Lipogenesis in experimental chronic renal failure in rats.

Hyperlipidemia is a common occurance in patients with chronic renal failure (CRF) and has been the subject of many clinical and experimental studies. Despite this, the role of lipogenesis in the development of hyperlipidemia is still obscure. The present study is based on a rat model of CRF involving a two-stage subtotal nephrectomy. In this study, we measured the activity of fatty acid synthase (FAS). This is the rate-limiting enzyme of lipogenesis and is present in liver and white adipose tissue (WAT). Using isotopic methods, we also determined the rate of lipogenesis in vivo in liver and WAT. In both liver and WAT, the results of the analyses were similar. In the uremic rats, there was a tendency for the FAS activity to rise. However, the difference was not statistically significant. Furthermore, there was no increase in the rate of lipogenesis in vivo in either tissue. In summary, the results of our study confirm the thesis that lipogenesis does not play a role in the development of hypertriglyceridemia seen in an experimental CRF in rats.     

LIPOGENESIS IN EXPERIMENTAL CHRONIC RENAL FAILURE IN RATS

Hyperlipidemia is a common occurance in patients with chronic renal failure (CRF) and has been the subject of many clinical and experimental studies. Despite this, the role of lipogenesis in the development of hyperlipidemia is still obscure. The present study is based on a rat model of CRF involving a two-stage subtotal nephrectomy. In this study, we measured the activity of fatty acid synthase (FAS). This is the rate-limiting enzyme of lipogenesis and is present in liver and white adipose tissue (WAT). Using isotopic methods, we also determined the rate of lipogenesis in vivo in liver and WAT. In both liver and WAT, the results of the analyses were similar. In the uremic rats, there was a tendency for the FAS activity to rise. However, the difference was not statistically significant. Furthermore, there was no increase in the rate of lipogenesis in vivo in either tissue. In summary, the results of our study confirm the thesis that lipogenesis does not play a role in the development of hypertriglyceridemia seen in an experimental CRF in rats.     

Overexpressing TNF-Alpha in Pancreatic Ductal Adenocarcinoma Cells and Fibroblasts Modifies Cell Survival and Reduces Fatty Acid Synthesis via Downregulation of Sterol Regulatory Element Binding Protein-1 and Activation of Acetyl CoA Carboxylase

The effect of tumor necrosis factor-alpha (TNF-α) gene delivery has been suggested as a potentially useful therapeutic approach to improve the chemotherapeutic treatment of patients with pancreatic ductal adenocarcinoma (PDA), but the exact mechanism of its action is not clearly understood. In this study, we analyzed the expression profile of TNF-α in PDA tissue and explored its potential role in fatty acid synthase (FAS) regulation in PDA cells and in fibroblasts. Quantitative real-time polymerase chain reaction was used to examine the expression of TNF-α in PDA, matching adjacent tissues, and benign lesions. Logistic regression models with robust variance were used to analyze the gene expression levels, and Kaplan–Meier survival curves were generated. In vitro, we overexpressed the TNF-α gene in PDA cells and fibroblasts and analyzed its effect on cell survival, migration, and on members of the FAS signaling pathway. We also evaluated TNF-α effects on a panel of inflammation-, angiogenesis-, and metastasis-related markers. In the tumor tissue of PDA patients, compared with their matched adjacent tissue, expression levels of TNF-α were not statistically different and did not correlate with survival or any other examined clinicopathological features. Overexpression of TNF-α significantly (p?<?0.05) reduced PDA and fibroblast cell migration. In PDA cells that highly overexpress TNF-α, this was associated with a significant reduction of FAS mRNA and protein expression levels and significant (p?<?0.05) reduction of SREBP-1 and ACC mRNA. Reduction of FAS by TNF-α was inhibited when either SREBP-1 or ACC was knocked down by siRNA. PDA cells and fibroblasts that overexpress TNF-α displayed differential regulation of several inflammation-related markers and reduced levels of metastasis-related genes. Our data demonstrate a previously unknown multi-targeted involvement of TNF-α in PDA lipogenesis and inflammation and metastasis and suggest that intratumoral introduction of TNF-α may have the potential as a novel therapeutic approach in human PDA.     

