Effect of carbohydrate overfeeding on whole body and adipose tissue metabolism in humans

OBJECTIVE: To evaluate the effect of a 4-day carbohydrate overfeeding on whole body net de novo lipogenesis and on markers of de novo lipogenesis in subcutaneous adipose tissue of healthy lean humans. RESEARCH METHODS AND PROCEDURES: Nine healthy lean volunteers (five men and four women) were studied after 4 days of either isocaloric feeding or carbohydrate overfeeding. On each occasion, they underwent a metabolic study during which their energy expenditure and net substrate oxidation rates (indirect calorimetry), and the fractional activity of the pentose-phosphate pathway in subcutaneous adipose tissue (subcutaneous microdialysis with 1,6(13)C2,6,6(2)H2 glucose) were assessed before and after administration of glucose. Adipose tissue biopsies were obtained at the end of the experiments to monitor mRNAs of key lipogenic enzymes. RESULTS: Carbohydrate overfeeding increased basal and postglucose energy expenditure and net carbohydrate oxidation. Whole body net de novo lipogenesis after glucose loading was markedly increased at the expense of glycogen synthesis. Carbohydrate overfeeding also increased mRNA levels for the key lipogenic enzymes sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase. The fractional activity of adipose tissue pentose-phosphate pathway was 17% to 22% and was not altered by carbohydrate overfeeding. DISCUSSION: Carbohydrate overfeeding markedly increased net de novo lipogenesis at the expense of glycogen synthesis. An increase in mRNAs coding for key lipogenic enzymes suggests that de novo lipogenesis occurred, at least in part, in adipose tissue. The pentose-phosphate pathway is active in adipose tissue of healthy humans, consistent with an active role of this tissue in de novo lipogenesis.     

Hormonal and nutritional regulation of lipogenic enzyme mRNA levels in rat primary white and brown adipocytes

Hyroid hormone stimulates hepatic lipogenesis in the rat by increasing the expression of relevant genes, including acetyl-CoA carboxylase and fatty acid synthase. S14 mRNA, which encodes a protein thought to be involved in lipogenesis, responds in parallel. The effects of thyroid hormone on lipogenesis in white and brown adipose tissue are less clear, and may be complicated by indirect effects of the hormone. Rat white and brown preadipocytes were therefore isolated, grown to confluence, and used to test direct effects of thyroid hormone, insulin, and glucose. Lipogenesis was assessed by tritiated water incorporation, and acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and S14 mRNAs were measured by Northern analysis. Insulin (1 nM) increased lipogenesis about 9-fold in both white and brown adipocytes. Similar increases were seen in the levels of the three mRNAs. Thyroid hormone (1 microM) stimulated lipogenesis and acetyl-CoA carboxylase, fatty acid synthase, and S14 mRNA levels up to 2-fold in both types of adipocyte in the presence or absence of insulin. A high carbohydrate level (25 mM glucose) had no effect on lipogenesis compared to a low carbohydrate level (5 mM glucose) in white and brown adipocytes. There was no synergistic effect on lipogenesis by the combination of thyroid hormone and high carbohydrate level in both types of adipocytes. These experiments have shown that T3 has small, direct stimulatory effects on lipogenesis in adipocytes. These effects are seen at a pre-translational level, through the coordinate induction of ACC, FAS, and S14 mRNAs. Although lipogenic rates were usually higher in brown adipocytes than white adipocytes, very similar patterns of regulation were seen in the two cell types. These data support the idea that the divergent results seen concerning T3 regulation of the lipogenic pathway in both brown and white adipose tissue in vivo arise from secondary effects of the alteration of thyroid status.     

Increased hepatic de novo lipogenesis and mitochondrial efficiency in a model of obesity induced by diets rich in fructose

Purpose

To assess hepatic de novo lipogenesis and mitochondrial energetics as well as whole-body energy homeostasis in sedentary rats fed a fructose-rich diet.

