Laurence Portois

Hepatic n-3 polyunsaturated fatty acid depletion promotes steatosis and insulin resistance in mice : genomic analysis of cellular targets.

Patients with non-alcoholic fatty liver disease are characterised by a decreased n-3/n-6 polyunsaturated fatty acid (PUFA) ratio in hepatic phospholipids. The metabolic consequences of n-3 PUFA depletion in the liver are poorly understood. We have reproduced a drastic drop in n-3 PUFA among hepatic phospholipids by feeding C57Bl/6J mice for 3 months with an n-3 PUFA depleted diet (DEF) versus a control diet (CT), which only differed in the PUFA content. DEF mice exhibited hepatic insulin resistance (assessed by euglycemic-hyperinsulinemic clamp) and steatosis that was associated with a decrease in fatty acid oxidation and occurred despite a higher capacity for triglyceride secretion. Microarray and qPCR analysis of the liver tissue revealed higher expression of all the enzymes involved in lipogenesis in DEF mice compared to CT mice, as well as increased expression and activation of sterol regulatory element binding protein-1c (SREBP-1c). Our data suggest that the activation of the liver X receptor pathway is involved in the overexpression of SREBP-1c, and this phenomenon cannot be attributed to insulin or to endoplasmic reticulum stress responses. In conclusion, n-3 PUFA depletion in liver phospholipids leads to activation of SREBP-1c and lipogenesis, which contributes to hepatic steatosis.

Prebiotic approach alleviates hepatic steatosis: Implication of fatty acid oxidative and cholesterol synthesis pathways

SCOPE Recent data suggest that gut microbiota contributes to the regulation of host lipid metabolism. We report how fermentable dietary fructo-oligosaccharides (FOS) control hepatic steatosis induced by n-3 PUFA depletion, which leads to hepatic alterations similar to those observed in non-alcoholic fatty liver disease patients. METHODS AND RESULTS C57Bl/6J mice fed an n-3 PUFA-depleted diet for 3 months were supplemented with FOS during the last 10 days of treatment. FOS-treated mice exhibited higher caecal Bifidobacterium spp. and lower Roseburia spp. content. Microarray analysis of hepatic mRNA revealed that FOS supplementation reduced hepatic triglyceride accumulation through a proliferator-activated receptor α-stimulation of fatty acid oxidation and lessened cholesterol accumulation by inhibiting sterol regulatory element binding protein 2-dependent cholesterol synthesis. Cultured precision-cut liver slices confirmed the inhibition of fatty acid oxidation. FOS effects were related to a decreased hepatic micro-RNA33 expression and to an increased colonic glucagon-like peptide 1 production. CONCLUSIONS The changes in gut microbiota composition by n-3 PUFA-depletion and prebiotics modulate hepatic steatosis by changing gene expression in the liver, a phenomenon that could implicate micro-RNA and gut-derived hormones. Our data underline the advantage of targeting the gut microbiota by colonic nutrients in the management of liver disease.

Hepatic steatosis in n-3 fatty acid depleted mice: focus on metabolic alterations related to tissue fatty acid composition.

BackgroundThere are only few data relating the metabolic consequences of feeding diets very low in n-3 fatty acids. This experiment carried out in mice aims at studying the impact of dietary n-3 polyunsaturated fatty acids (PUFA) depletion on hepatic metabolism.Resultsn-3 PUFA depletion leads to a significant decrease in body weight despite a similar caloric intake or adipose tissue weight. n-3 PUFA depleted mice exhibit hypercholesterolemia (total, HDL, and LDL cholesterol) as well as an increase in hepatic cholesteryl ester and triglycerides content. Fatty acid pattern is profoundly modified in hepatic phospholipids and triglycerides. The decrease in tissue n-3/n-6 PUFA ratio correlates with steatosis. Hepatic mRNA content of key factors involved in lipid metabolism suggest a decreased lipogenesis (SREBP-1c, FAS, PPARγ), and an increased β-oxidation (CPT1, PPARα and PGC1α) without modification of fatty acid esterification (DGAT2, GPAT1), secretion (MTTP) or intracellular transport (L-FABP). Histological analysis reveals alterations of liver morphology, which can not be explained by inflammatory or oxidative stress. However, several proteins involved in the unfolded protein response are decreased in depleted mice.Conclusionn-3 PUFA depletion leads to important metabolic alterations in murine liver. Steatosis occurs through a mechanism independent of the shift between β-oxidation and lipogenesis. Moreover, long term n-3 PUFA depletion decreases the expression of factors involved in the unfolded protein response, suggesting a lower protection against endoplasmic reticulum stress in hepatocytes upon n-3 PUFA deficiency.

