A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice

Intracellular lipids and their synthesis contribute to the mechanisms and complications of obesity-associated diseases. We describe an NMR approach that provides an abbreviated lipidomic analysis with concurrent lipid biosynthetic fluxes. Following deuterated water administration, positional isotopomer analysis by deuterium NMR of specific lipid species was used to examine flux through de novo lipogenesis (DNL), FA elongation, desaturation, and TG-glycerol synthesis. The NMR method obviated certain assumptions regarding sites of enrichment and exchangeable hydrogens required by mass isotope methods. The approach was responsive to genetic and pharmacological gain or loss of function of DNL, elongation, desaturation, and glyceride synthesis. BDF1 mice consuming a high-fat diet (HFD) or matched low-fat diet for 35 weeks were examined across feeding periods to determine how flux through these pathways contributes to diet induced fatty liver and obesity. HFD mice had increased rates of FA elongation and glyceride synthesis. However DNL was markedly suppressed despite insulin resistance and obesity. We conclude that most hepatic TGs in the liver of HFD mice were formed from the reesterification of existing or ingested lipids, not DNL.     

Intramuscular lipid metabolism, insulin action, and obesity

With the increasing prevalence of obesity, research has focused on the molecular mechanism(s) linking obesity and skeletal muscle insulin resistance. Metabolic alterations within muscle, such as changes in the cellular location of fatty acid transporter proteins, decreased mitochondrial enzyme activity, and defects in mitochondrial morphology, likely contribute to obesity and insulin resistance. These defects are thought to play a role in the reduced skeletal muscle fatty acid oxidation and increased intramuscular lipid (IMCL) accumulation that is apparent with obesity and other insulin-resistant states such as type 2 diabetes. Intramuscular triacylglycerol does not appear to be a ubiquitous marker of insulin resistance, although specific IMCL intermediates such as long-chain fatty acyl-CoAs, ceramide, and diacylglycerol may inhibit insulin signal transduction. In this review, we will briefly summarize the defects in skeletal muscle lipid metabolism associated with obesity, and discuss the proposed mechanisms by which these defects may contribute to insulin resistance.     

Liver-specific expression of carboxylesterase 1g/esterase-x reduces hepatic steatosis,counteracts dyslipidemia and improves insulin signaling

Ces1g/Es-x deficiency in mice results in weight gain, insulin resistance, fatty liver and hyperlipidemia through upregulation of de novo lipogenesis and oversecretion of triacylglycerol (TG)-rich lipoproteins. Here, we show that restoration of Ces1g/Es-x expression only in the liver significantly reduced hepatic TG concentration accompanied by decreased size of lipid droplets, reduced secretion of very low-density lipoproteins and improved insulin-mediated signal transduction in the liver. Collectively, these results demonstrate that hepatic Ces1g/Es-x plays a critical role in limiting hepatic steatosis, very low-density lipoprotein assembly and in augmenting insulin sensitivity.     

Increased lipid accumulation and insulin resistance in transgenic mice expressing DGAT2 in glycolytic (type II) muscle

Insulin resistance and type 2 diabetes are frequently accompanied by lipid accumulation in skeletal muscle. However, it is unknown whether primary lipid deposition in skeletal muscle is sufficient to cause insulin resistance or whether the type of muscle fiber, oxidative or glycolytic fiber, is an important determinant of lipid-mediated insulin resistance. Here we utilized transgenic mice to test the hypothesis that lipid accumulation specifically in glycolytic muscle promotes insulin resistance. Overexpression of DGAT2, which encodes an acyl-CoA:diacylglycerol acyltransferase that catalyzes triacylglycerol (TG) synthesis, in glycolytic muscle of mice increased the content of TG, ceramides, and unsaturated long-chain fatty acyl-CoAs in young adult mice. This lipid accumulation was accompanied by impaired insulin signaling and insulin-mediated glucose uptake in glycolytic muscle and impaired whole body glucose and insulin tolerance. We conclude that DGAT2-mediated lipid deposition specifically in glycolytic muscle promotes insulin resistance in this tissue and may contribute to the development of diabetes.     

