Development of in vitro model of insulin receptor cleavage induced by high glucose in HepG2 cells.

Soluble insulin receptor (sIR), the ectodomain of IR, has been detected in human plasma, and its concentration parallels that of blood glucose in patients with diabetes. IR has a pivotal role in glucose homeostasis and diabetes development; therefore, cleavage of IR promoted by hyperglycemia is involved in insulin resistance and glucose toxicity. To elucidate the physiology of sIR, we developed an in vitro model mimicking the changes in sIR levels in plasma from patients with diabetes. Among four human cell lines that expressed IR, spontaneous cleavage of IR occurred only in HepG2 cells. The molecular characteristics of sIR derived from HepG2 cells were similar to those of sIR detected in human plasma. The concentration of sIR in the medium did not differ between basal and high-glucose conditions in the initial 24-h period, but increasing the duration of pre-stimulation (>48 h) led to a significant increase in sIR levels in cells exposed to high glucose. Additionally, glucose-dependent increment of sIR was reversible in this model. These results are consistent with the observation of plasma sIR in patients with diabetes. Using this model, O-linked N-acetylglucosamine modification was determined to be involved in high-glucose-induced IR cleavage. A calcium-dependent protease was shown to cleave IR extracellularly. These findings show that this in vitro model could be useful for determining the molecular mechanism underlying IR cleavage.

Dietary combination of sucrose and linoleic acid causes skeletal muscle metabolic abnormalities in Zucker fatty rats through specific modification of fatty acid composition.

A dietary combination of sucrose and linoleic acid strongly contributes to the development of metabolic disorders in Zucker fatty rats. However, the underlying mechanisms of the metabolic disorders are poorly understood. We hypothesized that the metabolic disorders were triggered at a stage earlier than the 8 weeks we had previously reported. In this study, we investigated early molecular events induced by the sucrose and linoleic acid diet in Zucker fatty rats by comparison with other combinations of carbohydrate (sucrose or palatinose) and fat (linoleic acid or oleic acid). Skeletal muscle arachidonic acid levels were significantly increased in the sucrose and linoleic acid group compared to the other dietary groups at 4 weeks, while there were no obvious differences in the metabolic phenotype between the groups. Expression of genes related to arachidonic acid synthesis was induced in skeletal muscle but not in liver and adipose tissue in sucrose and linoleic acid group rats. In addition, the sucrose and linoleic acid group exhibited a rapid induction in endoplasmic reticulum stress and abnormal lipid metabolism in skeletal muscle. We concluded that the dietary combination of sucrose and linoleic acid primarily induces metabolic disorders in skeletal muscle through increases in arachidonic acid and endoplasmic reticulum stress, in advance of systemic metabolic disorders.

Induction of the hepatic stearoyl-CoA desaturase 1 gene in offspring after isocaloric administration of high fat sucrose diet during gestation.

Adverse early nutrition leads to metabolic aberrations in adulthood. Molecular and cellular mechanisms responsible are emerging; specific nutritional causes remain unclarified. We investigated gestational dietary intake and its influences on metabolism in offspring. Three groups of pregnant Sprague-Dawley rats were fed either AIN93G standard diet as control, isocaloric high fat sucrose diet or calorie restriction diet (50% of control) until delivery. All dams were fed control diet ad libitum during lactation. Offsprings' metabolic parameters were assessed at three weeks. Visceral fat and plasma triglycerides of high fat sucrose diet offspring were significantly higher than those of control diet and calorie restriction diet offspring. Plasma leptin level was higher in high fat sucrose diet than control offspring. Conversely, plasma adiponectin was lower in high fat sucrose diet and calorie restriction diet offspring compared to controls. Significant inductions of hepatic mRNA expression of stearoyl-CoA desaturase1 and Δ-5 desaturase genes, were observed in high fat sucrose diet and calorie restriction diet offspring. Gestational high sugar and fat intake even without over energy intake would be more detrimental to metabolisms of offspring compared to calorie restriction.

Protective effects of dipeptidyl peptidase-4 (DPP-4) inhibitor against increased ß cell apoptosis induced by dietary sucrose and linoleic acid in mice with diabetes.

Chronic exposure to high glucose and fatty acid levels caused by dietary sugar and fat intake induces ß cell apoptosis, leading to the exacerbation of type 2 diabetes. Oleic acid and linoleic acid are two major dietary fatty acids, but their effects in diabetes are unclear. We challenged ß cell-specific glucokinase haploinsufficient (Gck(+/-)) mice with a diet containing sucrose and oleic acid (SO) or sucrose and linoleic acid (SL) and analyzed ß cell apoptosis. In Gck(+/-) but not wild-type mice, SL significantly decreased the ß cell mass and ß cell proportion in islet cells arising from increased apoptosis to a greater degree than did SO. The mRNA expression of SREBP-1c was significantly higher, and that of E-cadherin was significantly lower in the islets of Gck(+/-) mice fed SL compared with mice fed SO. We next evaluated monotherapy with desfluorositagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, in these mouse groups. DPP-4 inhibitor protected against ß cell apoptosis, restored the ß cell mass, and normalized islet morphology in Gck(+/-) mice fed SL. DPP-4 inhibition normalized the changes in the islet expression of SREBP-1c and E-cadherin mRNA induced by the SL diet. Furthermore, linoleic acid induced ß cell apoptosis to a greater degree in the presence of high glucose levels than in the presence of low glucose levels in vitro in islets and MIN6 cells, whereas a GLP-1 receptor agonist prevented apoptosis. In conclusion, SL exacerbated ß cell apoptosis in diabetic Gck(+/-) mice but not in euglycemic wild-type mice, and DPP-4 inhibition protected against these effects.