Acute bidirectional manipulation of muscle glucose uptake by in vivo electrotransfer of constructs targeting glucose transporter genes

Analysis of conventional germ-line or tissue-specific gene manipulation in vivo is potentially confounded by developmental adaptation of animal physiology. We aimed to adapt the technique of in vivo electrotransfer (IVE) to alter local gene expression in skeletal muscle of rodents as a means of investigating the role of specific proteins in glucose metabolism in vivo. We utilized a square-wave electroporator to induce intracellular electrotransfer of DNA constructs injected into rat or mouse muscles and investigated the downstream effects. In initial studies, expression of green fluorescent protein reporter was induced in 53 +/- 10% of muscle fibers peaking at 7 days, and importantly, the electrotransfer procedure itself did not impact upon the expression of stress proteins or our ability to detect a reduction in 2-deoxyglucose tracer uptake by electroporated muscle of high-fat-fed rats during hyperinsulinemic-euglycemic clamp. To demonstrate functional effects of electrotransfer of constructs targeting glucose transporters, we administered vectors encoding GLUT-1 cDNA and GLUT-4 short hairpin RNAs (shRNAs) to rodent muscles. IVE of the GLUT-1 gene resulted in a 57% increase in GLUT-1 protein, accompanied by a proportionate increase in basal 2-deoxyglucose tracer uptake into muscles of starved rats. IVE of vectors expressing two shRNAs for GLUT-4 demonstrated to reduce specific protein expression and 2-deoxyglucose tracer uptake in 3T3-L1 adipocytes into mouse muscle caused a 51% reduction in GLUT-4 protein, associated with attenuated clearance of tracer to muscle after a glucose load. These results confirm that glucose transporter expression is largely rate limiting for glucose uptake in vivo and highlight the utility of IVE for the acute manipulation of muscle gene expression in the study of the role of specific proteins in glucose metabolism.     

Chronic exposure of human glomerular epithelial cells to high glucose concentration results in modulation of high-affinity glucose transporters expression

INTRODUCTION: GLUTs are specific membrane proteins that transport glucose down a concentration gradient. There have been few studies on their expression in the kidney. The aim of this study was to identify the expression of GLUTs 1, 3, and 4 in HGEC and their regulation under diabetic milieu. MATERIAL AND METHODS: An immortalized cell line of HGEC was used. Cells were cultured in medium containing 5 or 25 mM D-glucose. Western blotting and flow cytometry were used to examine the presence of GLUTs (1, 3, 4) and alterations in expression. RESULTS: Western blotting analysis revealed that GLUT-1 levels were increased by 53% in HGEC cultured under experimental diabetes compared to cells grown in 5mM glucose. GLUT-3 levels were also increased by 15% under diabetic conditions. GLUT-4 levels were decreased by 20% in diabetes. Fluorescence Activated Cell Sorting (FACS) analysis demonstrated that cell surface expression of GLUT-1 was increased by 28% in cells grown in 25mM glucose. High glucose concentration did not affect cell surface expression of GLUT-3 and GLUT-4. DISCUSSION: These findings suggest that depressed GLUT4 expression in glomerulus and overexpression of GLUT-1 and in a lesser extent of GLUT-3 may alter the glucose uptake in these cells. It has been suggested that the overexpression of GLUT-1 in glomerulus, being the major isoform, may lead to the initial pathologic hallmarks of diabetic nephropathy.     

