Chronic Elevated Glucose Research Articles

Augmented mitochondrial energy metabolism is an early response to chronic glucose stress in human pancreatic beta cells

Aims/hypothesisIn islets from individuals with type 2 diabetes and in islets exposed to chronic elevated glucose, mitochondrial energy metabolism is impaired. Here, we studied early metabolic changes and mitochondrial adaptations in human beta cells during chronic glucose stress.MethodsRespiration and cytosolic ATP changes were measured in human islet cell clusters after culture for 4 days in 11.1 mmol/l glucose. Metabolomics was applied to analyse intracellular metabolite changes as a result of glucose stress conditions. Alterations in beta cell function were followed using insulin secretion assays or cytosolic calcium signalling after expression of the calcium probe YC3.6 specifically in beta cells of islet clusters.ResultsAt early stages of glucose stress, mitochondrial energy metabolism was augmented in contrast to the previously described mitochondrial dysfunction in beta cells from islets of diabetic donors. Following chronic glucose stress, mitochondrial respiration increased (by 52.4%, p < 0.001) and, as a consequence, the cytosolic ATP/ADP ratio in resting human pancreatic islet cells was elevated (by 27.8%, p < 0.05). Because of mitochondrial overactivation in the resting state, nutrient-induced beta cell activation was reduced. In addition, chronic glucose stress caused metabolic adaptations that resulted in the accumulation of intermediates of the glycolytic pathway, the pentose phosphate pathway and the TCA cycle; the most strongly augmented metabolite was glycerol 3-phosphate. The changes in metabolites observed are likely to be due to the inability of mitochondria to cope with continuous nutrient oversupply. To protect beta cells from chronic glucose stress, we inhibited mitochondrial pyruvate transport. Metabolite concentrations were partially normalised and the mitochondrial respiratory response to nutrients was markedly improved. Furthermore, stimulus–secretion coupling as assessed by cytosolic calcium signalling, was restored.Conclusion/interpretationWe propose that metabolic changes and associated mitochondrial overactivation are early adaptations to glucose stress, and may reflect what happens as a result of poor blood glucose control. Inhibition of mitochondrial pyruvate transport reduces mitochondrial nutrient overload and allows beta cells to recover from chronic glucose stress.Graphical abstract

Anchored Gs‐coupled purinergic receptor regulation of L‐type CaV1.2 and vascular tone in diabetic hyperglycemia

Enhanced arterial myocyte contractility in response to elevated extracellular glucose (hyperglycemia) has been associated with protein kinase A (PKA) dependent phosphorylation of the CaV1.2 pore‐forming subunit (α1C) at serine 1928 (Ser1928), but how PKA is activated is unknown. Considering that glucose stimulates extracellular nucleotide release, we hypothesized that a Gs‐coupled purinergic receptor is a key upstream activator of PKA‐mediated L‐type Ca2+ channel (LTCC) potentiation and vasoconstriction by elevated glucose. Using human and murine arteries and arterial myocytes, we found protein expression of predicted molecular weight and sarcolemmal distribution for the only Gs‐coupled P2Y receptor P2Y11, as well as nanometer proximity of this receptor to CaV1.2 and PKA. A FRET‐based reporter confirmed that both elevated glucose and a specific P2Y11 activator stimulate cAMP production, and this was blocked in the presence of a specific P2Y11 inhibitor. Consistent with our hypothesis, the ectonucleotidase apyrase and a P2Y11 inhibitor, but not a Gq‐coupled P2Y6 inhibitor, prevented increased Ser1928 phosphorylation and LTCC activity in human and murine arterial myocytes and augmented vasoconstriction in murine arteries by elevated glucose. Similar results were observed by knocking down the expected P2Y11 protein. Comparable to glucose effects, a P2Y11 activator potentiated Ser1928 phosphorylation, LTCC activity, and vasoconstriction. Moreover, enhanced Ser1928 phosphorylation and LTCC activity upon chronic elevated glucose exposure were prevented in cells pretreated with the P2Y11 inhibitor. Altogether, our findings suggest a key role for a Gs‐coupled P2Y receptor that fits the profile of P2Y11 in regulation of LTCCs and vascular tone during diabetic hyperglycemia, with important clinical and therapeutic implications for diabetic hypertension.Support or Funding InformationThis work is supported by NIH R01HL098200 and R01HL121059.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 reduces pancreatic β-cells apoptosis in glucotoxicity through activation of autophagy