Methods

Male rats of 90 days of age were fed a high-fructose or control diet for 8 weeks. Body composition, energy balance, oxygen consumption, carbon dioxide production, non-protein respiratory quotient, de novo lipogenesis and insulin resistance were measured. Determination of specific activity of hepatic enzymes of de novo lipogenesis, mitochondrial mass, oxidative capacity and degree of coupling, together with parameters of oxidative stress and antioxidant defence, was also carried out.

Results

Body energy and lipid content as well as plasma insulin and non-esterified fatty acids were significantly higher in fructose-fed than in control rats. Significantly higher rates of net de novo lipogenesis and activities of hepatic lipogenic enzymes fatty acid synthase and stearoyl CoA desaturase-1 were found in fructose-fed rats compared to controls. Mitochondrial protein mass and degree of coupling were significantly higher in fructose-fed rats compared to controls. Hepatic mitochondria showed oxidative damage, both in the lipid and in the protein component, together with decreased activity of antioxidant defence.

Conclusion

Liver mitochondrial compartment is highly affected by fructose feeding. The increased mitochondrial efficiency allows liver cells to burn less substrates to produce ATP for de novo lipogenesis and gluconeogenesis. In addition, increased lipogenesis gives rise to whole body and ectopic lipid deposition, and higher mitochondrial coupling causes mitochondrial oxidative stress.     

The effect of chronic ethanol administration on lipogenesis in liver and adipose tissue in the rat

Rates of lipogenesis de novo have been studied in liver and epididymal fat pads of male rats chronically treated with ethanol. A solution of ethanol (150 ml/l) was administered as the only drinking fluid for 3 months with a standard solid diet; both food and drink were available ad lib. Lipogenesis in vivo was measured by the incorporation of tritiated water into lipid fractions: non-saponifiable lipid and fatty acids. Non-saponifiable lipid, both in liver and in adipose tissue, was unaffected by ethanol treatment. However, fatty acid synthesis de novo was significantly enhanced in both liver and adipose tissue, by 150 and 300% respectively. Plasma triacylglycerol and non-esterified fatty acid levels were unchanged and plasma glucose concentration slightly increased by ethanol administration. The rate of lipogenesis increased when insulin: glucagon increased twofold due to the effect of ethanol.     

The carbon pathway for lipogenesis in isolated adipocytes from rat, guinea pig, and human adipose tissue.

The synthesis of fatty acids from a variety of labeled substrates by isolated adipocytes of the rat, guinea pig, and human was investigated. The incorporation of radioactive glucose and pyruvate into triglyceride fatty acids was considerably lower in human than either rat or guinea pig adipose tissue. By contrast, the incorporation of palmitate into adipose tissue triglycerides was approximately the same in all three species. End carbon analysis of fatty acids isolated from adipocytes incubated with pyruvate-U-14C indicated that although the synthesis of fatty acids in human adipose tissue was extremely low compared to that of the rat and guinea pig, it represented de novo biosynthesis rather than chain elongation of existing fatty acids. It is suggested that in the human, fatty acids are synthetised de novo primarily in the liver. In adipose tissue, lipogenesis consists essentially of the esterification of fatty acids, obtained from plasma, into triglycerides.     

Resistance of lung fatty acid synthesis to inhibition by dietary fat in the meal-fed rat

One-half of the palmitate utilized by the lung for production of the surfactant phospholipid, dipalmitoyl phosphatidylcholine, originates from de novo palmitate synthesis in the lung. In this report the lung was examined for the influence of dietary fat on the lung de novo fatty acid synthesis pathway. Lung lipogenesis was reduced by fasting and accelerated by carbohydrate refeeding or insulin injection. However, in general lung fatty acid synthesis was unaffected by dietary fat. Supplementing one meal (high glucose diet) with as much as 36% additional fat kilocalories did not suppress lung fatty acid synthesis. An inhibition of fatty acid synthesis resulted from a fat supplement of +60 and +120% of meal kilocalories, but this inhibition was likely due to an attenuated rate of glucose absorption. Ingestion of a high carbohydrate diet supplemented with 10, 17, or 30% added kilocalories as safflower oil or palmitate had no effect on lipogenesis after 10 days. On the other hand, liver fatty acid synthesis and acetyl-CoA carboxylase were selectively suppressed by safflower oil, whereas dietary palmitate was ineffective as an inhibitor of lipogenesis. These data clearly demonstrate that the well-characterized preferential suppression of liver lipogenesis by dietary polyunsaturated fats does not extend to lung tissue, and, more importantly, the inhibition of liver lipogenesis is not secondary to an essential fatty acid deficiency. The marked resistance of lung fatty acid synthesis to inhibition by dietary fat might be a biological protective mechanism to ensure adequate palmitate for dipalmitoyl phosphatidylcholine synthesis.     