Mechanical forces in diabetic kidney disease: a trigger for impaired glucose metabolism

Nephropathy is one of the major microvascular complications of diabetes, and both hemodynamic and metabolic stimuli participate in its development and progression toward ESRD. There is now a greater understanding of the molecular pathways that are activated by high glomerular capillary pressure and hyperglycemia and how they interplay to produce kidney pathology. The observation that overexpression of glucose transporter 1 (GLUT-1) in mesangial cells could induce a "diabetic cellular phenotype" has led to the postulation that the expression of GLUT-1 could be upregulated in glomeruli that are exposed to high pressure. This review suggests a mechanism by which mechanical forces may aggravate a metabolic insult by stimulating excessive cellular glucose uptake. Proposed is the existence of a self-maintaining cycle whereby a hemodynamic stimulus on glomerular cells induces GLUT-1 overexpression followed by greater glucose uptake and activation of intracellular glucose metabolic pathways, resulting in excess TGF-beta1 production. TGF-beta1 in turn, maintains overexpression of GLUT-1, perpetuating a signaling sequence that has, as its ultimate effect, increased extracellular matrix synthesis. This mechanical and metabolic coupling suggests a novel pathophysiologic mechanism of injury in the kidney in diabetes and possibly other glomerular diseases.     

Chronic Exposure of Human Glomerular Epithelial Cells to High Glucose Concentration Results in Modulation of High-Affinity Glucose Transporters Expression

Introduction. GLUTs are specific membrane proteins that transport glucose down a concentration gradient. There have been few studies on their expression in the kidney. The aim of this study was to identify the expression of GLUTs 1, 3, and 4 in HGEC and their regulation under diabetic milieu. Material and Methods. An immortalized cell line of HGEC was used. Cells were cultured in medium containing 5 or 25 mM D-glucose. Western blotting and flow cytometry were used to examine the presence of GLUTs (1, 3, 4) and alterations in expression. Results. Western blotting analysis revealed that GLUT-1 levels were increased by 53% in HGEC cultured under experimental diabetes compared to cells grown in 5mM glucose. GLUT-3 levels were also increased by 15% under diabetic conditions. GLUT-4 levels were decreased by 20% in diabetes. Fluorescence Activated Cell Sorting (FACS) analysis demonstrated that cell surface expression of GLUT-1 was increased by 28% in cells grown in 25mM glucose. High glucose concentration did not affect cell surface expression of GLUT-3 and GLUT-4. Discussion. These findings suggest that depressed GLUT4 expression in glomerulus and overexpression of GLUT-1 and in a lesser extent of GLUT-3 may alter the glucose uptake in these cells. It has been suggested that the overexpression of GLUT-1 in glomerulus, being the major isoform, may lead to the initial pathologic hallmarks of diabetic nephropathy.     

Postnatal increase in insulin-sensitive glucose transporter expression is associated with improved recovery of postischemic myocardial function

OBJECTIVE: Glucose is an important substrate for energy production in the developing heart. Increased glucose uptake rate and metabolism during ischemia and reperfusion are closely linked to postischemic myocardial recovery. The initial rate-limiting step for glycolysis is the transport of glucose across the plasma membrane by glucose transporters (GLUT-1 and GLUT-4). We hypothesized that changes in GLUT-1 and GLUT-4 expression in developing hearts lead to age-dependent adaptive changes in glucose uptake capacity and influence tolerance to ischemia. METHODS: Western-immunoblotting was performed to determine GLUT-1 and GLUT-4 expression in myocardial tissue from 1, 2, and 3-week-old and adult rabbits. Glucose uptake rate was measured with (31)P-nuclear magnetic resonance spectroscopy using 2-deoxyglucose as substrate in isolated perfused hearts. Hearts from same age rabbits were perfused in the Langendorff mode with crystalloid buffer or buffer plus a GLUT-4 specific antibody in order to determine GLUT-4 mediated effects on myocardial protection. The hearts were subjected to 30 minutes of normothermic ischemia followed by reperfusion. Cardiac contractile function measurements were obtained pre- and postischemia. Tissue lactate accumulation was measured in all groups at end-ischemia CONCLUSIONS: Insulin-regulated glucose transporter (GLUT-4) expression in the heart increased gradually after birth reaching nearly adult levels by 3 weeks of age. Corresponding with the higher amount of GLUT-4 protein, improved recovery of postischemic contractile function was seen in older hearts in association with increased anaerobic glycolytic capacity. Interventions to accelerate postnatal GLUT-4 expression may improve ischemic tolerance in the neonatal heart.     