Attenuation of inflammation and cellular stress‐related pathways maintains insulin sensitivity in obese type I interleukin‐1 receptor knockout mice on a high‐fat diet

The development of insulin resistance in the obese is associated with chronic, low‐grade inflammation. We aimed to identify novel links between obesity, insulin resistance and the inflammatory response by comparing C57BL/6 with type I interleukin‐1 receptor knockout (IL‐1RI^?/?) mice, which are protected against diet‐induced insulin resistance. Mice were fed a high‐fat diet for 16 wk. Insulin sensitivity was measured and proteomic analysis was performed on adipose, hepatic and skeletal muscle tissues. Despite an equal weight gain, IL‐1RI^?/? mice had lower plasma glucose, insulin and triacylglycerol concentrations, compared with controls, following dietary treatment. The higher insulin sensitivity in IL‐1RI^?/? mice was associated with down‐regulation of antioxidant proteins and proteasomes in adipose tissue and hepatic soluble epoxide hydrolase, consistent with a compromised inflammatory response as well as increased glycolysis and decreased fatty acid β‐oxidation in their muscle. Their lower hepatic triacylglycerol concentrations may reflect decreased flux of free fatty acids to the liver, decreased hepatic fatty acid‐binding protein expression and decreased lipogenesis. Correlation analysis revealed down‐regulation of classical biomarkers of ER stress in their adipose tissue, suggesting that disruption of the IL‐1RI‐mediated inflammatory response may attenuate cellular stress, which was associated with significant protection from diet‐induced insulin resistance, independent of obesity.     

Mechanism of glucose intolerance in mice with dominant negative mutation of CEACAM1

Mice with liver-specific overexpression of dominant negative phosphorylation-defective S503A-CEACAM1 mutant (L-SACC1) developed chronic hyperinsulinemia resulting from blunted hepatic clearance of insulin, visceral obesity, and glucose intolerance. To determine the underlying mechanism of altered glucose homeostasis, a 2-h hyperinsulinemic euglycemic clamp was performed, and tissue-specific glucose and lipid metabolism was assessed in awake L-SACC1 and wild-type mice. Inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) caused insulin resistance in liver that was mostly due to increased expression of fatty acid synthase and lipid metabolism, resulting in elevated intrahepatic levels of triglyceride and long-chain acyl-CoAs. Whole body insulin resistance in the L-SACC1 mice was further attributed to defects in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Insulin resistance in peripheral tissues was associated with significantly elevated intramuscular fat contents that may be secondary to increased whole body adiposity (assessed by (1)H-MRS) in the L-SACC1 mice. Overall, these results demonstrate that L-SACC1 is a mouse model in which chronic hyperinsulinemia acts as a cause, and not a consequence, of insulin resistance. Our findings further indicate the important role of CEACAM1 and hepatic insulin clearance in the pathogenesis of obesity and insulin resistance.     

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans

Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity.     

Key role for ceramides in mediating insulin resistance in human muscle cells

Elevated non-esterified fatty acids, triglyceride, diacylglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide.     

Treatment with cinnamaldehyde reduces the visceral adiposity and regulates lipid metabolism,autophagy and endoplasmic reticulum stress in the liver of a rat model of early obesity

Nutrition at early stages of life contributes to the alarming incidence of childhood obesity, insulin resistance and hepatoesteatosis. Cinnamaldehyde, major component of cinnamon, increases insulin sensitivity and modulates adiposity and lipid metabolism. The aim of this study was to analyze the impact of cinnamaldehyde treatment during adolescence in a rat model of early obesity. Litter size reduction was used to induce overfeeding and early obesity. At postnatal day 30 (adolescence), the male Wistar rats received cinnamaldehyde by gavage (40 mg/kg of body weight/day) for 29 days and were studied at the end of treatment at 60 days old or 4 months thereafter (180 days old). At 60 days of age, the treatment with cinnamaldehyde promoted reduced visceral adiposity, serum triacylglycerol, and attenuation of energy efficiency and insulin resistance. In the liver, it reduced lipid synthesis, stimulated autophagy and reduced ER stress. At 180 days of age, animals treated with cinnamaldehyde during the adolescence exhibited normalization of visceral adiposity and energy efficiency, and attenuation of hyperphagia, serum hypertriglyceridemia and hepatic triacylglycerol content, with molecular markers indicative of reduced hepatic synthesis. However, the beneficial effect observed at 60 days of age on glucose homeostasis, autophagy and ER stress was lost. Therefore, the cinnamaldehyde supplementation during the adolescence has short- and long-term metabolic beneficial effects, highlighting its potential as an adjuvant in the treatment of early obesity.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Metformin and exercise reduce muscle FAT/CD36 and lipid accumulation and blunt the progression of high-fat diet-induced hyperglycemia