4,8-Sphingadienine and 4-hydroxy-8-sphingenine activate ceramide production in the skin

ABSTRACT: BACKGROUND: Ingestion of glucosylceramide improves transepidermal water loss (TEWL) from the skin, but the underlying mechanism by which a small amount of dietary glucosylceramide can vastly improve skin conditions remains unclear. In a previous report, glucosylceramides were shown to be digested to sphingoids, which were shown to be absorbed through the intestinal epithelium. Based on these observations, we hypothesized that sphingoids are the key molecules facilitating endogenous ceramide production. In this study, we assessed the effect of 4,8-sphingadienine (d18:2) and 4-hydroxy-8-sphingenine (t18:1), derived from konjac glucosylceramide, on stimulating ceramide production. METHODS: Konjac glucosylceramide acidolysis was performed using hydrochloric acid; the resulting d18:2 and t18:1 were fractionated by column chromatography. Real-time quantitative RT-PCR was performed to assess the effect of d18:2 and t18:1 on gene expression in normal human epidermal keratinocytes, while their effect on the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)gamma, was measured using a receptor-cofactor assay system. The effect of d18:2 and t18:1 on stimulating ceramide production was evaluated using HPTLC analysis in a 3-dimensional human skin model. RESULTS: We noted the upregulation of genes related to de novo ceramide synthesis as well as of those encoding the elongases of very long-chain fatty acids by d18:2 and t18:1, but not by glucosylceramide and 4-sphingenine. Both these sphingoids also facilitated the expression of PPARbeta/delta and PPARgamma; moreover, they also demonstrated ligand activity for PPARgamma. These results indicated that d18:2 and t18:1 promote the differentiation of keratinocytes. Analysis of the lipids within the 3-dimensional human skin model indicated that treatment with d18:2 and t18:1 not only upregulated gene expression but also increased ceramide production. CONCLUSIONS: The sphingoids d18:2 and t18:1 activated genes related to de novo ceramide synthesis and increased ceramide production, whereas glucosylceramide and 4-sphingenine could not. These results suggest that the effect of dietary glucosylceramides on the skin is mediated by d18:2 and t18:1.     

Comparison among the lipogenic potential of various substrates in rat hepatocytes: the differential effects of fructose-containing diets on hepatic lipogenesis

Fructose feeding has been reported to cause hypertriglyceridemia in rats. Apparently this is due to increased hepatic fatty acid synthesis. In hepatocytes from female rats fed a 60% sucrose or fructose diet, the rate of lipogenesis was two times higher than in cells from rats fed a 60% glucose diet and three times higher than in cells from rats fed a commercial nonpurified diet. In hepatocytes from rats fed the fructose-containing diets, lactate was a better substrate than either butyrate or acetate, whereas in cells from rats fed either the glucose diet or the non-purified diet, butyrate was the best lipogenic substrate, and the lipogenic potential of lactate and acetate was similar. In all cases, 1 mM fructose caused a 30-40% stimulation of lipogenesis, while 10 mM glucose did not enhance fat synthesis above the endogenous rates. These results suggest that the differential effect of fructose-containing diets on hepatic lipogenesis results from activation of pyruvate dehydrogenase, thereby increasing the efficiency by which lactate is used as a carbon source for fatty acid synthesis. The differences in lipogenic potential of the various substrates tested is discussed.     

