Regulation of the promoter region of the human adiponutrin/PNPLA3 gene by glucose and insulin.

The adiponutrin/PNPLA3 gene is highly expressed in adipose tissue and liver. Its expression is down-regulated by fasting and rapidly induced by refeeding a high carbohydrate diet. We aimed to determine whether the promoter region of adiponutrin is regulated by glucose and insulin. Endogenous adiponutrin mRNA was increased in mouse 3T3-L1 and human SGBS adipocytes and in human HepG2 cells cultured in 25 mM glucose compared to absence of glucose. A 3100 bp 5'-upstream region of the human adiponutrin gene was cloned into a luciferase reporter plasmid and used in transient transfection studies. Promoter activity was up-regulated by 25 mM glucose, 4.7-fold in HepG2 cells and 2-fold in CHO cells. The effect was shown in CHO cells to be concentration dependent and to depend on glucose metabolism as a non-metabolisable analogue was without effect. In CHO cells constitutively expressing human insulin receptor (CHO-IR), there was a concentration dependent increase of promoter activity by insulin in the presence of glucose. Cotransfection with an expression plasmid for upstream stimulatory factor 2 (USF2), increased promoter activity 1.6-fold in CHO-IR cells. The combined effect of insulin and USF2 (2.3-fold) was greater than the individual effects. Cotransfection of carbohydrate-response element binding protein did not elicit any induction of promoter activity. These results point to potential mechanisms for the observed in vivo nutritional regulation of adiponutrin expression and its up-regulation in fatty liver and by obesity.

A nitrogen balance experiment using simulated urine samples.

We describe an undergraduate laboratory experiment that combines the advantages of problem-based learning with the need for biochemistry students to become proficient in practical laboratory skills. It also avoids the need to obtain ethical approval for recruiting volunteers and eliminates any possible biosafety issues with the handling and disposal of large amounts of urine. Simulated human urine samples are prepared that contain urea, uric acid, and creatinine at concentrations that represent the levels expected in 2 liters of urine collected over 24 h from subjects on various protein diets or during different physiological states. The students measure the nitrogen-containing compounds in the "urine samples" using specific colorimetric assays and use the data they generate to derive knowledge about nitrogen balance and the excretory metabolism of amino acids.

Zinc protection of HepG2 cells from sporidesmin toxicity does not require de novo gene transcription.

Sporidesmin is an epidithiodioxopiperazine mycotoxin secreted by the saprophytic fungus Pithomyces chartarum. Ingestion of sporidesmin by ruminants grazing on the saprophyte infested pasture causes severe liver and bile duct damage leading to secondary photosensitisation. Zinc supplementation is used as an effective prophylaxis against sporidesmin toxicity in ruminants, however, the mechanism by which zinc protects is unknown. This study used the human hepatoma cell line, HepG2, as a model to examine the mechanism of zinc protection against sporidesmin toxicity. Treatment of cells with various concentrations of sporidesmin (0-10 microg/ml) resulted in a sigmoidal dose response curve with an LC50 of 5 microg/ml. Cells were protected from sporidesmin toxicity by pre-treatment for 2h or 16 h with zinc sulphate in a concentration dependent manner, with significant protection at 50 microM zinc and maximal protection at 200 microM zinc. To determine whether zinc protection required de novo gene transcription, cells were treated with the transcriptional inhibitor actinomycin D for one hour prior to and throughout the zinc pre-treatment. The presence of actinomycin D did not significantly reduce the zinc protection against sporidesmin cytotoxicity (80% protection without actinomycin D versus 71% protection with actinomycin D). Therefore, de novo gene transcription does not play a major role in the mechanism of zinc protection against sporidesmin toxicity in HepG2 cells.

Links between fatty acids and expression of UCP2 and UCP3 mRNAs.

Physiological and pathological states that are associated with elevated plasma fatty acids (FAs) increase uncoupling protein 2 (UCP2) mRNA in white adipose tissue and UCP3 mRNA in skeletal muscle and heart. A direct effect of unsaturated fatty acids from all classes has been shown in various cultured cells. There is evidence that FAs could induce expression of UCPs by acting as ligands for peroxisome proliferator-activated receptors, influencing the function of sterol responsive element binding protein or activating 5'-AMP-activated protein kinase. Oleic acid has been shown to stimulate the activity of the promoter regions of UCP2 and UCP3 genes and the FA responsive regions are beginning to be characterised.

