In addition to its antiatherogenic role, HDL reportedly modulates energy metabolism at the whole-body level. HDL functionality is associated with its structure and composition, and functional ...activities can differ between HDL subclasses. Therefore, we studied if HDL
and HDL
, the two major HDL subclasses, are able to modulate energy metabolism of skeletal muscle cells. Differentiated mouse and primary human skeletal muscle myotubes were used to investigate the influences of human HDL
and HDL
on glucose and fatty uptake and oxidation. HDL-induced changes in lipid distribution and mRNA expression of genes related to energy substrate metabolism, mitochondrial function, and HDL receptors were studied with human myotubes. Additionally, we examined the effects of apoA-I and discoidal, reconstituted HDL particles on substrate metabolism. In mouse myotubes, HDL subclasses strongly enhanced glycolysis upon high and low glucose concentrations. HDL
caused a minor increase in ATP-linked respiration upon glucose conditioning but HDL
improved complex I-mediated mitochondrial respiration upon fatty acid treatment. In human myotubes, glucose metabolism was attenuated but fatty acid uptake and oxidation were markedly increased by both HDL subclasses, which also increased mRNA expression of genes related to fatty acid metabolism and HDL receptors. Finally, both HDL subclasses induced incorporation of oleic acid into different lipid classes. These results, demonstrating that HDL subclasses enhance fatty acid oxidation in human myotubes but improve anaerobic metabolism in mouse myotubes, support the role of HDL as a circulating modulator of energy metabolism. Exact mechanisms and components of HDL causing the change, require further investigation.
A fatty acid analogue, 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), was previously shown to have hypolipidemic effects in rats by targeting mitochondrial activity predominantly in liver. This ...study aimed to determine if 1-triple TTA could influence carbohydrate metabolism. Male Wistar rats were treated for three weeks with oral supplementation of 100 mg/kg body weight 1-triple TTA. Blood glucose and insulin levels, and liver carbohydrate metabolism gene expression and enzyme activities were determined. In addition, human myotubes and Huh7 liver cells were treated with 1-triple TTA, and glucose and fatty acid oxidation were determined. The level of plasma insulin was significantly reduced in 1-triple TTA-treated rats, resulting in a 32% reduction in the insulin/glucose ratio. The hepatic glucose and glycogen levels were lowered by 22% and 49%, respectively, compared to control. This was accompanied by lower hepatic gene expression of phosphenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, and Hnf4A, a regulator of gluconeogenesis. Gene expression of pyruvate kinase, catalysing the final step of glycolysis, was also reduced by 1-triple TTA. In addition, pyruvate dehydrogenase activity was reduced, accompanied by 10-15-fold increased gene expression of its regulator pyruvate dehydrogenase kinase 4 compared to control, suggesting reduced entry of pyruvate into the TCA cycle. Indeed, the NADPH-generating enzyme malic enzyme 1 (ME1) catalysing production of pyruvate from malate, was increased 13-fold at the gene expression level. Despite the decreased glycogen level, genes involved in glycogen synthesis were not affected in livers of 1-triple TTA treated rats. In contrast, the pentose phosphate pathway seemed to be increased as the hepatic gene expression of glucose-6-phosphate dehydrogenase (G6PD) was higher in 1-triple TTA treated rats compared to controls. In human Huh7 liver cells, but not in myotubes, 1-triple-TTA reduced glucose oxidation and induced fatty acid oxidation, in line with previous observations of increased hepatic mitochondrial palmitoyl-CoA oxidation in rats. Importantly, this work recognizes the liver as an important organ in glucose homeostasis. The mitochondrially targeted fatty acid analogue 1-triple TTA seemed to lower hepatic glucose and glycogen levels by inhibition of gluconeogenesis. This was also linked to a reduction in glucose oxidation accompanied by reduced PHD activity and stimulation of ME1 and G6PD, favouring a shift from glucose- to fatty acid oxidation. The reduced plasma insulin/glucose ratio indicate that 1-triple TTA may improve glucose tolerance in rats.
Obesity and physical inactivity have a profound impact on skeletal muscle metabolism. In the present work, we have investigated differences in protein expression and energy metabolism in primary ...human skeletal muscle cells established from lean donors (BMI<25 kg/m
2
) and individuals with obesity (BMI>30 kg/m
2
). Furthermore, we have studied the effect of fatty acid pretreatment on energy metabolism in myotubes from these donor groups. Alterations in protein expression were investigated using proteomic analysis, and energy metabolism was studied using radiolabeled substrates. Gene Ontology enrichment analysis showed that glycolytic, apoptotic, and hypoxia pathways were upregulated, whereas the pentose phosphate pathway was downregulated in myotubes from donors with obesity compared to myotubes from lean donors. Moreover, fatty acid, glucose, and amino acid uptake were increased in myotubes from individuals with obesity. However, fatty acid oxidation was reduced, glucose oxidation was increased in myotubes from subjects with obesity compared to cells from lean. Pretreatment of myotubes with palmitic acid (PA) or eicosapentaenoic acid (EPA) for 24 h increased glucose oxidation and oleic acid uptake. EPA pretreatment increased the glucose and fatty acid uptake and reduced leucine fractional oxidation in myotubes from donors with obesity. In conclusion, these results suggest that myotubes from individuals with obesity showed increased fatty acid, glucose, and amino acid uptake compared to cells from lean donors. Furthermore, myotubes from individuals with obesity had reduced fatty acid oxidative capacity, increased glucose oxidation, and a higher glycolytic reserve capacity compared to cells from lean donors. Fatty acid pretreatment enhances glucose metabolism, and EPA reduces oleic acid and leucine fractional oxidation in myotubes from donor with obesity, suggesting increased metabolic flexibility after EPA treatment.
