Prevention of diet-induced obesity by dietary isomerized hop extract containing isohumulones, in rodents.

OBJECTIVE: Isomerized hop extract (IHE), which consists mainly of isohumulones and is required in the beer brewing process, was investigated for its effects on diet-induced obesity in two strains of mice. DESIGN: C57BL/6N and KK-A(y) mice were fed a standard or high-fat diet containing IHE and their body and tissue weights were measured at various time points. Oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were carried out in high-fat diet-fed C57BL/6N mice. The effects of IHE on intestinal lipid absorption were examined in Wistar rats using a plasma triacylglycerol assay after oral administration of a lipid emulsion. Fecal lipid levels were also measured in these animals after they were fed a high-fat diet containing IHE for 15 days. The effects of IHE on pancreatic lipase activity and the expression of genes involved in hepatic lipid metabolism were also examined using an in vitro assay and quantitative RT-PCR, respectively. RESULTS: Supplementation of high-fat-containing chow with IHE reduced body weight gain and improved glucose tolerance in our experimental mice. A reduction in body weight gain was also observed in C57BL/6N mice fed a standard diet containing IHE. Wistar rats fed a high-fat diet containing IHE showed reduced plasma triacylglycerol levels and an increase in their fecal lipid excretion. Similarly, their pancreatic lipase activity was inhibited and their elevation in plasma triacylglycerol levels seen after the oral administration of lipid emulsion was significantly suppressed. IHE-fed mice showed an increased expression in their lipid oxidation genes and a decreased expression in genes involved in triacylglycerol biosynthesis. CONCLUSION: The inhibition of intestinal dietary fat absorption may be the mechanism by which IHE induces its weight-lowering effects in high-fat diet-fed mice. The modulatory effect of IHE on lipid metabolism may also, at least partly, be responsible for its beneficial effects on body weight gain. These results suggest that IHE may be helpful in humans in preventing diet-induced obesity and perhaps even metabolic syndrome, the latter of which is known to be associated with obesity.

Inhibition of RXR and PPARgamma ameliorates diet-induced obesity and type 2 diabetes.

PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.

Adiponectin is an adipocyte-derived hormone. Recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where the gene encoding adiponectin is located. Here we show that decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin decreases insulin resistance by decreasing triglyceride content in muscle and liver in obese mice. This effect results from increased expression of molecules involved in both fatty-acid combustion and energy dissipation in muscle. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin, but only partially by either adiponectin or leptin alone. We conclude that decreased adiponectin is implicated in the development of insulin resistance in mouse models of both obesity and lipoatrophy. These data also indicate that the replenishment of adiponectin might provide a novel treatment modality for insulin resistance and type 2 diabetes.

Mitochondrial uncoupling protein 3 (UCP3) in skeletal muscle.

Mitochondrial uncoupling protein (UCP), mitochondrial transporters, function as a proton channel and increase thermogenesis. UCP1 is expressed in brown adipose tissues (BAT), UCP2 is widely expressed in multiple tissues, while UCP3 is expressed in skeletal muscle. Thus, UCPs, especially UCP3, in skeletal muscles is a good candidates for prevention of obesity and diabetes. However, the role of UCP3 in skeletal muscle for energy expenditure and obesity has been controversial. There is some evidence that the UCP3 is possibly regulated by energy substrate, such as lipid and glucose. These observations suggest that increased energy substrate entry in muscle results in an increase in UCP3 expression which leads to an increase in energy expenditure.

Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice.

Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acid found in beef and dairy products. CLA has been reported to reduce body fat. To examine the mechanism(s) of CLA reduction of fat mass, female C57BL/6J mice were fed standard semipurified diets (10% fat of total energy) with or without CLA (1% wt/wt). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling (TUNEL) and DNA fragmentation analysis revealed that fat-mass decrease by CLA was mainly due to apoptosis. Tumor necrosis factor (TNF)-alpha and uncoupling protein (UCP)-2 mRNA levels increased 12- and 6-fold, respectively, in isolated adipocytes from CLA-fed mice compared with control mice. Because it is known that TNF-alpha induces apoptosis of adipocytes and upregulates UCP2 mRNA, a marked increase of TNF-alpha mRNA with an increase of UCP2 in adipocytes caused CLA-induced apoptosis. However, with a decrease of fat mass, CLA supplementation resulted in a state resembling lipoatrophic diabetes: ablation of brown adipose tissue, a marked reduction of white adipose tissue, marked hepatomegaly, and marked insulin resistance. CLA supplementation decreased blood leptin levels, but continuous leptin infusion reversed hyperinsulinemia, indicating that leptin depletion contributes to the development of insulin resistance. These results demonstrate that intake of CLA reduces adipose tissue by apoptosis and results in lipodystrophy, but hyperinsulinemia by CLA can be normalized by leptin administration.