Glucose transporter-1 gene expression is associated with pancreatic cancer invasiveness and MMP-2 activity

BACKGROUND: Overexpression of the facilitative glucose transporter-1 (GLUT-1) has been observed for a wide range of human cancers, with the degree of overexpression generally being inversely correlated with prognosis. We tested the effects of modulating GLUT-1 expression on pancreatic cancer cellular invasiveness. METHODS: GLUT-1 expression in MIAPaCa-2, PANC-1, BXPC-3, and CAPAN-2 cells was assayed using Western blotting. Cells were stably transfected with a GLUT-1 expression vector or a GLUT-1 RNA interference vector to alter GLUT-1 expression. Matrix metalloproteinase-2 (MMP-2) activity and expression were assayed using zymography and Western blotting, respectively. In vitro cellular invasiveness was assayed using Matrigel Boyden chambers, and in vivo metastatic potential was assessed using a nude mouse xenograft model. RESULTS: Variable baseline GLUT-1 expression levels were detected among the cell lines. Forced overexpression of GLUT-1 induced increases in MMP-2 expression and activity and in cellular invasiveness. GLUT-1 silencing induced reductions in MMP-2 expression and activity, cellular invasiveness, and metastatic potential in vivo. CONCLUSION: GLUT-1 promotes pancreatic cellular invasiveness. The therapeutic implications of this finding warrant further study.     

创伤后大鼠胰岛素信号转导与葡萄糖转运关系的研究

目的 探讨创伤后大鼠骨骼肌组织中胰岛素受体后信号转导通路的变化及其与葡萄糖转运能力之间的关系.方法 通过对大鼠实施小肠部分切除手术建立创伤模型,观察胰岛素受体后信号转导通路上关键蛋白--胰岛素受体底物-1(IRS-1)和蛋白激酶B(PKB又名Akt)的含量及磷酸化状态的改变.采用3H标记葡萄糖示踪法检测创伤后骨骼肌葡萄糖转运能力的变化,并分别测定大鼠骨骼肌组织中葡萄糖转运蛋白-4(GLUT-4)的mRNA和蛋白表达水平.结果 两组大鼠骨骼肌组织中IRS-1和PKB/Akt蛋白总量没有明显差异,但是创伤组骨骼肌中酪氨酸位点磷酸化的IRS-1蛋白含最较对照组下降了31%(P=0.018),而丝氨酸位点磷酸化的IRS-1蛋白含量较对照组增加了63%(P=0.000),相应的创伤组骨骼肌中磷酸化的PKB/Akt蛋白含量较对照组下降了48%(P=0.000).创伤组大鼠骨骼肌对葡萄糖的转运能力明显低于对照组(P<0.01).两组大鼠骨骼肌中GLUT-4 mRNA和蛋白总量的表达没有明显差异(P=0.805和P=0.702),但是创伤组大鼠骨骼肌细胞膜上的GLUT-4蛋白含量明显少于对照组(P=0.028).结论 创伤后大鼠骨骼肌组织中IRS-1蛋白酪氨酸位点磷酸化减弱而丝氨酸位点磷酸化增强,导致下游PKB/Akt蛋白活性降低,从而使得骨骼肌细胞膜上GLUT-4分布减少以及对葡萄糖的转运能力降低,这可能是导致大鼠创伤后胰岛素抵抗的机制之一.     

