Effect of maternal high-fat diet on embryonic muscle development

Attenuation of myostatin signalling has been reported to increase fatty acid metabolism. Here, we investigated the effect of high fat on muscle development on a genetic background that lacks myostatin (Mstn−/−); (Fig. 1a–f). Muscle morphology from littermate embryos was performed at the developmental stage E18.5, when secondary myogenesis is considered to be approaching postnatal levels. As reported previously [21], total myotube number of the EDL was significantly higher in the Mstn−/− embryos compared to the WT ones. However, we found a significantly compromised myotube number of 15 % in the EDL of Mstn−/− embryos from mothers raised on a HF diet (Fig. 1a, b). Concordantly, a 30 and 12 % reduction in total myotube number was evident in the soleus and TA muscles, respectively, of Mstn−/− embryos from mothers on a HF diet in the absence of any differences in WT embryos (Fig. 1e,f). EDL primary and secondary myotube cross-sectional area (CSA) was significantly reduced by 10 % in WT embryos from mothers kept on a HF diet. Most importantly, a significant interaction between genotype and diet revealed a significantly higher CSA reduction in both primary (slow) and in particular secondary (fast) EDL myotubes of Mstn−/− embryos by 20 and 35 %, respectively (Fig. 1c). In addition, the number of myotubes with centrally located nuclei, as an indication of myotube remodelling and regeneration were significantly higher in the Mstn−/− HF mice (Fig. 1d). We examined the level of inflammation as a means to possibly explain the decrease in muscle development. Histological staining for F4/80, a macrophage marker revealed a significant increase for both genotypes in response to high-fat diet (Fig. 2a). We also noticed a significant increase in activated NF-κB immunostaining only in the Mstn−/− HF embryos (Fig. 2b). Taken together, these novel findings suggest that maternal subjection to HF diet has more severe and detrimental impact on muscle development on Mstn−/− compared to WT embryos possibly through an induction of an inflammatory response.

Fig. 1 Effect of high fat on embryonic muscle development. EDL muscle morphology at developmental stage E18.5 from wild type (WT) and myostatin null (Mstn−/−) embryos from mice subjected to maternal normal (ND) or a high-fat (HF) diet. a Representative immunofluorescence images depicting primary (slow; red) and secondary (fast; green outline) myotubes from EDL. Scale 100 μm. b Total myotube number in EDL muscle at E18.5. c Myotube cross-sectional area and d myotubes with central nuclei per unit area. e, f Total myotube number of the TA and soleus muscles at E18.5 from WT and Mstn−/− embryos from mothers under a ND or HF diet. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. Mstn−/− ND. Data are from N = 6–8 embryos per group Full size image

Fig. 2 Effect of high-fat diet on inflammatory markers of embryonic muscle. a Evidence of intramuscular macrophages stained for F4/80 and b NF-κB immunofluorescence in embryonic muscle. Scale 100 μm, ANOVA; (*) P < 0.05 vs. WT ND; (**) P < 0.05 vs. Mstn−/− ND; (#) P < 0.05 vs. WT HF. Data are from N = 5 embryos per group Full size image