Bisphenol A diglycidyl ether induces adipogenic differentiation of multipotent stromal stem cells through a peroxisome proliferator-activated receptor gamma-independent mechanism

Background: Bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE), used in manufacturing coatings and resins, leach from packaging materials into food. Numerous studies suggested that BPA and BADGE may have adverse effects on human health, including the possibility that exposure to such chemicals can be superimposed on traditional risk factors to initiate or exacerbate the development of obesity. BPA is a suspected obesogen, whereas BADGE, described as a peroxisome proliferator–activated receptor gamma (PPARγ) antagonist, could reduce weight gain.Objectives: We sought to test the adipogenic effects of BADGE in a biologically relevant cell culture model.Methods: We used multipotent mesenchymal stromal stem cells (MSCs) to study the adipogenic capacity of BADGE and BPA and evaluated their effects on adipogenesis, osteogenesis, gene expression, and nuclear receptor activation.Discussion: BADGE induced adipogenesis in human and mouse MSCs, as well as in mouse 3T3-L1 preadipocytes. In contrast, BPA failed to promote adipogenesis in MSCs, but induced adipogenesis in 3T3-L1 cells. BADGE exposure elicited an adipogenic gene expression profile, and its ability to induce adipogenesis and the expression of adipogenic genes was not blocked by known PPARγ antagonists. Neither BADGE nor BPA activated or antagonized retinoid “X” receptor (RXR) or PPARγ in transient transfection assays.Conclusions: BADGE can induce adipogenic differentiation in both MSCs and in preadipocytes at low nanomolar concentrations comparable to those that have been observed in limited human biomonitoring. BADGE probably acts through a mechanism that is downstream of, or parallel to, PPARγ.     

Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets

Background

Adipocyte volume (fat accumulation) and cell number (adipogenesis) is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation.

Methods

C3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics^® ArrayScan^® VTI HCS reader and SpotDetector^® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY^® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1β, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation.

Results

The ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, β-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1β.

Conclusion

This in vitro assay in differentiating adipocytes enables automated detection and quantification of changes in lipid droplet number, size and intensity. The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.     

Brain lipogenesis and regulation of energy metabolism

PURPOSE OF REVIEW: To revise current available information related to the role of brain lipogenic pathways in the regulation of energy homeostasis. RECENT FINDINGS: The 'classical' hypothalamic neuropeptide view of feeding regulation has been extensively reviewed and revised during the past few years. Accumulating evidence indicates that the modulation of lipogenesis de novo in the hypothalamus, through selective pharmacologic and genetic manipulation of acetyl-CoA carboxylase, AMP-activated protein kinase, carnitine palmitoyltransferase 1, fatty acid synthase and malonyl-CoA decarboxylase enzymes, has a severe impact on food intake and body weight homeostasis. Furthermore, as these manipulations alter the hypothalamic pool of lipids, such as malonyl-CoA or long chain fatty acyl-CoA or both, the concept of lipids as signals of nutrient abundance able to modulate feeding in the hypothalamus has recently re-emerged. SUMMARY: In this review, we summarize what is known about brain lipogenesis and energy balance and propose further avenues of research. Defining these novel mechanisms could offer new targets for the treatment of obesity and metabolic syndrome.     

Raphani Semen (Raphanus sativus L.) Ameliorates Alcoholic Fatty Liver Disease by Regulating De Novo Lipogenesis

In this study, we investigated the pharmacological effect of a water extract of Raphani Semen (RSWE) on alcoholic fatty liver disease (AFLD) using ethanol-induced AFLD mice (the NIAAA model) and palmitic acid (PA)-induced steatosis HepG2 cells. An RSWE supplement improved serum and hepatic triglyceride (TG) levels of AFLD mice, as well as their liver histological structure. To explore the molecular action of RSWE in the improvement of AFLD, we investigated the effect of RSWE on four major pathways for lipid homeostasis in the liver: free fatty acid transport, lipogenesis, lipolysis, and β-oxidation. Importantly, RSWE decreased the mRNA expression of de novo lipogenesis-related genes, such as Srebf1, Cebpa, Pparg, and Lpin1, as well as the protein levels of these factors, in the liver of AFLD mice. That these actions of RSWE affect lipogenesis was confirmed using PA-induced steatosis HepG2 cells. Overall, our findings suggest that RSWE has the potential for improvement of AFLD by inhibiting de novo lipogenesis.     