Effects of glucose deprivation or glucose instability on mesangial cells in culture

Mesangial cell (MC) abnormalities play a central role in diabetic nephropathy. Variations in plasma glucose levels may contribute to MC dysfunction. This study evaluated the effects of glucose deprivation or fluctuations on MC viability, glucose uptake, and mesangial matrix production. The expression levels of fibronectin and glucose transporters (GLUT-1 and GLUT-4) were determined. MCs were exposed to normal (NG, 10 mM), low (LG, 1 mM) or high (HG, 30 mM) glucose concentrations for 1, 4, 12, 24 and 72 h. Glucose oscillation was achieved by alternating the glucose concentration (LG, NG or HG) in the medium every 8, 12, and 4 h over a total of 24 h. LG induced a significant increase (90%) in glucose uptake dependent of GLUT-1, which was not followed by alteration in fibronectin expression. Fluctuations in glucose levels induced a rise in glucose uptake also mediated by GLUT-1. Fibronectin did not change in any oscillation group. Results suggest that, in spite of a rise in glucose uptake by MCs under low glucose availability, or exposed to glucose fluctuations, mesangial matrix overproduction did not occur. Thus, neither glucose deprivation nor glucose instability, at least during short periods, is involved in the mesangial matrix overproduction observed in diabetes.     

Overexpression of 1-acyl-glycerol-3-phosphate acyltransferase-alpha enhances lipid storage in cellular models of adipose tissue and skeletal muscle

Plasma nonesterified fatty acids (NEFA) at elevated concentrations antagonize insulin action and thus may play a critical role in the development of insulin resistance in type 2 diabetes. Plasma NEFA and glucose concentrations are regulated, in part, by their uptake into peripheral tissues. Cellular energy uptake can be increased by enhancing either energy transport or metabolism. The effects of overexpression of 1-acylglycerol-3-phosphate acyltransferase (AGAT)-alpha, which catalyzes the second step in triglyceride formation from glycerol-3-phosphate, was studied in 3T3-L1 adipocytes and C2C12 myotubes. In myotubes, overexpression of AGAT-alpha did not affect total [14C]glucose uptake in the presence or absence of insulin, whereas insulin-stimulated [14C]glucose conversion to cellular lipids increased significantly (33%, P = 0.004) with a concomitant decrease (-30%, P = 0.005) in glycogen formation. [3H]oleic acid (OA) uptake in AGAT-overexpressing myotubes increased 34% (P = 0.027) upon insulin stimulation. AGAT-alpha overexpression in adipocytes increased basal (130%, P = 0.04) and insulin-stimulated (27%, P = 0.01) [3H]OA uptake, increased insulin-stimulated glucose uptake (56%, P = 0.04) and conversion to cellular lipids (85%, P = 0.007), and suppressed basal (-44%, P = 0.01) and isoproterenol-stimulated OA release (-45%, P = 0.03) but not glycerol release. Our data indicate that an increase in metabolic flow to triglyceride synthesis can inhibit NEFA release, increase NEFA uptake, and promote insulin-mediated glucose utilization in 3T3-L1 adipocytes. In myotubes, however, AGAT-alpha overexpression does not increase basal cellular energy uptake, but can enhance NEFA uptake and divert glucose from glycogen synthesis to lipogenesis upon insulin stimulation.     

Metabolic response to glucose overload in surgical stress: Energy disposal in brown adipose tissue

The metabolic response to total parenteral nutrition (TPN) overload was investigated in clinical studies of surgically stressed humans and in experimental studies of surgically stressed animals. In both studies, all non-protein calories were administered as glucose, and subjects were divided into two groups, classified according to the amount of caloric supplementation after surgical stress, i.e., either overfed or appropriate (groups C1 and C2, respectively, in the clinical study, and groups E1 and E2, respectively, in the experimental study). In the clinical study, the postoperative energy expenditure in group C1 increased from the preoperative value, becoming significantly more elevated than that in group C2. The respiratory quotient (RQ) in group C1 exceeded 1.0, representing lipogenesis from carbohydrate, and the plasma norepinephrine concentration was also higher in group C1, indicating that lipolysis was likely to have been enhanced in this milieu. These findings imply that lipogenesis and lipolysis can occur simultaneously, constituting a futile cycle, and that this activated cycle could be a reason for the increased energy expenditure associated with overfeeding after surgical stress. To gather further evidence, we evaluated the rate of lipogenesis and lipolytic activity in white and brown adipose tissue (WAT; BAT) in an experimental study of surgically stressed rats. In BAT, both lipogenesis and lipolysis were activated in group E1. The results of both studies suggest that glucose overload in surgically stressed individuals increases energy expenditure by activating a futile cycle and that BAT is more involved in this cycle than WAT.     