Effect of high-fat diet on mouse gross anatomy

The above findings demonstrated that a maternal high-fat diet had a detrimental effect on the foetal muscle development programme. We also noted that pregnant as well as virgin male and female Mstn−/− mice raised on a high-fat diet developed precocious levels of visceral fat. The effect of high-fat diet on percent body mass revealed a significant increase in WT mice by 20–22 % that was evident throughout the study (Fig. 3a). Surprisingly, Mstn−/− mice (irrespective of sex) subjected to a high-fat diet for 10 weeks elicited a 37–76 % increase in body mass. Moreover, Mstn−/− mice are known for their hypermuscular phenotype compared to wild-type littermates and individual hind limb muscles were heavier compared to wild-type cohorts. Curiously, high-fat diet did not affect individual muscle masses in either genotype, with the exception of TA and vastus lateralis in WT and Mstn−/−, respectively (Fig. 3b). Importantly, high-fat diet compromised Mstn−/− mice survival curves by 33–45 % in male (Fig. 3c) and female mice (data not shown). These phenotypic findings indicate that HF diet has deleterious effects on body mass changes and life span in the Mstn−/− mice. Histological examination of heart did not reveal any fibrotic lesions (Fig. 3d). We furthermore looked for transcript levels of key factors playing a role in pathological heart hypertrophy and fibrosis as a possible explanation for mortality (i.e. smooth muscle α actin, Acta2; β myosin heavy chain, βMHC; lectin, galactoside-binding soluble 3, Lgals3; connective tissue growth factor, Ctgf; procollagen C-endopeptidase enhancer, Pcolce; and sarcoplasmic reticulum Ca2+ ATPase, Serca2a). Acta2 mRNA were elevated in Mstn−/− mice compared to WT and were further increased in Mstn−/− mice in response to HF diet (Fig. 3e). Mstn−/− ND mice showed significantly higher mRNA levels for βMHC and Lgals3 compared to WT ND and high-fat diet led to increased levels for both genes only in WT HF mice. No changes were found for Ctgf, Pcolce and Serca2a.

Fig. 3 Effect of high-fat diet on body mass, skeletal muscle masses, animal survival curve and cardiac muscle. a Percent changes of body mass in wild type (WT) and myostatin null (Mstn−/−) mice subjected to either a normal (ND) or a high-fat (HF) diet for 10 weeks. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF; (**) P < 0.005 vs. Mstn−/− ND. b Effect of high-fat (HF) diet on EDL, soleus, plantaris, tibialis anterior (TA), gastrocnemius, rectus femoris and vastus lateralis muscle masses. c Animal survival curve in wild type (WT) and myostatin null (Mstn−/−) mice subjected to either a normal (ND) or a high-fat (HF) diet. ANOVA; (*) P < 0.05 vs. WT normal diet (ND); (#) P < 0.05 vs. Mstn−/− ND. A 33 and 45 % reduction of animal survival in Mstn−/− HF diet cohort was observed at the 8-week and 10-week time point, respectively. d Trichrome staining on the heart muscle. Scale 100 μm. e mRNA levels of key markers involved in cardiac hypertrophy and fibrosis. ANOVA; (*) P < 0.05 vs. WT ND, (#) P < 0.05 vs. WT HF; (**) P < 0.005 vs. Mstn−/− ND ; N = 5 male mice per group Full size image

Effect of high-fat diet on blood lipids, liver function markers and cellular damage markers

Plasma lipids were profiled with a Beckman Coulter AU680 clinical chemistry analyser. High-fat diet led to a significant increase of total-HDL and LDL-cholesterol and glycerol in both WT and Mstn−/− mice of either sex (Fig. 4). Triglyceride levels were twice the levels in Mstn−/− mice on a normal diet compared to similarly fed WT animals. In addition, triglycerides and free fatty acids were significantly increased only in the Mstn−/− HF mice. Liver function markers were unaffected in WT HF mice, while Mstn−/− HF mice showed an increase for bilirubin and aspartate aminotransferase as well as a decrease for alanine aminotransferase, respectively (Fig. 4). Interestingly, markers of cellular damage (i.e. lactate dehydrogenase, amylase and creatine kinase) were significantly increased only in the Mstn−/− HF mice (Fig. 4) and Additional file 1: Table S1.

Fig. 4 Effect of high-fat diet on blood lipids, liver function markers and markers of cellular damage. ANOVA; (*) P < 0.05 vs. WT ND; (**) P < 0.05 vs. Mstn−/− ND; (#) P < 0.05 vs. WT HF. Data are from N = 5 male mice per group Full size image

Effect of high-fat diet on muscle metabolic properties

Fat catabolism takes place in the mitochondria. Given the impaired mitochondrial contents that characterise the muscles of Mstn−/− mice [27], we next focused our analysis on mitochondrial respiration in skeletal muscle from WT and Mstn−/− mice in response to high-fat diet. Mitochondrial activity estimated via SDH histological staining showed a significant increase in SDH positive fibres in WT mice after both 4 and 10 weeks of HF diet by 5 % (Fig. 5a, b). As expected, Mstn−/− mice had fewer SDH positive fibres compared to WT cohorts but subjection to high-fat diet did not affect SDH levels in Mstn−/− mice. This finding may be taken as functional compensation in the WT mice to metabolise the excess of fat supplied by nutrition or alternatively as failure to appropriately augment mitochondrial activity in response to high-fat diet by the Mstn−/− mice. We next analysed muscle fibre composition of EDL muscle in order to decipher the role of skeletal muscle morphology on total body metabolism and the development of obesity.