IS CARBOHYDRATE CONVERTED TO FAT IN HUMANS?

A noninvasive, stable-isotopic method was developed for measuring de novo synthesis of fatty acids in humans. After a carbohydrate load was given to non-overfed, nonobese men, de novo hepatic lipogenesis was found to be insignificant.     

Changes in fat synthesis influenced by dietary macronutrient content

DE NOVO: lipogenesis is the biological process by which C2 precursors of acetyl-CoA are synthesized into fatty acids. In human subjects consuming diets higher in fat (> 30 % energy), lipogenesis is down regulated and extremely low; typically < 10 % of the fatty acids secreted by the liver. This percentage will increase when dietary fat is reduced and replaced by carbohydrate, although the extent of carbohydrate-induced lipogenesis is dependent on the type of carbohydrate (monosaccharide v. polysaccharide) and the form in which the carbohydrate is fed (liquid meals, solid less-processed food). Clearly, massive overconsumption of carbohydrate can also increase lipogenesis. A second related phenomenon that occurs when dietary fat is reduced is hypertriacylglycerolaemia. This rise in blood triacylglycerol concentration could be due to increased de novo lipogenesis or to reduced clearance of lipid from the blood. The present paper will review the metabolic mechanisms leading to the elevations in blood triacylglycerol concentration that occur with dietary fat reduction. Studies considered will be those investigating fatty acid synthesis in subjects chronically fed low-fat high-carbohydrate diets and studies in which data were obtained in both the fasted and fed states. Also summarized will be data from subjects who had consumed diets of different carbohydrate types, as well as the most recent data from postprandial studies investigating factors that affect the magnitude of the rise in blood lipids following a meal. Given the changing availability of carbohydrate in the food supply, it will be important to understand how the balance of fat and carbohydrate in the diet influences lipogenesis, and the relative contribution of the process of de novo lipogenesis to the escalating incidence of obesity observed around the world.     

Increased lipogenesis and fatty acid reesterification contribute to hepatic triacylglycerol stores in hyperlipidemic Txnip-/- mice

The effect of decreased fatty acid oxidation on liver lipid metabolism in HcB-19 mice, a mouse model of hyperlipidemia (Txnip(-/-)), was investigated using metabolic labeling. De novo cholesterol synthesis and de novo lipogenesis were quantified using 1-(13)C(1) acetic acid, and liver triacylglycerol (TAG) derived from dietary fatty acids was quantified using dietary glyceryl tri(hexandecanoate-d(31)). Tissue samples were analyzed for TAG, free cholesterol (FC), and cholesterol ester (CE) content. Txnip(-/-) mice had significantly elevated (P < 0.05) serum nonesterified fatty acids compared with wild-type (WT) littermates; their livers weighed more and contained more TAG and total cholesterol. Txnip(-/-) liver also contained measurable CE; CE was not detectable in WT mice. Liver CE content was elevated despite lower cholesterol fractional synthesis rates (16 vs. 31%/d in Txnip(-/-) and WT mice, respectively). FC absolute synthesis rate (ASR) in WT mice (0.28 +/- 0.0 micromol/d) was similar to the combined synthesis rates of FC (0.13 +/- 0.10 micromol/d) and CE (0.10 +/- 0.00 micromol/d) in Txnip(-/-) mice. Lipogenesis, as assessed by TAG-palmitate ASR, was significantly greater in Txnip(-/-) mice (1.47 +/- 0.08 vs. 0.49 +/- 0.06 micro mol/d) and liver fatty acid synthase activity was also higher (7.96 +/- 2.53 vs. 4.83 +/- 1.44 U/mg protein). Both elevated lipogenesis and increased fatty acid reesterification to glycerol and cholesterol contributed to fat in the livers of Txnip(-/-) mice. These data support elevated fatty acid synthesis as the primary contributor to liver TAG in Txnip(-/-) mice, although increased esterification of fatty acids also contributed to excess liver TAG. The absolute total cholesterol synthesis rate was not altered, but esterification of fatty acids to cholesterol provided an additional means to buffer physiologically the negative results of excess fatty acid availability.