Insulin regulation of hepatic glucose transporter protein is impaired in chronic pancreatitis.

OBJECTIVE: The effect of chronic pancreatitis and insulin on the expression of the hepatic facilitative glucose transporter protein (GLUT-2) was determined in rats. SUMMARY BACKGROUND DATA: Chronic pancreatitis is associated with diabetes mellitus or impaired glucose tolerance. Suppression of hepatic glucose production (HGP) by insulin is impaired, although the mechanism is unknown. METHODS: Normal rats, rats with chronic pancreatitis induced 12 to 16 weeks earlier by oleic acid injection into the pancreatic ducts, and sham-operated rats were studied. Isolated, single-pass liver perfusion was performed, during which glucagon (1.2 pM) was infused, with or without insulin (0.6 or 1.2 nM). The suppression of HGP production by insulin was compared with changes in GLUT-2 in the membrane fraction of liver biopsies obtained before and after hormone perfusion. RESULTS: Glycogen-rich (fed) livers of normal rats (n = 16) demonstrated a dose-dependent suppression of hepatic glucose production by insulin (50 +/- 5% HGP induced by glucagon alone during 1.2-nM insulin perfusion) and a dose-dependent decrease in GLUT-2 (30 +/- 13% of basal level during 1.2-nM insulin perfusion). Sham-operated rats (n = 6) also showed reductions in HGP (51 +/- 4%) and GLUT-2 (14 +/- 10%) during 1.2-nM insulin perfusion. In contrast, rats with chronic pancreatitis (n = 6) showed no suppression of HGP during 1.2-nM insulin perfusion, and an increase in GLUT-2 (+20 +/- 6%) after insulin perfusion (p < 0.02 vs. sham). CONCLUSIONS: Insulin suppresses glucagon-stimulated HGP in normal and sham-operated rats, and this reduction in HGP is associated with a decrease in the membrane-bound quantity of GLUT-2. In chronic pancreatitis, insulin suppression of HGP is absent, and this is accompanied by an increase in GLUT-2 in the hepatocyte membrane. The authors conclude that the insulin-mediated change in the level of hepatocyte GLUT-2 is impaired in chronic pancreatitis, and may contribute to the altered glucose metabolism observed commonly in this disease.     

Insulin-independent induction of sterol regulatory element-binding protein-1c expression in the livers of streptozotocin-treated mice

Insulin and glucose together have been previously shown to regulate hepatic sterol regulatory element-binding protein (SREBP)-1c expression. We sought to explore the nutritional regulation of lipogenesis through SREBP-1c induction in a setting where effects of sugars versus insulin could be distinguished. To do so, mice were insulin depleted by streptozotocin (STZ) administration and subjected to a fasting-refeeding protocol with glucose, fructose, or sucrose. Unexpectedly, the insulin-depleted mice exhibited a marked induction of SREBP-1c on all sugars, and this increase in SREBP-1c was even more dramatic than in the non-STZ-administered controls. The time course of changes in SREBP-1 induction varied depending on the type of sugars in both control and STZ-administered mice. Glucose refeeding gave a peak of SREBP-1c induction, whereas fructose refeeding caused slow and gradual increments, and sucrose refeeding fell between these two responses. Expression of various lipogenic enzymes were also gradually increased over time, irrespective of the types of sugars, with greater intensities in STZ-administered than in nontreated mice. In contrast, induction of hepatic glucokinase and suppression of phoshoenolpyruvate carboxykinase were insulin dependent in an early refed state. These data clearly demonstrate that nutritional regulation of SREBP-1c and lipogenic genes may be completely independent of insulin as long as sufficient carbohydrates are available.