Upregulated PDK4 expression is a sensitive marker of increased fatty acid oxidation.

Fatty acid oxidation is a central fueling pathway for mitochondrial ATP production. Regulation occurs through multiple nutrient- and energy-sensitive molecular mechanisms. We explored if upregulated mRNA expression of the mitochondrial enzyme pyruvate dehydrogenase kinase 4 (PDK4) may be used as a surrogate marker of increased mitochondrial fatty acid oxidation, by indicating an overall shift from glucose to fatty acids as the preferred oxidation fuel. The association between fatty acid oxidation and PDK4 expression was studied in different contexts of metabolic adaption. In rats treated with the modified fatty acid tetradecylthioacetic acid (TTA), Pdk4 was upregulated simultaneously with fatty acid oxidation genes in liver and heart, whereas muscle and white adipose tissue remained unaffected. In MDA-MB-231 cells, fatty acid oxidation increased nearly three-fold upon peroxisome proliferator-activated receptor α (PPARα, PPARA) overexpression, and four-fold upon TTA-treatment. PDK4 expression was highly increased under these conditions. Further, overexpression of PDK4 caused increased fatty acid oxidation in these cells. Pharmacological activators of PPARα and AMPK had minor effects, while the mTOR inhibitor rapamycin potentiated the effect of TTA. There were minor changes in mitochondrial respiration, glycolytic function, and mitochondrial biogenesis under conditions of increased fatty acid oxidation. TTA was found to act as a mild uncoupler, which is likely to contribute to the metabolic effects. Repeated experiments with HeLa cells supported these findings. In summary, PDK4 upregulation implies an overarching metabolic shift towards increased utilization of fatty acids as energy fuel, and thus constitutes a sensitive marker of enhanced fatty acid oxidation.

Loss of perilipin 2 in cultured myotubes enhances lipolysis and redirects the metabolic energy balance from glucose oxidation towards fatty acid oxidation.

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.

Synthesis, in vitro and in vivo biological evaluation of new oxysterols as modulators of the liver X receptors.

Liver X Receptor (LXR) modulators have shown potential as drugs since they target genes affecting metabolism and fatty acid synthesis. LXR antagonists are of particular interest since they are able to reduce the synthesis of complex fatty acids and glucose uptake. Based on molecular modeling, five new cholesterol mimics were synthesized, where four contained a hydroxyl group in the 22-S-position. The new compounds were screened in vitro against several genes affecting lipid metabolism. The compound that performed best in vitro was a dimethylamide derivative of 22(S)-hydroxycholesterol and it was chosen for in vivo testing. However, the blood plasma analysis from the in vivo tests revealed a concentration lower than needed to give any response, indicating either rapid metabolism or low bioavailability.

Development of new LXR modulators that regulate LXR target genes and reduce lipogenesis in human cell models.

Four new mimics of 22-S-hydroxycholesterol (22SHC) were synthesized and evaluated using molecular modeling and tested in human muscle cells (primary myotubes) and hepatocytes (HepG2 cells). The new compounds (9, 12, 15a and 15b) showed good interrelationship between docking scores, to both LXRα and LXRß, and in vitro results. The LXR agonist T0901317 increased the expressions of genes involved in lipogenesis (SCD1, FAS) and cholesterol efflux (ABCA1), but only 22SHC counteracted the up-regulation of SCD1 and FAS by T0901317. Compound 9 and 12 decreased the expression of SCD1, while 9 also decreased the expression of FAS. Compounds 15a showed a significant antagonistic effect on ABCA1 expression, but neither 15a nor 15b were able to counteract the effect of T0901317 on all genes examined. Lipogenesis was increased after T0901317 treatment and only 22SHC significantly counteracted this effect. Treatment with 22SHC and compound 12 reduced lipogenesis compared to control. An increased glucose uptake was observed for all compounds, except for 15b. In summary, the new synthetic 22SHC mimics showed antagonistic effects similar to that of 22SHC, but the new substances were less potent. The sulfonamide 12 showed similar effects to 22SHC and the best effect on gene expression of the new mimics, however, it was not able to reduce the effect of T0901317 as observed for 22SHC.

