Effects of Totum-63 on glucose homeostasis and postprandial glycemia: a translational study.

Global prevalence of type 2 diabetes (T2D) is rising and may affect 700 million people by 2045. Totum-63 is a polyphenol-rich natural composition developed to reduce the risk of T2D. We first investigated the effects of Totum-63 supplementation in high-fat diet (HFD)-fed mice for up to 16 wk and thereafter assessed its safety and efficacy (2.5 g or 5 g per day) in 14 overweight men [mean age 51.5 yr, body mass index (BMI) 27.6 kg·m-2] for 4 wk. In HFD-fed mice, Totum-63 reduced body weight and fat mass gain, whereas lean mass was unchanged. Moreover, fecal energy excretion was higher in Totum-63-supplemented mice, suggesting a reduction of calorie absorption in the digestive tract. In the gut, metagenomic analyses of fecal microbiota revealed a partial restoration of HFD-induced microbial imbalance, as shown by principal coordinate analysis of microbiota composition. HFD-induced increase in HOMA-IR score was delayed in supplemented mice, and insulin response to an oral glucose tolerance test was significantly reduced, suggesting that Totum-63 may prevent HFD-related impairments in glucose homeostasis. Interestingly, these improvements could be linked to restored insulin signaling in subcutaneous adipose tissue and soleus muscle. In the liver, HFD-induced steatosis was reduced by 40% (as shown by triglyceride content). In the subsequent study in men, Totum-63 (5 g·day-1) improved glucose and insulin responses to a high-carbohydrate breakfast test (84% kcal carbohydrates). It was well tolerated, with no clinically significant adverse events reported. Collectively, these data suggest that Totum-63 could improve glucose homeostasis in both HFD-fed mice and overweight individuals, presumably through a multitargeted action on different metabolic organs.NEW & NOTEWORTHY Totum-63 is a novel polyphenol-rich natural composition developed to reduce the risk of T2D. Totum-63 showed beneficial effects on glucose homeostasis in HFD-fed mice, presumably through a multitargeted action on different metabolic organs. Totum-63 was well tolerated in humans and improved postprandial glucose and insulin responses to a high-carbohydrate breakfast test.

PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells.

UNLABELLED: Although non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease there is no pharmacological agent approved for its treatment. Since peroxisome proliferator-activated receptors (PPARs) are closely associated with hepatic lipid metabolism, they seem to play important roles in NAFLD. However, the effects of PPAR agonists on steatosis that is a common pathology associated with NAFLD, remain largely controversial. In this study, the effects of various PPAR agonists, i.e. fenofibrate, bezafibrate, troglitazone, rosiglitazone, muraglitazar and tesaglitazar on oleic acid-induced steatotic HepaRG cells were investigated after a single 24-hour or 2-week repeat treatment. Lipid vesicles stained by Oil-Red O and triglycerides accumulation caused by oleic acid overload, were decreased, by up to 50%, while fatty acid oxidation was induced after 2-week co-treatment with PPAR agonists. The greatest effects on reduction of steatosis were obtained with the dual PPARα/γ agonist muraglitazar. Such improvement of steatosis was associated with up-regulation of genes related to fatty acid oxidation activity and down-regulation of many genes involved in lipogenesis. Moreover, modulation of expression of some nuclear receptor genes, such as FXR, LXRα and CAR, which are potent actors in the control of lipogenesis, was observed and might explain repression of de novo lipogenesis. CONCLUSION: Altogether, our in vitro data on steatotic HepaRG cells treated with PPAR agonists correlated well with clinical investigations, bringing a proof of concept that drug-induced reversal of steatosis in human can be evaluated in in vitro before conducting long-term and costly in vivo studies in animals and patients.

Progressive endoplasmic reticulum stress contributes to hepatocarcinogenesis in fatty acyl-CoA oxidase 1-deficient mice.