Fig. 5 Effect of high-fat diet on EDL muscle mitochondrial activity, fibre type and contractile properties. a EDL succinate dehydrogenase (SDH) activity in wild type (WT) and myostatin null (Mstn−/−) mice subjected to a normal (ND) or high-fat (HF) diet for 4 and 10 weeks. Representative histochemical staining for SDH. Scale 100 μm. N = 6 mice per group. b Quantification of SDH-positive (SDH+) fibres among groups. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF. c Fibre type changes on EDL from wild type (WT) and myostatin null (Mstn−/−) mice subjected to a normal (ND) or high-fat (HF) diet for 4 and 10 weeks. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF. N = 6 mice per group. d EDL muscle contractile properties in wild type (WT) and myostatin null (Mstn−/−) mice subjected to a normal (ND) or high-fat (HF) diet. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF. N = 6 mice per group. Exercise fatigue test of from wild type (WT) and myostatin null (Mstn−/−) mice subjected to a normal (ND) or high-fat (HF) diet for 6 weeks. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. Mstn−/− ND; (**) P < 0.05 vs. WT HF. N = 5 male mice per group Full size image

In line, with the SDH findings, we observed a fibre type shift from glycolytic IIB to more oxidative types (i.e. IIX and IIA) after 4 and 10 weeks of HF diet in the wild-type mice. In contrast, no significant changes were found in EDL muscle fibre types for Mstn−/− mice subjected to HF diet (Fig. 5c). Taken together, the changes in SDH and MHC isoforms found in WT mice potentially indicate functional metabolic alterations that favour fat buffering and utilization in a system with excess fat content. However, this mechanism is not activated robustly in Mstn−/− animals.

Effect of high-fat diet on muscle contractile properties

We next determined the effect of HF diet on EDL muscle contractile properties. As previously reported by us and others, EDL tetanic and specific force were significantly lower in Mstn−/− EDL muscles compared to WT mice subjected to normal diet [28, 29]. Twitch tension revealed a significant interaction between diet and genotype originating mainly in a 30 % reduction for WT mice in response to HF diet (Fig. 5d). We also observed a significant reduction in tetanic tension for WT mice on HF diet, which exceeded the known low levels found in Mstn−/− mice (Fig. 5d). By normalising tetanic force to wet muscle mass, we found a sharp reduction in specific tension for WT mice reaching the known attenuated levels of Mstn−/− mice (Fig. 5d). Overall, HF diet did not have any impact on the contractile properties of Mstn−/− mice. These findings indicate that despite the metabolic remodelling of EDL muscle for WT mice, HF has detrimental effects on muscle contractile properties. In addition, the already compromised contractile properties of Mstn−/− EDL muscle were not further affected by HF diet. When animals were challenged by an exercise fatigue protocol, we noticed an attenuated exercise tolerance for both genotypes under HF diet which was more pronounced in the Mstn−/− mice (Fig. 5d).

Effect of high-fat diet on the expression of genes controlling metabolic activity in skeletal muscle