Dietary phosphorus acutely impairs endothelial function.

Excessive dietary phosphorus may increase cardiovascular risk in healthy individuals as well as in patients with chronic kidney disease, but the mechanisms underlying this risk are not completely understood. To determine whether postprandial hyperphosphatemia may promote endothelial dysfunction, we investigated the acute effect of phosphorus loading on endothelial function in vitro and in vivo. Exposing bovine aortic endothelial cells to a phosphorus load increased production of reactive oxygen species, which depended on phosphorus influx via sodium-dependent phosphate transporters, and decreased nitric oxide production via inhibitory phosphorylation of endothelial nitric oxide synthase. Phosphorus loading inhibited endothelium-dependent vasodilation of rat aortic rings. In 11 healthy men, we alternately served meals containing 400 mg or 1200 mg of phosphorus in a double-blind crossover study and measured flow-mediated dilation of the brachial artery before and 2 h after the meals. The high dietary phosphorus load increased serum phosphorus at 2 h and significantly decreased flow-mediated dilation. Flow-mediated dilation correlated inversely with serum phosphorus. Taken together, these findings suggest that endothelial dysfunction mediated by acute postprandial hyperphosphatemia may contribute to the relationship between serum phosphorus level and the risk for cardiovascular morbidity and mortality.

Hypocaloric high-protein diet improves fatty liver and hypertriglyceridemia in sucrose-fed obese rats via two pathways.

The mechanism by which replacement of some dietary carbohydrates with protein during weight loss favors lipid metabolism remains obscure. In this study, we investigated the effect of an energy-restricted, high-protein/low-carbohydrate diet on lipid metabolism in obese rats. High-sucrose-induced obese rats were assigned randomly to one of two energy-restricted dietary interventions: a carbohydrate-based control diet (CD) or a high-protein diet (HPD). Lean rats of the same age were assigned as normal control. There was significantly greater improvement in fatty liver and hypertriglyceridemia with the HPD diet relative to the CD diet. Expression of genes regulated by fibroblast growth factor-21 (FGF21) and involved in liver lipolysis and lipid utilitization, such as lipase and acyl-CoA oxidase, increased in obese rats fed the HPD. Furthermore, there was an inverse correlation between levels of FGF21 gene expression (regulated by glucagon/insulin balance) and increased triglyceride concentrations in liver from obese rats. Expression of hepatic stearoyl-CoA desaturase-1 (SCD1), regulated primarily by the dietary carbohydrate, was also markedly reduced in the HPD group (similar to plasma triglyceride levels in fasting animals) relative to the CD group. In conclusion, a hypocaloric high-protein diet improves fatty liver and hypertriglyceridemia effectively relative to a carbohydrate diet. The two cellular pathways at work behind these benefits include stimulation of hepatic lipolysis and lipid utilization mediated by FGF21 and reduction of hepatic VLDL-TG production by SCD1 regulation.

Liver X receptor negatively regulates fibroblast growth factor 21 in the fatty liver induced by cholesterol-enriched diet.

Cholesterol homeostasis is regulated by the liver X receptor (LXR) at the transcriptional level, but it remains unknown whether LXR can affect expression levels of intrahepatic lipolysis related gene. Recent evidence has demonstrated that fibroblast growth factor 21 (FGF21) regulates hepatic lipolysis and fatty acid utilization. In the present study, we examined the role of LXR in FGF21 gene expression associated with regulation of cross-talk signals between cholesterol and triglyceride metabolism in the liver. An in vivo cholesterol feeding test revealed that intake of excess cholesterol increased cholesterol catabolism related gene expression as well as fatty-acid biosynthesis related gene expression. Moreover, the accumulated cholesterol suppressed FGF21 and hormone-sensitive lipase (HSL) gene expression. After 15-day cholesterol feeding, hepatic triglyceride concentrations were negatively correlated with expression levels of the FGF21 and HSL genes in the liver. An LXR agonist (TO-901317) repressed the FGF21 gene expression in mouse primary hepatocytes and HepG2 cells. A promoter deletion study and electrophoretic mobility shift assay revealed that the human FGF21 promoter has at least one LXR response element located from -37 to -22 bp. In summary, LXR represses FGF21 gene expression at the transcription level and might suppress lipolysis and lipid utilization to protect the liver from excess accumulation of toxic cholesterol.