Derangements in skeletal muscle fatty acid (FA) metabolism associated with insulin resistance in obesity appear to involve decreased FA oxidation and increased accumulation of lipids such as ceramides and diacylglycerol (DAG). We investigated potential lipid-related mechanisms of metformin (Met) and/or exercise for blunting the progression of hyperglycemia/hyperinsulinemia and skeletal muscle insulin resistance in female Zucker diabetic fatty rats (ZDF), a high-fat (HF) diet-induced model of diabetes. Lean and ZDF rats consumed control or HF diet (48 kcal %fat) alone or with Met (500 mg/kg), with treadmill exercise, or with both exercise and Met interventions for 8 wk. HF-fed ZDF rats developed hyperglycemia (mean: 24.4 +/- 2.1 mM), impairments in muscle insulin-stimulated glucose transport, increases in the FA transporter FAT/CD36, and increases in total ceramide and DAG content. The development of hyperglycemia was significantly attenuated with all interventions, as was skeletal muscle FAT/CD36 abundance and ceramide and DAG content. Interestingly, improvements in insulin-stimulated glucose transport and increased GLUT4 transporter expression in isolated muscle were seen only in conditions that included exercise training. Reduced FA oxidation and increased triacylglycerol synthesis in isolated muscle were observed with all ZDF rats compared with lean rats (P < 0.01) and were unaltered by therapeutic intervention. However, exercise did induce modest increases in peroxisome proliferator-activated receptor-gamma coactivator-1alpha, citrate synthase, and beta-hydroxyacyl-CoA dehydrogenase activity. Thus reduction of skeletal muscle FAT/CD36 and content of ceramide and DAG may be important mechanisms by which exercise training blunts the progression of diet-induced insulin resistance in skeletal muscle.     

Lipid metabolism in skeletal muscle: generation of adaptive and maladaptive intracellular signals for cellular function

Fatty acids derived from adipose tissue lipolysis, intramyocellular triacylglycerol lipolysis, or de novo lipogenesis serve a variety of functions in skeletal muscle. The two major fates of fatty acids are mitochondrial oxidation to provide energy for the myocyte and storage within a variety of lipids, where they are stored primarily in discrete lipid droplets or serve as important structural components of membranes. In this review, we provide a brief overview of skeletal muscle fatty acid metabolism and highlight recent notable advances in the field. We then 1) discuss how lipids are stored in and mobilized from various subcellular locations to provide adaptive or maladaptive signals in the myocyte and 2) outline how lipid metabolites or metabolic byproducts derived from the actions of triacylglycerol metabolism or β-oxidation act as positive and negative regulators of insulin action. We have placed an emphasis on recent developments in the lipid biology field with respect to understanding skeletal muscle physiology and discuss unanswered questions and technical limitations for assessing lipid signaling in skeletal muscle.     

The critical role of atypical protein kinase C in activating hepatic SREBP-1c and NF��B in obesity