Human cysteine dioxygenase gene: structural organization, tissue-specific expression and downregulation by phorbol 12-myristate 13-acetate.

The organization of the human cysteine dioxygenase (CDO) gene was found to be similar to its rat counterpart, and the location of the introns in the protein structure was identical to the rat CDO gene. The major transcription start site, identified by primer extension, was located 260 bp upstream from the ATG codon. The sequence of the 5'-immediate upstream region was highly conserved between the human and rat CDO genes. The putative promoter region contained a TATA-box-like sequence, and many putative cis-acting elements including HNF5, GRE, TRE, CRE, CArG box, ARE, MBS, and NF-kB. A Northern blot analysis revealed that CDO mRNA was strongly expressed in the liver and placenta, and weakly in the heart, brain and pancreas. CDO mRNA was also detected in human hepatoblastoma HepG2 cells. The CDO mRNA level in HepG2 cells was decreased after 2 h and reached a minimum 6 h-8 h after a phorbol 12-myristate 13-acetate (PMA) treatment, and then gradually returned to the basal level.

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle.

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.

Up-regulation of liver uncoupling protein-2 mRNA by either fish oil feeding or fibrate administration in mice.

Fish oil feeding showed less obesity in rodents, relative to other dietary oils. N-3 fatty acids rich in fish oil and fibrate compounds are peroxisome proliferator-activated receptor alpha (PPARalpha) ligands that stimulate beta-oxidation of fatty acids in liver and are used for treatment of hypertriglycemic patients. Since UCP-2, a member of an uncoupling protein family, has been shown to express in hepatocytes, the effects of these agents on the expression of UCP2 mRNA were investigated. C57BL/6J mice were divided into three groups; the first group was given a high-carbohydrate diet, and the other two groups were given a high-fat diet (60% of total energy) as safflower oil or fish oil for 5 months. Safflower oil diet fed mice developed obesity, but those fed fish oil diet did not. Therefore, the effects of fish oil feeding on the expression of UCP1, UCP2 and UCP3 in liver, skeletal muscle (gastrocnemius), white adipose tissue (WAT) and brown adipose tissue (BAT) were assessed by Northern blotting. Compared with safflower oil feeding, fish oil feeding up-regulated liver UCP2, BAT UCP2 and skeletal muscle UCP3 mRNA, while down-regulated WAT UCP2 and BAT UCP3 mRNA. Among these alterations, 5-fold up-regulation of liver UCP2 mRNA, relative to carbohydrate feeding, was noteworthy. Fenofibrate administration (about 500 mg/kg BW/d) for 2 wks also induced liver UCP2 expression by 9-fold. These data indicated that fish oil feeding and fibrate administration each up-regulated UCP2 mRNA expression in liver possibly via PPARalpha and hence each has the potential of increasing energy expenditure for prevention of obesity.

Up-regulation of uncoupling protein 3 (UCP3) mRNA by exercise training and down-regulation of UCP3 by denervation in skeletal muscles.

In skeletal muscles, increased utilization of lipids and carbohydrates accompanied with increased energy expenditure has been observed during and after exercise. UCP3, mitochondrial uncoupling protein, is expressed in skeletal muscles. We investigated UCP3 mRNA levels in exercise training mice which increased energy expenditure and in sciatic nerve-denervated mice which decreased energy expenditure. Mice exercised by 2 wk swimming had 14- to 18-fold increases of UCP3 mRNA in skeletal muscles 3 h after the last swimming, but no increases of UCP1 mRNA in BAT and of UCP2 mRNA in WAT. However, 22 h after exercise, UCP3 mRNA increases observed in skeletal muscles 3 h after exercise returned to sedentary levels. Similar transient increases of UCP3 mRNA were observed in 1 wk treadmill running training or a single exercise bout. In denerved gastrocnemius, GLUT4 and UCP3 mRNA decreased by 58 and 45%, respectively. These data indicate that UCP3 may have a role for fine adjustments of energy expenditure and that up-regulation of UCP3 mRNA may be a defense mechanism against extra energy supply to consume extra energy in skeletal muscles.