We next determined the gene expression patterns of Mstn and key metabolic regulators in EDL. HF diet did not affect EDL Mstn mRNA levels in the WT mice and as expected Mstn transcript was not detectable in the Mstn−/− mice (Fig. 6a). We determined the transcript levels of key genes involved in fatty acid uptake (i.e. Cd36, Fatp1, Cpt1b, Slc25a20 and Cpt2), mitochondrial fatty acid oxidation (i.e. Acadl and Acadm) as well as glucose metabolism (i.e. Pdk4, Glut1 and Glut 4; Fig. 6b–d). High-fat diet resulted in a significant induction of gene products that regulate fatty acid uptake (i.e. Fatp1, Cpt1b and Slc25a20) in EDL muscle in both WT and Mstn−/− mice (Fig. 6b). However, a significant interaction was evident between diet and genotype originating in a more robust induction of mRNA levels in the WT mice compared to Mstn−/− with regard to fatty acid uptake genes Cd36 (i.e. 1.3 in WT vs. 0.3-fold change in Mstn−/−) and Cpt2 (i.e. 0.9-fold in WT vs. 0.2-fold change in Mstn−/−) as well as the fatty acid oxidation genes Acadl (i.e. 1.6-fold in WT vs. 0.5-fold change in Mstn−/−) and Acadm (i.e. 1.1-fold in WT vs. 0.4-fold change in Mstn−/−; Fig. 6b, c). A significant main effect of genotype was apparent on mRNA levels of genes that regulate glucose metabolism (i.e. Pdk4, Glut1 and Glut4), due to the predominant glycolytic muscle phenotype of the Mstn−/− mice. Moreover, HF diet did not affect expression of glucose metabolism genes with the exception of a significant increase on Glut1 (constitutive glucose transporter in the fasting state [30]) levels only in the WT cohort (Fig. 6d). We also found that wild-type mice subjected to HF diet show a 4-fold upregulation of ucp1 in the EDL muscle, which is blunted in the Mstn−/− HF diet mice (Fig. 6c). A similar profile of gene expression was discovered when we examined transcripts in the soleus muscle (Additional file 2: Figure S1). Collectively, these data show a sub-optimal transcriptional adaptation of muscle HF in the absence of myostatin.

Fig. 6 Effect of high-fat diet on EDL muscle gene expression. EDL gene expression levels of a Myostatin, b key factors regulating fatty acid uptake (i.e. Cd36, Fatp1, Cpt1b, Slc25a20 and Cpt2), c fatty acid oxidation (i.e. Acadl and Acadm) as well as Uucp1 and d glucose metabolism (i.e. Pdk4, Glut1 and Glut 4). ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF; (**) P < 0.05 vs. Mstn−/− ND. N = 6 male mice per group Full size image

Effect of high-fat diet on the expression of genes controlling metabolic activity in liver

As the EDL of Mstn−/− mice showed a blunted response to HF, we next determined the gene expression patterns of key metabolic regulators in the liver, another major site regulating adiposity. We measured mRNA levels of genes that regulate fatty acid uptake (i.e. Cd36, Cpt1b, Slc25a20 and Cpt2), fatty acid oxidation (i.e. Acadl and Acadm), and glucose metabolism (i.e. Pdk4, Glut1 and Glut4) as well as transcriptional regulators of energy metabolism from the family nuclear receptors (i.e. Ppara, Ppard, Pgc1a and Pgc1b). We found that HF diet induced genes involved in fatty acid uptake and oxidation only in the WT cohort (Fig. 7a–d). Conversely, gene expression level changes were blunted in the liver of Mstn−/− HF mice for all genes regulating fatty acid uptake, except Acadl and Acadm (involved in fatty acid oxidation). With regard to genes that control glucose metabolism, HF diet increased mRNA levels of glucose transporters Glut1 and Glut4 and decreased Pdk4 levels in the WT mice, all changes suggesting an increased metabolic response and glucose utilization (Fig. 7c). HF diet significantly reduced Pdk4, did not affect Glut1 and increased Glut4 mRNA levels in the Mstn−/− mice (Fig. 7c). With the exception of Glut1, the changes on mRNA levels of Pdk4 and Glut4 also denote increased glucose metabolism and transport within the liver. We also examined the expression of Ppara, a master regulator of lipid metabolism in the liver and adipose tissue [31]. Ppara was upregulated in WT mice in response to a high-fat diet. However, the gene was induced less robustly at significant levels by high fat in gene Mstn−/− mice (Fig. 7d). Taken together, this data indicates that the transcriptional regulation of fat metabolism by the liver in Mstn−/− mice is compromised. This finding suggests that Mstn−/− mice do not handle the excess of dietary fats like WT mice. We therefore conducted a profiling of tissue fat content for fat droplet deposition in the liver (Fig. 7e). Oil Red O staining revealed a pronounced fat accumulation in the liver of WT mice maintained on a HF diet, a profile that differed greatly from the control condition. Significantly and in contrast to WT mice, we found that there was no fat deposition in the livers of Mstn−/− mice raised on a high-fat diet. Indeed, there was no change in Oil Red profiles between Mstn−/− mice raised on HF compared to normal diet.