Fatty acyl-coenzyme A oxidase 1 (ACOX1) knockout (ACOX1(-/-)) mice manifest hepatic metabolic derangements that lead to the development of steatohepatitis, hepatocellular regeneration, spontaneous peroxisome proliferation, and hepatocellular carcinomas. Deficiency of ACOX1 results in unmetabolized substrates of this enzyme that function as biological ligands for peroxisome proliferator-activated receptor-α (PPARα) in liver. Here we demonstrate that sustained activation of PPARα in ACOX1(-/-) mouse liver by these ACOX1 substrates results in endoplasmic reticulum (ER) stress. Overexpression of transcriptional regulator p8 and its ER stress-related effectors such as the pseudokinase tribbles homolog 3, activating transcription factor 4, and transcription factor CCAAT/-enhancer-binding protein homologous protein as well as phosphorylation of eukaryotic translation initiation factor 2α, indicate the induction of unfolded protein response signaling in the ACOX1(-/-) mouse liver. We also show here that, in the liver, p8 is a target for all three PPAR isoforms (-α, -ß, and -γ), which interact with peroxisome proliferator response elements in p8 promoter. Sustained activation of p8 and unfolded protein response-associated ER stress in ACOX1(-/-) mouse liver contributes to hepatocyte apoptosis and liver cell proliferation culminating in the development of hepatocarcinogenesis. We also demonstrate that human ACOX1 transgene is functional in ACOX1(-/-) mice and effectively prevents metabolic dysfunctions that lead to ER stress and carcinogenic effects. Taken together, our data indicate that progressive PPARα- and p8-mediated ER stress contribute to the hepatocarcinogenesis in ACOX1(-/-) mice.

Transcription coactivator mediator subunit MED1 is required for the development of fatty liver in the mouse.

UNLABELLED: Peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor, when overexpressed in liver stimulates the induction of adipocyte-specific and lipogenesis-related genes and causes hepatic steatosis. We report here that Mediator 1 (MED1; also known as PBP or TRAP220), a key subunit of the Mediator complex, is required for high-fat diet-induced hepatic steatosis as well as PPARγ-stimulated adipogenic hepatic steatosis. Mediator forms the bridge between transcriptional activators and RNA polymerase II. MED1 interacts with nuclear receptors such as PPARγ and other transcriptional activators. Liver-specific MED1 knockout (MED1(ΔLiv) ) mice, when fed a high-fat (60% kcal fat) diet for up to 4 months failed to develop fatty liver. Similarly, MED1(ΔLiv) mice injected with adenovirus-PPARγ (Ad/PPARγ) by tail vein also did not develop fatty liver, whereas mice with MED1 (MED1(fl/fl) ) fed a high-fat diet or injected with Ad/PPARγ developed severe hepatic steatosis. Gene expression profiling and northern blot analyses of Ad/PPARγ-injected mouse livers showed impaired induction in MED1(ΔLiv) mouse liver of adipogenic markers, such as aP2, adipsin, adiponectin, and lipid droplet-associated genes, including caveolin-1, CideA, S3-12, and others. These adipocyte-specific and lipogenesis-related genes are strongly induced in MED1(fl/fl) mouse liver in response to Ad/PPARγ. Re-expression of MED1 using adenovirally-driven MED1 (Ad/MED1) in MED1(ΔLiv) mouse liver restored PPARγ-stimulated hepatic adipogenic response. These studies also demonstrate that disruption of genes encoding other coactivators such as SRC-1, PRIC285, PRIP, and PIMT had no effect on hepatic adipogenesis induced by PPARγ overexpression. CONCLUSION: We conclude that transcription coactivator MED1 is required for high-fat diet-induced and PPARγ-stimulated fatty liver development, which suggests that MED1 may be considered a potential therapeutic target for hepatic steatosis. (HEPATOLOGY 2011;).

Reversal of mouse Acyl-CoA oxidase 1 (ACOX1) null phenotype by human ACOX1b isoform [corrected].