Macronutrient composition of the diet differentially affects leptin and adiponutrin mRNA expression in response to meal feeding.

A number of adipose-specific genes, including adiponutrin and the adipocytokines, appear to be involved in regulating overall energy balance, as their expression is dysregulated in various obese states and is responsive to feeding. This study determined the effect of meal-feeding diets of markedly different macronutrient composition (70% by weight protein or fat) on the expression of adiponutrin and several adipocytokines in white adipose tissue of rats. Adiponutrin mRNA rapidly increased by at least 8-fold within 3 hours after the high-protein meal. This response was similar to that seen after a high-sucrose meal (70% by weight of sucrose). In contrast, leptin mRNA was unchanged after the high-protein meal, whereas it increased more than 5-fold after a high-sucrose meal. On the high-protein diet the leptin mRNA did not decline upon fasting after the meal, whereas on the high-sucrose diet fasting brought about a rapid decline in leptin mRNA, suggesting that the composition of the diet had altered mRNA turnover. In rats on diets high in either saturated or polyunsaturated fats, adiponutrin mRNA remained at fasting levels even after the meals. Leptin mRNA was unchanged and was maintained at post-meal levels. Resistin and acrp30/adiponectin mRNAs remained unchanged regardless of the macronutrient composition of the diet. The mechanism by which macronutrient composition of the diet is able to differentially influence the expression of adiponutrin and the adipocytokines, leptin, resistin, and acrp30/adiponectin remains to be determined.

Fatty acids and hypoxia stimulate the expression and secretion of the adipokine ANGPTL4 (angiopoietin-like protein 4/ fasting-induced adipose factor) by human adipocytes.

BACKGROUND/AIMS: Hypoxia occurs in white adipose tissue in obesity, modulating the expression and release of specific inflammation-related adipokines. ANGPTL4 (angiopoietin-like protein 4/fasting-induced adipose factor), which is implicated in angiogenesis, lipid metabolism and glucose homeostasis, is a major hypoxia-sensitive gene; recent studies indicate that ANGPTL4 expression is also regulated by fatty acids. We have examined the effects of hypoxia and fatty acids, alone and together, on the expression and release of ANGPTL4 by human adipocytes. METHODS: Human adipocytes were differentiated and incubated with fatty acids (250 µM) in normoxia (21% O(2)) or hypoxia (1% O(2)). ANGPTL4 mRNA was measured by real-time PCR and the protein in the medium determined by ELISA. RESULTS: In normoxia, ANGPTL4 gene expression was upregulated by palmitic, oleic, arachidonic and eicosapentaenoic acids, and ANGPTL4 release was increased. In contrast, there was no effect of lauric or myristic acids. Hypoxia alone increased the expression and secretion of ANGPTL4, and lauric, myristic, arachidonic and eicosapentaenoic acids each further increased expression and release in hypoxic adipocytes. CONCLUSION: The expression and secretion of ANGPTL4 by human adipocytes is upregulated by both hypoxia and fatty acids. The stimulatory effect of fatty acids on ANGPTL4 production is augmented under hypoxic conditions.

Adiponutrin mRNA expression in white adipose tissue is rapidly induced by meal-feeding a high-sucrose diet.

Adiponutrin is a recently identified gene of unknown function that is expressed exclusively in adipose tissues. To provide information about its physiological regulation and possible function, the effect of meal-feeding rats on the expression of adiponutrin mRNA in white adipose tissue was studied. A high-sucrose meal increased adiponutrin mRNA levels by at least 5-fold within 3 h. Post-meal levels returned to pre-meal levels with a half-life of about 5 h. The induction was prevented by injection of actinomycin-D prior to the meal. This pattern of expression was very similar to that seen for leptin mRNA. There were only minimal, or no, effects on acrp30/adiponectin, resistin, adipsin, or stearoyl-CoA desaturase 1. Adiponutrin appears more like leptin with respect to its acute regulation by meal-feeding than to any of the other adipokines or to enzymes directly involved in lipogenesis. This suggests that adiponutrin could be involved in overall energy homeostasis, as is leptin.