Effects of xylitol on metabolic parameters and visceral fat accumulation.

Xylitol is widely used as a sweetener in foods and medications. Xylitol ingestion causes a small blood glucose rise, and it is commonly used as an alternative to high-energy supplements in diabetics. In previous studies, a xylitol metabolite, xylulose-5-phosphate, was shown to activate carbohydrate response element binding protein, and to promote lipogenic enzyme gene transcription in vitro; however, the effects of xylitol in vivo are not understood. Here we investigated the effects of dietary xylitol on lipid metabolism and visceral fat accumulation in rats fed a high-fat diet. Sprague-Dawley rats were fed a high-fat diet containing 0 g (control), 1.0 g/100 kcal (X1) or 2.0 g/100 kcal (X2) of xylitol. After the 8-week feeding period, visceral fat mass and plasma insulin and lipid concentrations were significantly lower in xylitol-fed rats than those in high-fat diet rats. Gene expression levels of ChREBP and lipogenic enzymes were higher, whereas the expression of sterol regulatory-element binding protein 1c was lower and fatty acid oxidation-related genes were significantly higher in the liver of xylitol-fed rats as compared with high-fat diet rats. In conclusion, intake of xylitol may be beneficial in preventing the development of obesity and metabolic abnormalities in rats with diet-induced obesity.

Paradoxical regulation of human FGF21 by both fasting and feeding signals: is FGF21 a nutritional adaptation factor?

Fibroblast growth factor 21 (FGF21) has recently emerged as a metabolic hormone involved in regulating glucose and lipid metabolism in mouse, but the regulatory mechanisms and actions of FGF21 in humans remain unclear. Here we have investigated the regulatory mechanisms of the human FGF21 gene at the transcriptional level. A deletion study of the human FGF21 promoter (-1672 to +230 bp) revealed two fasting signals, including peroxisome proliferator-activated receptor α (PPARα) and glucagon signals, that independently induced human FGF21 gene transcription in mouse primary hepatocytes. In addition, two feeding signals, glucose and xylitol, also dose-dependently induced human FGF21 gene transcription and mRNA expression in both human HepG2 cells and mouse primary hepatocytes. FGF21 protein expression and secretion were also induced by high glucose stimulation. The human FGF21 promoter (-1672 to +230 bp) was found to have a carbohydrate-responsive element at -380 to -366 bp, which is distinct from the PPAR response element (PPRE). Knock-down of the carbohydrate response element binding protein by RNAi diminished glucose-induced human FGF21 transcription. Moreover, we found that a region from -555 to -443 bp of the human FGF21 promoter region exerts an important role in the activation of basic transcription. In conclusion, human FGF21 gene expression is paradoxically and independently regulated by both fasting and feeding signals. These regulatory mechanisms suggest that human FGF21 is increased with nutritional crisis, including starvation and overfeeding.

Diet-induced adipose tissue inflammation and liver steatosis are prevented by DPP-4 inhibition in diabetic mice.

OBJECTIVE: Diet composition alters the metabolic states of adipocytes and hepatocytes in diabetes. The effects of dipeptidyl peptidase-4 (DPP-4) inhibition on adipose tissue inflammation and fatty liver have been obscure. We investigated the extrapancreatic effects of DPP-4 inhibition on visceral fat and the liver. RESEARCH DESIGN AND METHODS: We investigated diet-induced metabolic changes in ß-cell-specific glucokinase haploinsufficient (Gck(+/-)) diabetic mice. We challenged animals with a diet containing a combination of sucrose and oleic acid (SO) or sucrose and linoleic acid (SL). Next, we assessed the effects of a DPP-4 inhibitor, des-fluoro-sitagliptin, on adipose tissue inflammation and hepatic steatosis. RESULTS: The epididymal fat weight and serum leptin level were significantly higher in Gck(+/-) mice fed SL than in mice fed SO, although no significant differences in body weight or adipocyte size were noted. Compared with SO, SL increased the numbers of CD11c(+) M1 macrophages and CD8(+) T-cells in visceral adipose tissue and the expression of E-selectin, P-selectin, and plasminogen activator inhibitor-1 (PAI-1). DPP-4 inhibition significantly prevented adipose tissue infiltration by CD8(+) T-cells and M1 macrophages and decreased the expression of PAI-1. The production of cytokines by activated T-cells was not affected by DPP-4 inhibition. Furthermore, DPP-4 inhibition prevented fatty liver in both wild-type and Gck(+/-) mice. DPP-4 inhibition also decreased the expressions of sterol regulatory element-binding protein-1c, stearoyl-CoA desaturase-1, and fatty acid synthase, and increased the expression of peroxisome proliferator-activated receptor-α in the liver. CONCLUSIONS: Our findings indicated that DPP-4 inhibition has extrapancreatic protective effects against diet-induced adipose tissue inflammation and hepatic steatosis.