Disruption of the peroxisomal acyl-CoA oxidase 1 (Acox1) gene in the mouse results in the development of severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-alpha (PPARalpha). These mice manifest spontaneous massive peroxisome proliferation in regenerating hepatocytes and eventually develop hepatocellular carcinomas. Human ACOX1, the first and rate-limiting enzyme of the peroxisomal beta-oxidation pathway, has two isoforms including ACOX1a and ACOX1b, transcribed from a single gene. As ACOX1a shows reduced activity toward palmitoyl-CoA as compared with ACOX1b, we used adenovirally driven ACOX1a and ACOX1b to investigate their efficacy in the reversal of hepatic phenotype in Acox1(-/-) mice. In this study, we show that human ACOX1b is markedly effective in reversing the ACOX1 null phenotype in the mouse. In addition, expression of human ACOX1b was found to restore the production of nervonic (24:1) acid and had a negative impact on the recruitment of coactivators to the PPARalpha-response unit, which suggests that nervonic acid might well be an endogenous PPARalpha antagonist, with nervonoyl-CoA probably being the active form of nervonic acid. In contrast, restoration of docosahexaenoic (22:6) acid level, a retinoid-X-receptor (RXRalpha) agonist, was dependent on the concomitant hepatic expression of both ACOX1a and ACOX1b isoforms. This is accompanied by a specific recruitment of RXRalpha and coactivators to the PPARalpha-response unit. The human ACOX1b isoform is more effective than the ACOX1a isoform in reversing the Acox1 null phenotype in the mouse. Substrate utilization differences between the two ACOX1 isoforms may explain the reason why ACOX1b is more effective in metabolizing PPARalpha ligands.

Nuclear receptors and transcription factors in the development of fatty liver disease.

Liver regulates certain key aspects of lipid metabolism including de novo lipogenesis, fatty acid oxidation, and lipoprotein uptake and secretion. Disturbances in these hepatic functions can contribute to the development of fatty liver disease. An understanding of the regulatory mechanisms influencing hepatic lipid homeostasis and systemic energy balance is therefore of paramount importance in gaining insights that might be useful in the management of fatty liver disease. In this regard, emerging evidence indicates that certain members of the nuclear receptor superfamily and some key transcription coactivators function as intracellular sensors to orchestrate hepatic lipid metabolism. Dysregulation of nuclear receptor-mediated transcriptional signaling and perturbations in the levels of their cognate endogenous ligands play a prominent role in the development of fatty liver disease. The potential of nuclear receptors, transcription coactivators as well as enzymes that participate in the synthesis and degradation of endogenous nuclear receptor ligands, as effective therapeutic targets for fatty liver disease needs evaluation.

Coactivators in PPAR-Regulated Gene Expression.

Peroxisome proliferator-activated receptor (PPAR)alpha, beta (also known as delta), and gamma function as sensors for fatty acids and fatty acid derivatives and control important metabolic pathways involved in the maintenance of energy balance. PPARs also regulate other diverse biological processes such as development, differentiation, inflammation, and neoplasia. In the nucleus, PPARs exist as heterodimers with retinoid X receptor-alpha bound to DNA with corepressor molecules. Upon ligand activation, PPARs undergo conformational changes that facilitate the dissociation of corepressor molecules and invoke a spatiotemporally orchestrated recruitment of transcription cofactors including coactivators and coactivator-associated proteins. While a given nuclear receptor regulates the expression of a prescribed set of target genes, coactivators are likely to influence the functioning of many regulators and thus affect the transcription of many genes. Evidence suggests that some of the coactivators such as PPAR-binding protein (PBP/PPARBP), thyroid hormone receptor-associated protein 220 (TRAP220), and mediator complex subunit 1 (MED1) may exert a broader influence on the functions of several nuclear receptors and their target genes. Investigations into the role of coactivators in the function of PPARs should strengthen our understanding of the complexities of metabolic diseases associated with energy metabolism.

Biochemical characterization of two functional human liver acyl-CoA oxidase isoforms 1a and 1b encoded by a single gene.

Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.