Chronic elevated glucose is harmful to pancreatic β-cells, resulting in pancreatic β-cells dysfunction and apoptosis. Understanding the molecular mechanisms associated with β-cells survival is pivotal for the prevention of β-cells injury caused by glucotoxicity. The role of Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) in the fate of pancreatic β-cells constantly exposed to high glucose was studied. Sustained high glucose increased PINK1 protein expression both in rat pancreatic β-cells and INS-1 β-cells, and that this increase can be inhibited by PINK1 knockdown and further enhanced by PINK1 over-expression. PINK1 deficiency aggravated glucotoxicity-induced pancreatic β-cells apoptosis and inhibition of autophagy whereas PINK1 could reverse these adverse effects. This study provides fundamental data supporting the potential protective role of PINK1 as a new therapeutic target necessary to preserve β-cells survival under non-physiological hyperglycemia conditions.

The hyperglycemic and hyperinsulinemic combo gives you diabetic kidney disease immediately. Focus on “Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney”

diabetic kidney disease (DKD) is the leading cause of end-stage kidney failure in the United States (11). Clinically, nephropathy induced by diabetes is characterized by albumin in the urine and declining glomerular filtration rate. On histological examination, diabetic kidney disease is a glomerular disease defined by mesangial expansion, nodular sclerosis, arterial hyalinosis (arteriolar thickening by deposition of pink hyaline material) leading to glomerulosclerosis and tubulointerstitial fibrosis. DKD is caused by hyperglycemia (HG) as reducing the glycohemoglobin levels from 9 to 7% in patients with type 1 diabetes resulted in a greater than 50% decrease in nephropathy development (3). However, recent studies of patients with type 2 diabetes failed to show a significant reduction in DKD progression when the glycohemoglobin target was reduced below 7% (4). Many of us would like to understand the reason for this surprising difference in nephropathy development and progression. One interpretation is that hyperglycemia is just one of the factors that play a role in the development of diabetic complications in type 2 diabetes and that hyperinsulinemia (HI), insulin resistance, and adiponectin could be equally important. In the current edition of the journal, Mariappan et al. (8) examine the effect of acute (7 h) combined hyperglycemia and hyperinsulinemia on profibrotic and proinflammatory gene expression in rat kidneys (8). The authors describe an approximately twofold increase in urinary isoprostane levels indicative of increased renal oxidative stress under these conditions. They observed a significant increase in renal TGF-β expression and its downstream mediator phospho-Smad3 (8). The expression of fibronectin and β1-laminin was also increased even after this short period of HI/HG. Increased expression of phosphorylated mTOR, S6 kinase, ERK, eIF4E, eIF4A are likely responsible for the increased matrix protein synthesis and kidney enlargement. The authors went on to identify the TLR4/Myd88 and NF-κB pathways as responsible for the increased cytokine expression in the context of hyperglycemia and hyperinsulinemia. In summary, the authors provide compelling evidence that acute HI combined with HG regulate the expression of multiple pathways known to play roles in DKD development. Unfortunately, the authors neglected to provide results from control rats exposed to hyperglycemia or hyperinsulinemia alone; thus, we cannot fully conclude whether these effects are mediated by glucose or the increased insulin levels. For example, we are left to wonder whether reactive oxygen species release is higher in HG/HI animals or lower compared with HG rats. In addition, the length of the “acute” treatment warrants further scrutiny of the time course of these changes. Do changes occur only acutely or are they still evident after prolonged hyperglycemia? Are changes reversible after periods of normoglycemia or are changes sustained? Acute changes in cellular metabolism can cause changes in the epigenome by providing more or less substrate for the key histone or DNA modifying enzymes (7). Cells cultured in high glucose-containing medium sustain changes in their histone modification patterns, for example, at the NF-κB locus, that are likely responsible for its sustained increased expression (5, 6, 9). Future studies will be necessary to determine whether the observed changes are reversible or sustained once euglycemic environment is reestablished. The study by Mariappan et al. is significant as it highlights the critical role of hyperinsulinemia in the development of diabetic kidney disease. Indeed, on a cellular level, it is possible that in the setting of both HI and HG, insulin shifts even more glucose into the intracellular compartment providing substrate for the mitochondria and increasing the reactive oxygen species release. Furthermore, the study provides experimental evidence for the notion that acute fluctuations of glucose and insulin levels may be just as damaging to cells as chronic elevated glucose levels (2). For example, in animal models of diabetes, increased reactive oxygen species release and podocyte apoptosis are observed right around the time when hyperglycemia develops and neither of these changes is sustained (10). As we do not routinely treat the hyperglycemia in diabetic mouse models, glucose levels are relatively constant in these animals, which could contribute to the fact that the diabetic mouse models do not develop progressive renal function decline (1). Clinical studies also support the notion that glycemic variability is a critical factor in the development of complications. In summary, this study provides the first description of acute HI/HG-induced kidney damage. In the future, this new model can be used to examine the patho-mechanism of DKD and develop new therapeutic interventions.