Identification of a Glucose Response Element in the Promoter of the Rat Glucagon Receptor Gene

We cloned the 5′ upstream region of the rat glucagon receptor gene, demonstrating that the 5′ noncoding domain of the glucagon receptor mRNA contained two untranslated exons of 131 and 166 nucleotides (nt), respectively, separated by two introns of 0.6 and 3.2 kilobase pairs. We also observed an alternative splicing involving the 166-base pair exon. Cloning of up to 2 kilobase pairs of the newly identified genomic domain and transfection of various constructs driving a reporter gene, in pancreatic islet cell line INS-1, uncovered a strong glucose regulation of the promoter activity of plasmids containing up to nucleotide −868, or more, upstream from the transcriptional start point. This promoter activity displayed threshold-like behavior, with low activity of the promoter below 5 mm glucose, and maximal activation as of 10 mm glucose. This glucose regulation was mapped to a highly palindromic 19-nucleotide region between nt −545 and −527. Indeed, deletion or mutation of this sequence abolished the glucose regulation. This domain contained two palindromic “E-boxes” CACGTG and CAGCTG separated by 3 nt, a feature similar to the “L4 box” found in the pyruvate kinase L gene promoter. This is the first description of a G protein-coupled receptor gene promoter regulated by glucose.

Rapid cellular enrichment of eicosapentaenoate after a single intravenous injection of a novel medium-chain triacylglycerol:fish-oil emulsion in humans

BACKGROUND Dietary deficiency in n-3 (omega-3) polyunsaturated fatty acids (PUFAs) prevails in Western populations and potentially results in adverse health outcomes. To circumvent the slow n-3 PUFA incorporation in phospholipids of key cells after oral supplementation, a new preparation for intravenous bolus injection was developed with 20 g triacylglycerols/100 mL of a mixture of 80% medium-chain triacylglycerols (MCTs) and 20% fish oil (FO) (wt:wt), and 0.4 g alpha-tocopherol/100 mL of the same mixture. OBJECTIVE Our objective was to document the enrichment of n-3 PUFAs in leukocyte and platelet phospholipids after a bolus intravenous injection of MCT:FO in men. DESIGN Twelve healthy male subjects received injections over a 5-min period of 50 mL of either MCT:FO or a control MCT:long-chain triacylglycerol (MCT:LCT) emulsion containing 20 g triacylglycerols/100 mL with equal amounts (wt:wt) of MCT and soybean triacylglycerols (LCT) and containing 0.02 g alpha-tocopherol/100 mL; after an 8-wk interval, the subjects received injections of the other preparation. RESULTS Clinical and biological variables that assessed tolerance and safety remained unchanged. Plasma elimination was faster for MCT:FO than for MCT:LCT (half-life: 24.5 +/- 3.5 min compared with 32.9 +/- 3.0 min; P < 0.025). This was associated with a greater increase in the plasma nonesterified fatty acid concentration. The content of n-3 PUFAs, specifically eicosapentaenoic acid (20:5n-3), increased in leukocyte and platelet phospholipids within 60 min and > or =24 h after MCT:FO injection. CONCLUSION Bolus intravenous injection of a novel MCT:FO emulsion allows rapid enrichment of cells with n-3 PUFAs.