Physical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular ...mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes.
Electrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting.
High-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTracker®Red FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells.
Our results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise.
Skeletal muscle plays an important role in glycaemic control and metabolic homeostasis, making it a tissue of interest with respect to type 2 diabetes mellitus. The aim of the present study was to ...determine if ligands of Toll-like receptors (TLRs) could have an impact on energy metabolism and myokine expression and secretion in cultured human skeletal muscle cells. The myotubes expressed mRNA for TLRs 1-6. TLR3, TLR4, TLR5 and TLR6 ligands (TLRLs) increased glucose metabolism. Furthermore, TLR4L and TLR5L increased oleic acid metabolism. The metabolic effects of TLRLs were not evident until after at least 24 h pre-incubation of the cells and here the metabolic effects were more evident for the metabolism of glucose than oleic acid, with a shift towards effects on oleic acid metabolism after chronic exposure (168 h). However, the stimulatory effect of TLRLs on myokine expression and secretion was detected after only 6 h, where TLR3-6L stimulated secretion of interleukin-6 (IL-6). TLR5L also increased secretion of interleukin-8 (IL-8), while TLR6L also increased secretion of granulocyte-macrophage colony stimulating factor (GM-CSF). Pre-incubation of the myotubes with IL-6 for 24 h increased oleic acid oxidation but had no effect on glucose metabolism. Thus IL-6 did not mimic all the metabolic effects of the TLRLs, implying metabolic effects beyond the actions of this myokine.
Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study ...skeletal muscle energy metabolism. However, the transferability of the results to
might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism.
We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid.
These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.
Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The ...mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the Musculus obliquus internus abdominis of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with ¹⁴Cglucose or ³Hdeoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of legumain. Dysregulation of glucose metabolism and decreased legumain activity by simvastatin points out new knowledge about the effects of statins on skeletal muscle, and may contribute to the understanding of the myotoxicity observed by statins.
Primary human myotubes represent an alternative system to intact skeletal muscle for the study of human diseases related to changes in muscle energy metabolism. This work aimed to study if fatty acid ...and glucose metabolism in human myotubes in vitro were related to muscle of origin, donor gender, age, or body mass index (BMI). Myotubes from a total of 82 donors were established from three different skeletal muscles, i.e.,
musculus vastus lateralis
,
musculus obliquus internus abdominis
, and
musculi interspinales
, and cellular energy metabolism was evaluated. Multiple linear regression analyses showed that donor age had a significant effect on glucose and oleic acid oxidation after correcting for gender, BMI, and muscle of origin. Donor BMI was the only significant contributor to cellular oleic acid uptake, whereas cellular glucose uptake did not rely on any of the variables examined. Despite the effect of age on substrate oxidation, cellular mRNA expression of pyruvate dehydrogenase kinase 4 (
PDK4
) and peroxisome proliferator–activated receptor gamma coactivator 1 alpha (
PPARGC1A
) did not correlate with donor age. In conclusion, donor age significantly impacts substrate oxidation in cultured human myotubes, whereas donor BMI affects cellular oleic acid uptake.
Adipose tissue is one of the main regulative sites for energy metabolism. Excess lipid storage and expansion of white adipose tissue (WAT) is the primary contributor to obesity, a strong predisposing ...factor for development of insulin resistance. Sentrin-specific protease (SENP) 2 has been shown to play a role in metabolism in murine fat and skeletal muscle cells, and we have previously demonstrated its role in energy metabolism of human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism in primary human fat cells by using cultured primary human adipocytes to knock down the SENP2 gene. Glucose uptake and oxidation, as well as accumulation and distribution of oleic acid into complex lipids were decreased, while oleic acid oxidation was increased in SENP2-knockdown cells compared to control adipocytes. Furthermore, lipogenesis was reduced by SENP2-knockdown in adipocytes. Although TAG accumulation relative to total uptake was unchanged, there was increased mRNA expression of metabolically relevant genes such as UCP1 and PPARGC1A and mRNA and proteomic data revealed increased levels of mRNA and proteins related to mitochondrial function by SENP2-knockdown. In conclusion, SENP2 is an important regulator of energy metabolism in primary human adipocytes and its knockdown reduce glucose metabolism and lipid accumulation, while increasing lipid oxidation in human adipocytes.
Lipid droplet (LD) coating proteins are essential for the formation and stability of intracellular LDs. Plin2 is an abundant LD coating protein in skeletal muscle, but its importance for muscle ...function is unclear. We show that myotubes established from Plin2−/− mice contain reduced content of LDs and accumulate less oleic acid (OA) in triacylglycerol (TAG) due to elevated LD hydrolysis in comparison with Plin2+/+ myotubes. The reduced ability to store TAG in LDs in Plin2−/− myotubes is accompanied by a shift in energy metabolism. Plin2−/− myotubes are characterized by increased oxidation of OA, lower glycogen synthesis, and reduced glucose oxidation in comparison with Plin2+/+ myotubes, perhaps reflecting competition between FAs and glucose as part of the Randle cycle. In accord with these metabolic changes, Plin2−/− myotubes have elevated expression of Ppara and Ppargc1a, transcription factors that stimulate expression of genes important for FA oxidation, whereas genes involved in glucose uptake and oxidation are suppressed. Loss of Plin2 had no impact on insulin-stimulated Akt phosphorylation. Our results suggest that Plin2 is essential for protecting the pool of skeletal muscle LDs to avoid an uncontrolled hydrolysis of stored TAG and to balance skeletal muscle energy metabolism.
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