The embryonic genes Dkk3, Hoxd8, Hoxd9 and Tbx1 identify muscle types in a diet-independent and fiber-type unrelated way.

BACKGROUND: The mouse skeletal muscle is composed of four distinct fiber types that differ in contractile function, number of mitochondria and metabolism. Every muscle type has a specific composition and distribution of the four fiber types. To find novel genes involved in specifying muscle types, we used microarray analysis to compare the gastrocnemius with the quadriceps from mice fed a low fat diet (LFD) or high fat diet (HFD) for 8 weeks. Additional qPCR analysis were performed in the gastrocnemius, quadriceps and soleus muscle from mice fed an LFD or HFD for 20 weeks. RESULTS: In mice fed the 8-week LFD 162 genes were differentially expressed in the gastrocnemius vs. the quadriceps. Genes with the strongest differences in expression were markers for oxidative fiber types (e.g. Tnni1) and genes which are known to be involved in embryogenesis (Dkk3, Hoxd8,Hoxd9 and Tbx1). Also Dkk2, Hoxa5, Hoxa10, Hoxc9, Hoxc10, Hoxc6 and Tbx15 were detectably, but not differentially expressed in adult muscle tissue. Expression of differentially expressed genes was not influenced by an 8-week or 20-week HFD. Comparing gastrocnemius, quadriceps and soleus, expression of Hoxd8 and Hoxd9 was not related with expression of markers for the four different fiber types. We found that the expression of both Hoxd8 and Hoxd9 was much higher in the gastrocnemius than in the quadriceps or soleus, whereas the expression of Dkk3 was high in quadriceps, but low in both gastrocnemius and soleus. Finally, expression of Tbx1 was high in quadriceps, intermediate in soleus and low in gastrocnemius. CONCLUSIONS: We found that genes from the Dkk family, Hox family and Tbx family are detectably expressed in adult mouse muscle. Interestingly, expression of Dkk3, Hoxd8, Hoxd9 and Tbx1 was highly different between gastrocnemius, quadriceps and soleus. In fact, every muscle type showed a unique combination of expression of these four genes which was not influenced by diet. Altogether, we conclude that genes important for embryogenesis identify mouse muscle types in a diet-independent and fiber type-unrelated manner.

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance.

BACKGROUND: Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance. METHODOLOGY: C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR. PRINCIPAL FINDINGS: At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (-4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks. CONCLUSIONS/INTERPRETATION: Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.

Adipophilin protein expression in muscle--a possible protective role against insulin resistance.

Adipophilin is a 50 kDa protein that belongs to the PAT family (perilipin, adipophilin, TIP47, S3-12 and OXPAT), which comprises proteins involved in the coating of lipid droplets. Little is known about the functional role of adipophilin in muscle. Using the C2C12 cell line as a model, we demonstrate that palmitic acid-treated cells highly express the adipophilin protein in a dose-dependent way. Next, we show that oleic acid is a more potent inducer of adipophilin protein levels than palmitic acid. Cells treated with oleic acid have a higher adipophilin protein expression and higher triglyceride levels but less impairment of insulin signaling than cells treated with palmitic acid. Additionally, we show that peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta and PPARgamma agonists all increase the expression of the adipophilin protein in C2C12 cells. This effect was most pronounced for the PPARalpha agonist GW7647. Furthermore, the expression of adipophilin as a 37 kDa N-terminally truncated protein is higher in the gastrocnemius than in the quadriceps of C57BL/6J mice, especially after an 8-week high-fat diet. The expression of adipophilin was higher in the muscle of mice fed a 4-week high-fat diet based on olive oil or safflower oil than in mice fed a 4-week high-fat diet based on palm oil. After 2 weeks of intervention, plasma glucose, plasma insulin and the homeostasis model assessment of insulin resistance index were lower in mice fed a 4-week high-fat diet based on olive oil or safflower oil than in mice fed a 4-week high-fat diet based on palm oil. Taken together, the results obtained in the present study indicate that adipophilin protein expression in muscle is involved in maintaining insulin sensitivity.

An 8-week high-fat diet induces obesity and insulin resistance with small changes in the muscle transcriptome of C57BL/6J mice.

BACKGROUND: Skeletal muscle is responsible for most of the insulin-stimulated glucose uptake and metabolism. Therefore, it plays an important role in the development of insulin resistance, one of the characteristics of the metabolic syndrome (MS). As the prevalence of the MS is increasing, there is an urgent need for more effective intervention strategies. METHODS: C57BL/6J mice were fed an 8-week low-fat diet (10 kcal%; LFD) or high-fat diet (45 kcal%; HFD). Microarray analysis was performed by using two comparisons: (1) 8-week HFD transcriptome versus 8-week LFD transcriptome and (2) transcriptome of mice sacrificed at the start of the intervention versus 8-week LFD transcriptome and 8-week HFD transcriptome, respectively. RESULTS: Although an 8-week HFD induced obesity and impaired insulin sensitivity, HFD-responsive changes in the muscle transcriptome were relatively small (<1.3-fold). In fact, 8-weeks of aging induced more pronounced changes than an HFD. One comparison revealed the transcriptional downregulation of the mito- gen-activated protein kinase cascade, whereas both comparisons showed the upregulation of fatty acid oxidation, demonstrating that the two comparison strategies are confirmative as well as complementary. CONCLUSION: We suggest using complementary analysis strategies in the genome-wide search for gene expression changes induced by mild interventions, such as an HFD.