Pancreatic islet function in ω3 fatty acid-depleted rats

In order to gain information on the determinism of the perturbation of fuel homeostasis in situations characterized by a depletion in long-chain polyunsaturated ω3 fatty acids (ω3), the metabolic and hormonal status of ω3-depleted rats (second generation) was examined. When required, these rats were injected intravenously 120 min before sacrifice with a novel medium-chain triglyceride-fish oil emulsion able to provoke a rapid and sustained increase of the ω3 content in cell phospholipids. The measurement of plasma glucose, insulin, phospholipid, triglyceride, and unesterified fatty acid concentration indicated modest insulin resistance in the ω3-depleted rats. The plasma triglyceride and phospholipid concentrations were decreased in the ω3-depleted rats with abnormally low contribution of ω3 in both circulating and pancreatic islet lipids. The protein, insulin, and lipid content of the islets, as well as their intracellular and extracellular spaces, were little affected in the ω3-depleted rats. The metabolism of d-glucose in the islets of ω3-depleted rats was characterized by a lesser increase in d-[5-3H]glucose utilization and d-[U−14C]glucose oxidation in response to a given rise in hexose concentration and an abnormally low ratio between d-glucose oxidation and utilization. These abnormalities could be linked to an increased metabolism of endogenous fatty acids with resulting alteration of glucokinase kinetics. The release of insulin evoked by d-glucose, at a close-to-physiological concentration (8.3mM), was increased in the ω3-depleted rats, this being considered as consistent with their insulin resistance. Relative to such a release, that evoked by a further rise in d-glucose concentration or by non-glucidic nutrients was abnormally high in ω3-depleted rats, and restored to a normal level after of the intravenous injection of the ω3-rich medium-chain triglyceride-fish oil emulsion. Because the latter procedure failed to correct the perturbation of d-glucose metabolism in the islets of ω3-depleted rats, it is proposed that the anomalies in the secretory behaviour of islets in terms of their response to an increase in hexose concentration or non-nutrient secretagogues is mainly attributable to alteration in K+ and Ca2+ handling, as indeed recently documented in separate experiments.In order to gain information on the determinism of the perturbation of fuel homeostasis in situations characterized by a depletion in long-chain polyunsaturated omega3 fatty acids (omega3), the metabolic and hormonal status of omega3-depleted rats (second generation) was examined. When required, these rats were injected intravenously 120 min before sacrifice with a novel medium-chain triglyceride-fish oil emulsion able to provoke a rapid and sustained increase of the omega3 content in cell phospholipids. The measurement of plasma glucose, insulin, phospholipid, triglyceride, and unesterified fatty acid concentration indicated modest insulin resistance in the omega3-depleted rats. The plasma triglyceride and phospholipid concentrations were decreased in the omega3-depleted rats with abnormally low contribution of omega3 in both circulating and pancreatic islet lipids. The protein, insulin, and lipid content of the islets, as well as their intracellular and extracellular spaces, were little affected in the omega3-depleted rats. The metabolism of D-glucose in the islets of omega3-depleted rats was characterized by a lesser increase in D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation in response to a given rise in hexose concentration and an abnormally low ratio between D-glucose oxidation and utilization. These abnormalities could be linked to an increased metabolism of endogenous fatty acids with resulting alteration of glucokinase kinetics. The release of insulin evoked by D-glucose, at a close-to-physiological concentration (8.3 mM), was increased in the omega3-depleted rats, this being considered as consistent with their insulin resistance. Relative to such a release, that evoked by a further rise in D-glucose concentration or by non-glucidic nutrients was abnormally high in omega3-depleted rats, and restored to a normal level after of the intravenous injection of the omega3-rich medium-chain triglyceride-fish oil emulsion. Because the latter procedure failed to correct the perturbation of D-glucose metabolism in the islets of omega3-depleted rats, it is proposed that the anomalies in the secretory behaviour of islets in terms of their response to an increase in hexose concentration or non-nutrient secretagogues is mainly attributable to alteration in K+ and Ca2+ handling, as indeed recently documented in separate experiments.

Lipid peroxidation is not a prerequisite for the development of obesity and diabetes in high-fat-fed mice.

The mechanism, by which a high-fat (HF) diet could impair glucose metabolism, is not completely understood but could be related to inflammation, lipotoxicity and oxidative stress. Lipid peroxides have been proposed as key mediators of intracellular metabolic response. The purpose of the present study was to analyse, in mice fed with a HF diet, the possible association between obesity and glucose tolerance on the one hand, and between oxidative stress and lipid peroxidation on the other hand. The present results show that a HF diet (70 % energy as fat), v. a high-carbohydrate chow diet (control), increases body weight and fat mass development, and impairs glycaemia and insulinaemia within 4 weeks. It also promotes the expression of NADPH oxidase in the liver--signing both oxidative and inflammatory stress--but decreases thiobarbituric acid-reactive substances content in the liver as well as in epididymal, subcutaneous and visceral adipose tissues. HF diet, with elevated vitamin E content, induces high concentration of alpha-tocopherol in liver and adipose tissues, which contributes to the protection against lipid peroxidation. Thus, lipid peroxidation in key organs is not necessarily related to the development of metabolic disorders associated with diabetes and obesity.