Reduced Expression of ATP-Binding Cassette Transporter G1 Increases Cholesterol Accumulation in Macrophages of Patients With Type 2 Diabetes Mellitus

Patients with type 2 diabetes mellitus are at increased risk for the development of atherosclerosis. A pivotal event in the development of atherosclerosis is macrophage foam cell formation. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 regulate macrophage cholesterol efflux and hence play a vital role in macrophage foam cell formation. We have previously found that chronic elevated glucose reduces ABCG1 expression. In the present study, we examined whether patients with type 2 diabetes mellitus had decreased ABCG1 and/or ABCA1, impaired cholesterol efflux, and increased macrophage foam cell formation. Blood was collected from patients with and without type 2 diabetes mellitus. Peripheral blood monocytes were differentiated into macrophages, and cholesterol efflux assays, immunoblots, histological analysis, and intracellular cholesteryl ester measurements were performed. Macrophages from patients with type 2 diabetes mellitus had a 30% reduction in cholesterol efflux with a corresponding 60% increase in cholesterol accumulation relative to control subjects. ABCG1 was present in macrophages from control subjects but was undetectable in macrophages from patients with type 2 diabetes mellitus. In contrast, ABCA1 expression in macrophages was similar in both control subjects and patients with type 2 diabetes mellitus. Macrophage expression of ABCG1 in both patients and control subjects was induced by treatment with the liver X receptor agonist TO-901317. Upregulation of liver X receptor dramatically reduced foam cell formation in macrophages from patients with type 2 diabetes mellitus. ABCG1 expression and cholesterol efflux are reduced in patients with type 2 diabetes mellitus. This impaired ABCG1-mediated cholesterol efflux significantly correlates with increased intracellular cholesterol accumulation. Strategies to upregulate ABCG1 expression and function in type 2 diabetes mellitus could have therapeutic potential for limiting the accelerated vascular disease observed in patients with type 2 diabetes mellitus.

S100B content and secretion decrease in astrocytes cultured in high-glucose medium

S100B is an astrocyte calcium-binding protein that plays a regulatory role in the cytoskeleton and cell cycle. Moreover, extracellular S100B, a marker of glial activation in several conditions of brain injury, has a trophic or apoptotic effect on neurons, depending on its concentration. Hyperglycemic rats show changes in glial parameters, including S100B expression. Here, we investigated cell density, morphological and biochemical alterations in primary cortical astrocytes from rats and C6 glioma cells cultured in high-glucose medium. Astrocytes and C6 glioma cells have a reduced content of S100B and glial fibrillary acidic protein when cultured in a high-glucose environment, as well as a reduced content of glutathione and cell proliferation rate. Although these cells have been used indistinctly to study S100B secretion, we observed a contrasting profile of S100B secretion in a high-glucose medium: a decrease in primary astrocytes and an increase in C6 glioma cells. Based on the in vitro neurotrophic effects of the S100B protein, our data suggest that chronic elevated glucose levels affect astrocyte activity, reducing extracellular secretion of S100B and that this, in turn, could affect neuronal activity and survival. Such astrocyte alterations could contribute to cognitive deficit and other impairments observed in diabetic patients.

Glucose-stimulated DNA Synthesis through Mammalian Target of Rapamycin (mTOR) Is Regulated by KATP Channels: EFFECTS ON CELL CYCLE PROGRESSION IN RODENT ISLETS

The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the K(ATP) channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of K(ATP) channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10-100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 microm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2-4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an approximately 62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.

Glucose Regulates Interleukin-8 Production in Aortic Endothelial Cells through Activation of the p38 Mitogen-activated Protein Kinase Pathway in Diabetes