Fig. 7 Effect of high-fat diet on liver gene expression and liver fat deposition. Liver gene expression levels of a key factors regulating fatty acid uptake (i.e. Cd36, Cpt1b, Slc25a20 and Cpt2), b fatty acid oxidation (i.e. Acadl and Acadm), c glucose metabolism (i.e. Pdk4, Glut1 and Glut 4) as well as d transcriptional regulators (i.e. Ppara, Ppard, Pgc1a and Pgc1b). ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF; (**) P < 0.05 vs. Mstn−/− ND. N = 6 mice per group. e Representative histological images of liver fat contents by Oil Red O staining from wild type (WT) and myostatin null (Mstn−/−) male mice subjected to a normal (ND) or high-fat (HF) diet for 10 weeks. Scale 100 μm Full size image

Effect of high-fat diet on gene expression patterns of white fat

A qualitative evaluation of abdominal fat depots revealed a large increase in visceral fat contents in the Mstn−/− mice in response to HF diet (Fig. 8a). This finding was unexpected given previous evidence suggesting that Mstn−/− mice are protected against diet-induced obesity (e.g. [16, 17]). We sought to determine the gene expression patterns of the same key regulators of fatty acid uptake, oxidation, glucose metabolism and transcriptional regulators in white adipose tissue of WT and Mstn−/− mice subjected to HF diet with a view of developing an understanding of mechanisms underpinning the excessive fat deposits in the Mstn−/− mice fed on high-fat diet (Fig. 8b–d). A significant interaction between genotype and diet was evident for Cpt1b, Slc25a20, Cpt2, Fatp1 and Fabp3 originating in either a significant upregulation for the WT cohort or a blunted response for the Mstn−/− mice (Fig. 8b). Cd36 gene expression was induced in a similar manner for both genotypes. Similarly, fatty acid oxidation gene expression (i.e. Acadl and Acadm mRNA levels) was significantly upregulated in the WT mice and totally blunted in the Mstn−/− mice (Fig. 8c). Taken together, these data suggest that fatty acid metabolism within the adipose tissue of the Mstn−/− mice is transcriptionally compromised at least in two different levels; fatty acid uptake as well as fatty acid oxidation. On the contrary, WT mice gene expression changes suggest an increased capacity for both cellular uptake and use of fatty acids. Again, as for the liver, Ppara was more robustly activated in WT compared to Mstn−/− in response to high fat (Fig. 8d). Examination of the expression of Ucp1 in the fat revealed a striking finding. WT fat from mice raised on normal or high-fat diets expressed very little Ucp1. In contrast, Mstn−/− mice expressed elevated levels of Ucp1 in the normal state which was dramatically decreased following the introduction of a high-fat diet. Similar profiles were obtained for Ucp1 protein levels (Fig. 8c, e). These results suggest that the normal white fat of the Mstn−/− mice has a high activity of Ucp1 and thereby resembles brown fat. The browning of fat has been shown to be mediated by signalling initiated by Fndc5/irisin [32]. Recent studies have found that white fat expresses this protein and regulates its metabolic properties in an autocrine fashion [33]. We found that the expression of Fndc5/irisin decreased by 4-fold in WT mice following the introduction of a high-fat diet. In contrast, its levels dropped by 13-fold in Mstn−/− mice following the same intervention (Fig. 8d). These results show that the fatty acid uptake and fatty acid oxidation programmes are robustly induced by high fat in adipose tissues of WT mice by high fat but this response is minimal in Mstn−/− mice. Furthermore, we show that levels of Ucp1 which would act to metabolise fat are dramatically decreased in the adipose tissue of Mstn−/− mice in response to high fat possibly due to a decrease in the expression of FNdc5/irisin. Since the irisin-Ucp1 pathway that regulates the development of brown adipose tissue (BAT) was perturbed in Mstn−/− HF mice, we measured transcript levels of key genes that promote BAT (i.e. Cidea, PR domain zinc finger protein 16; Prdm16; proton assistant amino acid transporter-2, Pat2; Fig. 9a). There was a significant increase of all three genes in WT HF mice, while Mstn−/− HF mice showed a blunted response. Similarly, we measured the mRNA levels of adipophilin (Plin2) and perilipin (Plin5), two genes that regulate fat cell metabolism and lipid storage in white adipose tissue. Plin2 is known to have an adipogenic role, while Plin5 can be both adipogenic or lipolytic [34]. There was a significant increase in mRNA for both genes in WT but not in Mstn−/− in response to HF (Fig. 9b). Plin2 mRNA levels in the liver were not affected by diet, but there was a significant main effect of the genotype with Mstn−/− HF mice having lower levels vs. WT cohorts. At last, we found a significant increase in Plin4 and Lipe in the liver of WT HF mice without any change for the Mstn−/− HF mice (Fig. 9c).