Involvement of gut microbial fermentation in the metabolic alterations occurring in n-3 polyunsaturated fatty acids-depleted mice

BackroundWestern diet is characterized by an insufficient n-3 polyunsaturated fatty acid (PUFA) consumption which is known to promote the pathogenesis of several diseases. We have previously observed that mice fed with a diet poor in n-3 PUFA for two generations exhibit hepatic steatosis together with a decrease in body weight. The gut microbiota contributes to the regulation of host energy metabolism, due to symbiotic relationship with fermentable nutrients provided in the diet. In this study, we have tested the hypothesis that perturbations of the gut microbiota contribute to the metabolic alterations occurring in mice fed a diet poor in n-3 PUFA for two generations (n-3/- mice).MethodsC57Bl/6J mice fed with a control or an n-3 PUFA depleted diet for two generations were supplemented with prebiotic (inulin-type Fructooligosaccharides, FOS, 0.20 g/day/mice) during 24 days.Resultsn-3/-mice exhibited a marked drop in caecum weight, a decrease in lactobacilli and an increase in bifidobacteria in the caecal content as compared to control mice (n-3/+ mice). Dietary supplementation with FOS for 24 days was sufficient to increase caecal weight and bifidobacteria count in both n-3/+ and n-3/-mice. Moreover, FOS increased lactobacilli content in n-3/-mice, whereas it decreased their level in n-3/+ mice. Interestingly, FOS treatment promoted body weight gain in n-3/-mice by increasing energy efficiency. In addition, FOS treatment decreased fasting glycemia and lowered the higher expression of key factors involved in the fatty acid catabolism observed in the liver of n-3/-mice, without lessening steatosis.Conclusionsthe changes in the gut microbiota composition induced by FOS are different depending on the type of diet. We show that FOS may promote lactobacilli and counteract the catabolic status induced by n-3 PUFA depletion in mice, thereby contributing to restore efficient fat storage.

Perturbation of glycerol metabolism in hepatocytes from n3-PUFA-depleted rats

Second generation n3-PUFA-depleted rats represent a good animal model of metabolic syndrome as they display several features of the disease such as liver steatosis, visceral obesity and insulin resistance. The goal of our study was to investigate the influence of n3-PUFA deficiency on hepatic glycerol metabolism. Aquaglyceroporin 9 (AQP9) allows hepatic glycerol transport and consequently contributes to neoglucogenesis. AQP9 knockout mice display hypertriacyl-glycerolemia, one of the hallmarks of the metabolic syndrome. Our data show reduced AQP9 expression at the protein level in n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-¹⁴C]glycerol uptake was increased in hepatocytes from n3-PUFA-depleted animal cells. The apparent discrepancy between decreased AQP9 protein expression, and increased [U-¹⁴C]glycerol uptake could be explained by an observed increase in glycerol kinase activity.

Adverse effects of conjugated alpha-linolenic acids (CLnA) on lipoprotein profile on experimental atherosclerosis in hamsters

Conjugated linoleic acids (CLAs) such as rumenic acid (RA) have the potential to alter blood lipid profiles in animals and in humans. In contrast, physiological effects of conjugated α-linolenic acids (CLnAs), which concomitantly are omega-3 and conjugated fatty acids, are still unknown. The aim of this study was to evaluate the potential of CLnA to interfere in early steps of atherosclerosis by altering lipoprotein profiles and fatty streaks in the aortas. F1B hamsters were fed a control or one of the three hypercholesterolemic (HC) diets: HC-control, HC-RA (18:2 cis-9, trans-11) or HC-CLnA (CLnA: equimolar mixture of 18:3 cis-9, trans-11, cis-15 and cis-9, trans-13, cis-15) diet. In low-cholesterol control-fed hamsters, the proportion of high-density lipoprotein cholesterol (HDL-C) was around 45% while in HC-fed hamsters, HDL-C was around 10% and cholesterol was mostly (80%) carried by triglyceride-rich lipoproteins (TRL). Low-density lipoprotein (LDL) triglycerides (TGs) increased by approximately 60% in hamsters fed either HC-RA or HC-CLnA compared with HC-controls but not compared with the low-cholesterol control diet. HDL cholesterol decreased by 24% and 16% in hamsters fed HC-RA and HC-CLnA, respectively. Small dense LDL-cholesterol increased by approximately 60% in hamsters fed HC-RA and HC-CLnA compared with the HC-control group and by more than a 100% compared with hamsters on the control diet. The relative percentage of liver cholesteryl ester content increased by 88% in hamsters fed HC diets compared with the control diet. Significant differences in fatty streaks were observed between control and HC-diet-fed hamsters. However, no significant difference was observed among the HC-diet-fed hamsters. This study shows that animals fed any one of the HC diets developed an adverse lipoprotein profile compared with a normolipidic diet. Also, HC-RA or HC-CLnA diets altered lipoprotein profile compared with animals fed the HC-control diet but had no beneficial effects on atherosclerosis.