a potential role in regulating NLRP3, and may be involved in inflammation in DN. Aim. In this study, we aimed to explore the mechanisms mediated by RIPK2 in autophagy and the relationship with ROS-NLRP3 of DN, by investigating the levels of RIPK2 and autophagy in glomerular mesangial cells (GMCs) stimulated with high glucose. Material and Methods. GMCs were divided into the following groups: normal group (NC), high glucose group (HG), and RIPK2 siRNA group. RIPK2, LC3, caspase1, and IL-1β levels were measured by western blotting and RT-PCR. Autophagosomes were measured by GFP-RFP-LC3; ROS were detected by DCFH-DA. Results. High glucose upregulated RIPK2 and LC3 in GMCs during short periods (0-12 h) (p<0.01), while RIPK2 and LC3 were significantly downregulated in the long term (12-72 h) (p<0.01); these changes were positively correlated with glucose concentration (p<0.01). In addition, levels of ROS, caspase1, and IL-1β increased in a time- and dose-dependent manner in the high glucose group, even with an increased expression of LC3 (p<0.01). However, LC3 expression decreased in the siRIPK2 group, while levels of ROS, caspase1, and IL-1β increased (p<0.01). Conclusions. Autophagy was activated by high glucose at short time periods but was inhibited in the long term, demonstrating a dual role for high glucose in autophagy of GMCs. RIPK2 regulates ROS-NLRP3 inflammasome signaling through autophagy and may be involved in the pathogenesis of DN.

TLR7 mediates increased vulnerability to ischemic acute kidney injury in diabetes

SUMMARY OBJECTIVE Diabetes is a risk factor for acute kidney injury (AKI). However, its mechanism of pathogenesis has not been elucidated. The aim of the study was to investigate the role of inflammation and the toll-like receptor 7 (TLR7) in ischemic AKI for diabetes. METHODS A high glucose hypoxia-reoxygenation model of human renal tubular epithelial (HK-2) cells was used to generate AKI induced by ischemia-reperfusion in diabetes. The activity of cells was measured by CCK-8 assay and LDH activity. Inflammatory cytokines were assessed by ELISA. TLR7, MyD88, and NF-κB expressions were examined by western blotting. Apoptosis was evaluated by flow cytometry. RESULTS The high glucose group and low glucose group were subjected to hypoxia-reoxygenation. The low glucose group developed only mild cell damage, apoptosis, and inflammatory response. In contrast, an equivalent hypoxia-reoxygenation injury provoked severe cell damage, apoptosis, and inflammatory response in the high glucose group. Expression of TLR7 and its related proteins were measured in the high glucose group before and after hypoxia-reoxygenation. The high glucose group exhibited more significant increases in TLR7 expression following hypoxia-reoxygenation than the low glucose group. In addition, the expression of TLR7 and its related proteins after hypoxia-reoxygenation were higher in the high glucose group than in the low glucose group. Inhibition of TLR7 provides significant protection against ischemic injury in diabetes. CONCLUSION Our results suggest that diabetes increases the vulnerability to ischemia-induced renal injury. This increased vulnerability originates from a heightened inflammatory response involving the TLR7 signal transduction pathway.