We have shown that chronic elevated glucose (25 mm) increases monocyte adhesion to human aortic endothelial cells (EC). This increased adhesion is mediated primarily through induction of interleukin (IL)-8 via activation of the transcription factor AP-1 (Srinivasan, S., Yeh, M., Danziger, E. C., Hatley, M. E., Riggan, A. E., Leitinger, N., Berliner, J. A., and Hedrick, C. C. (2003) Circ. Res. 92, 371-377). In the current study, we identified the elements in the AP-1 transcriptional complex that are activated by glucose. These elements include c-Jun, c-Fos, and Fra-1. AP-1 is activated by cellular oxidative stress, and we have reported significant production of ROS by high glucose-cultured cells. We examined signaling pathways upstream of AP-1 in EC that lead to AP-1 activation by HG. EC cultured in 25 mm glucose had a 2-fold increase in p38 phosphorylation compared with control normal glucose-cultured EC. Inhibition of the p38 pathway using 5 microm SB203580 significantly reduced glucose-mediated IL-8 mRNA production by 60%. Furthermore, blocking p38 pathway activation using a dominant-negative p38 construct significantly reduced glucose-mediated monocyte adhesion by 50%. Thus, glucose-stimulated monocyte adhesion is primarily regulated through phosphorylation of p38 with subsequent activation of AP-1, leading to IL-8 production. To study this pathway in the setting of diabetes, we used the db/db mouse. P38 phosphorylation was increased in diabetic db/db mice compared with control mice. We found a dramatic elevation in plasma levels of KC, the mouse ortholog of IL-8 in diabetic db/db mice (1800 +/- 100 pg/ml KC in db/db versus 300 +/- 75 pg/ml in C57BL/6J control mice, p < 0.0001). Inhibition of the p38 pathway in diabetic db/db mice significantly reduced monocyte adhesion by 50%. Taken together, these data indicate that chronic elevated glucose in diabetes activates the p38 MAP kinase pathway to increase inflammatory IL-8 gene induction and monocyte/endothelial adhesion.

Induction of β-Cell Rest by a Kir6.2/SUR1-Selective KATP-Channel Opener Preserves β-Cell Insulin Stores and Insulin Secretion in Human Islets Cultured at High (11 mM) Glucose

In health, most insulin is secreted in pulses. Type 2 diabetes mellitus (TTDM) is characterized by impaired pulsatile insulin secretion with a defect in insulin pulse mass. It has been suggested that this defect is partly due to chronic overstimulation of beta-cells imposed by insulin resistance and hyperglycemia, which results in depletion of pancreatic insulin stores. It has been reported that in TTDM overnight inhibition of insulin secretion (induction of beta-cell rest) leads to quantitative normalization of pulsatile insulin secretion upon subsequent stimulation. Recently, decreased orderliness of insulin secretion has been recognized as another attribute of impaired insulin secretion in TTDM. In the current studies we sought to address at the level of the isolated islet whether chronic elevated glucose concentrations induce both defects involved in impaired insulin secretion in TTDM: deficiency and decreased orderliness of insulin secretion. We use the concept of beta-cell rest, induced by a novel beta-cell selective K(ATP)-channel opener (KCO), NN414 (6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide), to test whether preservation of insulin stores leads to normalization of both processes in response to glucose stimulation. Human islets were isolated from three cadaveric organ donors and studied in perifusion experiments and static incubation. Acute activation of K(ATP)-channels suppressed insulin secretion from perifused human islets by approximately 90% (P < 0.0001). This KCO also inhibited glucagon secretion in a dose-dependent manner (P = 0.01). Static incubation at 11 and 16 vs. 4 mM glucose for 96 h decreased islet insulin stores by approximately 80% and 85% (P < 0.0001, respectively). In subsequent perifusion experiments, total insulin secretion ( approximately 30%; P < 0.01) from these islets and insulin pulse mass ( approximately 40%; P < 0.05) were both decreased (11 vs. 4 mM). The inhibition of insulin secretion during static incubation with KCO reduced the loss of islet insulin stores in a dose-dependent manner (P < 0.0001) and resulted in increased total insulin secretion (2.6-fold; P < 0.01) and insulin pulse mass (2.5-fold; P < 0.05) during subsequent perifusion. The orderliness of insulin secretion was significantly reduced after chronic incubation of human islets at 11 mM glucose (P = 0.04), but induction of beta-cell rest at 11 mM failed to normalize the regularity of insulin secretion during subsequent perifusion. We conclude that physiological increased glucose concentrations (11 mM), which are frequently observed in diabetes, lead to a loss of islet insulin stores and defective pulsatile insulin secretion as well as reduced orderliness of insulin secretion. Induction of beta-cell rest by selective activation of beta-cell K(ATP)-channels preserves insulin stores and pulsatile insulin secretion without restoring the orderliness of insulin secretion. Therefore, the concept of beta-cell rest may provide a strategy to protect beta-cells from chronic overstimulation and to improve islet function. Impaired glucose-regulated insulin secretion in TTDM may, however, partially involve mechanisms that are distinct from insulin stores and insulin secretion rates.

Lisofylline, a novel antiinflammatory compound, protects mesangial cells from hyperglycemia- and angiotensin II-mediated extracellular matrix deposition.