Fig. 8 Effect of high-fat diet on body fat contents and on white adipose tissue gene expression. a Visceral fat deposition in WT and Mstn−/− mice subjected to either a ND or a HF diet. Note the excessive fat contents in the Mstn−/− HF cohort. White adipose tissue (WAT) gene expression levels of b key factors regulating fatty acid uptake (i.e. Cd36, Cpt1b, Slc25a20, Cpt2, Fatp1 and Fabp3), c fatty acid oxidation (i.e. Acadl and Acadm) and Ucp1. d Fatty acid transcriptional regulator Ppara and regulator of Upc1-Fndc5/irisin. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF; (**) P < 0.05 vs. Mstn−/− ND. N = 6 mice per group. e Representative immunoblot for ucp1 protein. N = 3 male mice per group Full size image

Fig. 9 Gene expression of brown adipose tissue (BAT) markers and fat cell metabolism in a, b white adipose tissue (WAT) and c in the liver. ANOVA; (*) P < 0.05 vs. WT ND; (#) P < 0.05 vs. WT HF; (**) P < 0.05 vs. Mstn−/− ND. N = 5 male mice per group Full size image

Metabonomic analysis of skeletal muscle

Metabolic profiles were obtained from the hydrophilic extracts of the gastrocnemius muscle by 1H NMR spectroscopy. Score plot from the pair-wise PCA model comparing WT and Mstn−/− control muscles revealed differences between the two genotypic groups driven by higher creatine/phosphocreatine in the muscles of WT mice and lower lactate compared to Mstn−/− muscle (Fig. 10a–e). Clear differences were observed in the metabolic profiles of the muscles when feeding WT mice a HF diet (Fig. 10a). From the corresponding loadings plot, the HF diet can be seen to increase muscular anserine and decrease lactate in the WT mice (Fig. 10d). Anserine is a histidine-related compound with established antioxidants properties in skeletal muscle and brain of several species that is believed to inhibit lipid oxidation by means of free radical scavenging or metal chelation [35, 36]. In contrast, the HF diet did not induce any robust metabolic perturbations in the muscles of Mstn−/− mice (Fig. 10b). Comparing the metabolic phenotypes of WT and Mstn−/− muscle fed, a HF diet revealed the Mstn−/− muscle to contain higher amounts of lactate while the WT muscle contained greater amounts of creatine/phosphocreatine and anserine (Fig. 10c). We complemented these studies by performing an extensive analysis in the lipid content in muscle. Triglyceride contents in cellular extracts from gastrocnemius showed a significant increase for both genotypes in response to HF diet, which however was more pronounced in KO HF mice (Fig. 10f). No major effects were observed for other lipid subclasses, except a significant decrease for phosphatidylethanolamines in HF mice for both genotypes (Additional file 3: Figure S2).