Obesity is frequently associated with systemic insulin resistance, glucose intolerance, and hyperlipidemia. Impaired insulin action in muscle and paradoxical diet/insulin-dependent overproduction of hepatic lipids are important components of obesity, but their pathogenesis and inter-relationships between muscle and liver are uncertain. We studied two murine obesity models, moderate high-fat-feeding and heterozygous muscle-specific PKC-λ knockout, in both of which insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but conserved in liver. In both models, activation of hepatic sterol receptor element binding protein-1c (SREBP-1c) and NFκB (nuclear factor-kappa B), major regulators of hepatic lipid synthesis and systemic insulin resistance, was chronically increased in the fed state. In support of a critical mediatory role of aPKC, in both models, inhibition of hepatic aPKC by adenovirally mediated expression of kinase-inactive aPKC markedly diminished diet/insulin-dependent activation of hepatic SREBP-1c and NFκB, and concomitantly improved hepatosteatosis, hypertriglyceridemia, hyperinsulinemia, and hyperglycemia. Moreover, in high-fat–fed mice, impaired insulin signaling to IRS-1–dependent phosphatidylinositol 3-kinase, PKB/Akt and aPKC in muscle and hyperinsulinemia were largely reversed. In obesity, conserved hepatic aPKC-dependent activation of SREBP-1c and NFκB contributes importantly to the development of hepatic lipogenesis, hyperlipidemia, and systemic insulin resistance. Accordingly, hepatic aPKC is a potential target for treating obesity-associated abnormalities.     

Development of leptin resistance in rat soleus muscle in response to high-fat diets

Direct evidence for leptin resistance in peripheral tissues such as skeletal muscle does not exist. Therefore, we investigated the effects of different high-fat diets on lipid metabolism in isolated rat soleus muscle and specifically explored whether leptin's stimulatory effects on muscle lipid metabolism would be reduced after exposure to high-fat diets. Control (Cont, 12% kcal fat) and high-fat [60% kcal safflower oil (n-6) (HF-Saff); 48% kcal safflower oil plus 12% fish oil (n-3)] diets were fed to rats for 4 wk. After the dietary treatments, muscle lipid turnover and oxidation in the presence and absence of leptin was measured using pulse-chase procedures in incubated resting soleus muscle. In the absence of leptin, phospholipid, diacylglycerol, and triacylglycerol (TG) turnover were unaffected by the high-fat diets, but exogenous palmitate oxidation was significantly increased in the HF-Saff group. In Cont rats, leptin increased exogenous palmitate oxidation (21.4 +/- 5.7 vs. 11.9 +/- 1.61 nmol/g, P = 0.019) and TG breakdown (39.8 +/- 5.6 vs. 27.0 +/- 5.2 nmol/g, P = 0.043) and decreased TG esterification (132.5 +/- 14.6 vs. 177.7 +/- 29.6 nmol/g, P = 0.043). However, in both high-fat groups, the stimulatory effect of leptin on muscle lipid oxidation and hydrolysis was eliminated. Partial substitution of fish oil resulted only in the restoration of leptin's inhibition of TG esterification. Thus we hypothesize that, during the development of obesity, skeletal muscle becomes resistant to the effects of leptin, resulting in the accumulation of intramuscular TG. This may be an important initiating step in the development of insulin resistance common in obesity.     

Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis

Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from beta-oxidation and toward glycerolipid synthesis. Adenoviral-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have >2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.     

Artificial selection for high-capacity endurance running is protective against high-fat diet-induced insulin resistance

Elevated oxidative capacity, such as occurs via endurance exercise training, is believed to protect against the development of obesity and diabetes. Rats bred both for low (LCR)- and high (HCR)-capacity endurance running provide a genetic model with inherent differences in aerobic capacity that allows for the testing of this supposition without the confounding effects of a training stimulus. The purpose of this investigation was to determine the effects of a high-fat diet (HFD) on weight gain patterns, insulin sensitivity, and fatty acid oxidative capacity in LCR and HCR male rats in the untrained state. Results indicate chow-fed LCR rats were heavier, hypertriglyceridemic, less insulin sensitive, and had lower skeletal muscle oxidative capacity compared with HCR rats. Upon exposure to an HFD, LCR rats gained more weight and fat mass, and their insulin resistant condition was exacerbated, despite consuming similar amounts of metabolizable energy as chow-fed controls. These metabolic variables remained unaltered in HCR rats. The HFD increased skeletal muscle oxidative capacity similarly in both strains, whereas hepatic oxidative capacity was diminished only in LCR rats. These results suggest that LCR rats are predisposed to obesity and that expansion of skeletal muscle oxidative capacity does not prevent excess weight gain or the exacerbation of insulin resistance on an HFD. Elevated basal skeletal muscle oxidative capacity and the ability to preserve liver oxidative capacity may protect HCR rats from HFD-induced obesity and insulin resistance.