Chronic elevated glucose levels and activation of the renal renin-angiotensin system have been implicated in the pathogenesis of diabetic nephropathy. We tested the ability of lisofylline (LSF), a novel antiinflammatory compound, to prevent extracellular matrix (ECM) accumulation and growth factor production by human mesangial cells (HMCs) cultured in chronic elevated glucose (HG) or angiotensin II (AngII). HMCs were cultured in normal glucose (NG) (5.5 mm) and in HG (25 mm) for 7 d or with 10-7 m AngII for 4 h with or without LSF. Levels of the ECM protein fibronectin and TGF-beta in media were shown to increase in HG compared with NG. LSF decreased HG-induced fibronectin and TGF-beta production to control levels. Increased expression of collagen type IV and laminin was observed in AngII-cultured HMCs. LSF protected HMCs from the AngII induction of these key matrix proteins. cAMP-responsive binding element phosphorylation was significantly higher in both HG and AngII-cultured HMCs. LSF reduced phosphorylation of both cAMP-responsive binding element and p38 MAPK compared with control. These data demonstrate that LSF protects HMCs from HG- and AngII-mediated ECM deposition by the reduction of matrix protein secretion possibly through regulation of TGF-beta production and modulation of the p38 MAPK pathway. These results suggest that LSF may provide therapeutic benefit for prevention or treatment of diabetic nephropathy.

Glucose Down-regulates the Expression of the Peroxisome Proliferator-activated Receptor-α Gene in the Pancreatic β-Cell

To better understand the action of glucose on fatty acid metabolism in the beta-cell and the link between chronically elevated glucose or fatty acids and beta-cell decompensation in adipogenic diabetes, we investigated whether glucose regulates peroxisomal proliferator-activated receptor (PPAR) gene expression in the beta-cell. Islets or INS(832/13) beta-cells exposed to high glucose show a 60-80% reduction in PPARalpha mRNA expression. Oleate, either in the absence or presence of glucose, has no effect. The action of glucose is dose-dependent in the 6-20 mm range and maximal after 6 h. Glucose also causes quantitatively similar reductions in PPARalpha protein and DNA binding activity of this transcription factor. The effect of glucose is blocked by the glucokinase inhibitor mannoheptulose, is partially mimicked by 2-deoxyglucose, and is not blocked by the 3-O-methyl or the 6-deoxy analogues of the sugar that are not phosphorylated. Chronic elevated glucose reduces the expression levels of the PPAR target genes, uncoupling protein 2 and acyl-CoA oxidase, which are involved in fat oxidation and lipid detoxification. A 3-day exposure of INS-1 cells to elevated glucose results in a permanent rise in malonyl-CoA, the inhibition of fat oxidation, and the promotion of fatty acid esterification processes and causes elevated insulin secretion at low glucose. The results suggest that a reduction in PPARalpha gene expression together with a rise in malonyl-CoA plays a role in the coordinated adaptation of beta-cell glucose and lipid metabolism to hyperglycemia and may be implicated in the mechanism of beta-cell "glucolipotoxicity."

Evidence that glucose increases monocyte binding to human aortic endothelial cells.

The rate of atherosclerosis is accelerated in humans with diabetes. The adhesion of monocytes to the vascular endothelium is a key event in the development of atherosclerosis. Alloxan (ALX)-induced diabetes in rabbits causes leukocyte accumulation on the arterial surface. However, the effect of glucose exposure on monocyte binding is not understood. We evaluated the effect of chronic elevated glucose on human monocyte binding to human aortic endothelial cells (HAEC) in culture. Monocyte binding to HAEC was significantly increased by chronic incubation of HAEC in high glucose for 7-10 days (CH-HG; 25 mM) compared with cells cultured for the same time in normal glucose (5.5 mM; CH-HG, 188 +/- 10 cells/field vs. normal glucose, 111 +/- 7; P < 0.0005). Use of mannitol at a concentration to stimulate the hyperosmolar effects of glucose did not significantly alter monocyte binding. Acute 20-min exposure of HAEC to high glucose did not alter monocyte binding. The adherence of HL-60 cells, a neutrophil-like cell line, or human neutrophils was not induced by CH-HG culture. High glucose-induced monocyte binding was not associated with induction of the major endothelial cell adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 (ICAM-1). A monoclonal antibody TS1-18 to the beta 2 integrin component that is involved in binding to ICAM-1 on endothelial cells significantly reduced monocyte binding, whereas anti-VLA-4 antibody was not effective. These results suggest that hyperglycemia can accelerate the rate of atherosclerosis in diabetics by increasing monocyte binding to the endothelium.