Simvastatin inhibits glucose metabolism and legumain activity in human myotubes.

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 [¹4C]glucose or [³H]deoxyglucose 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.

Remodeling of oxidative energy metabolism by galactose improves glucose handling and metabolic switching in human skeletal muscle cells.

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [(14)C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [(14)C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.

The liver X receptor modulator 22(S)-hydroxycholesterol exerts cell-type specific effects on lipid and glucose metabolism.

The aim of this study was to explore the effects of 22(S)-hydroxycholesterol (22(S)-HC) on lipid and glucose metabolism in human-derived cells from metabolic active tissues. Docking of T0901317 and 22(S)-HC showed that both substances fitted into the ligand binding domain of liver X receptors (LXR). Results show that while several lipogenic genes were induced by T0901317 in myotubes, HepG2 cells and SGBS cells, effect of 22(S)-HC varied more between cell types. In myotubes, most lipogenic genes were downregulated or unchanged by 22(S)-HC, whereas a more diverse pattern was found in HepG2 and SGBS cells. Treatment with 22(S)-HC induced sterol regulatory element binding transcription factor 1 in SGBS and HepG2 cells, but not in myotubes. Fatty acid synthase was downregulated by 22(S)-HC in myotubes, upregulated in SGBS and unchanged in HepG2 cells. De novo lipogenesis was increased by T0901317 in all cell models, whereas differently affected by 22(S)-HC depending on the cell type; decreased in myotubes and HepG2 cells, whereas increased in SGBS cells. Oxidation of linoleic acid was reduced by 22(S)-HC in all cell models while glucose uptake increased and tended to increase in myotubes and SGBS cells, respectively. Cholesterol efflux was unaffected by 22(S)-HC treatment. These results show that 22(S)-HC affects LXR-regulated processes differently in various cell types. Ability of 22(S)-HC to reduce lipogenesis and lipid accumulation in myotubes and hepatocytes indicate that 22(S)-HC might reduce lipid accumulation in non-adipose tissues, suggesting a potential role for 22(S)-HC or a similar LXR modulator in the treatment of type 2 diabetes.

Leukemia inhibitory factor reduces contractile function and induces alterations in energy metabolism in isolated cardiomyocytes.

Interleukin (IL)-6 related cytokines may be involved in the pathophysiology of heart failure. Leukemia inhibitory factor (LIF) is an IL-6 related cytokine, and elevated levels of LIF have been found in failing hearts. The aim of our study was to investigate how LIF may influence isolated cardiomyocytes. Adult cardiomyocytes were isolated from male Wistar rat hearts and treated with 1 nM LIF for 48 h. Contractile function was measured using a video-edge detection system. Fractional shortening was reduced at 0.25 Hz in LIF treated cells (7.4% +/- 0.5%) compared to control cells (9.0% +/- 0.7%). Gene expression analysis showed that expression of the mitochondrial ATP-synthase F(1) alpha subunit was reduced in cells exposed to LIF. The activity of the enzyme was also reduced in these cells (0.10 +/- 0.05 mumol/min per mg protein) compared to controls (1.23 +/- 0.40 mumol/min per mg protein). The levels of ATP and creatine phosphate were reduced by 15.0% +/- 3.0% and 11.2% +/- 2.7% in LIF treated cells. LIF increased both (3)H-deoxyglucose uptake and lactate levels, suggesting an increase in anaerobic energy metabolism. Beta-oxidation of (14)C-oleic acid was increased by 51.2% +/- 14.1% following LIF treatment, but no changes were found in cellular uptake or oxidation of (14)C-oleic acid to CO(2). In conclusion, LIF induces contractile dysfunction and changes in energy metabolism in isolated cardiomyocytes.