Effects of lipoprotein lipase and statins on cholesterol uptake into heart and skeletal muscle

Regulation of cholesterol metabolism in cultured cells and in the liver is dependent on actions of the LDL receptor. However, nonhepatic tissues have multiple pathways of cholesterol uptake. One possible pathway is mediated by LPL, an enzyme that primarily hydrolyzes plasma triglyceride into fatty acids. In this study, LDL uptake and tissue cholesterol levels in heart and skeletal muscle of wild-type and transgenic mice with alterations in LPL expression were assessed. Overexpression of a myocyte-anchored form of LPL in heart muscle led to increased uptake of LDL and greater heart cholesterol levels. Loss of LDL receptors did not alter LDL uptake into heart or skeletal muscle. To induce LDL receptors, mice were treated with simvastatin. Statin treatment increased LDL receptor expression and LDL uptake by liver and skeletal muscle but not heart muscle. Plasma creatinine phosphokinase as well as muscle mitochondria, cholesterol, and lipid droplet levels were increased in statin-treated mice overexpressing LPL in skeletal muscle. Thus, pathways affecting cholesterol balance in heart and skeletal muscle differ.     

Adipose, muscle and lung tissue lipoprotein lipase activities in young streptozotocin treated rats

Lipoprotein lipase (LPL) activity was studied in adipose, muscle and lung tissues of post-weanling rats 48 and 96 hours after experimentally induced diabetes by streptozotocin administration. Weight gain was reduced, and blood glucose level increased about 3-4 fold above the control level as an indication of the diabetic state. LPL activity in brown and white adipose tissues decreased in diabetic rats to 10-30% of the control level. In soleus muscle the LPL activity was slightly enhanced 96 hours after the streptozotocin injection. In cardiac muscle the LPL activity was markedly increased already 48 hours after the administration of streptozotocin and the increase remained significant until 96 hours. There was in the pulmonary tissue also an increase of LPL activity of diabetic rats, although this was significant only 96 hours after streptozotocin treatment. The results suggest marked tissue specific variation in the LPL activity. Moreover, tissue responses to experimentally induced diabetes vary. In adipose tissue the decrease in the LPL activity suggests that lipid transport to adipocytes is decreased while an increase in skeletal and cardiac muscles and in lung tissue proposes that their lipid utilization is enhanced.     

Mitochondrial dysfunction and inhibition of myoblast differentiation in mice with high-fat-diet-induced pre-diabetes

Pre-diabetes is characterized by impaired glucose tolerance (IGT) and/or impaired fasting glucose. Impairment of skeletal muscle function is closely associated with the progression of diabetes. However, the entire pathological characteristics and mechanisms of pre-diabetes in skeletal muscle remain fully unknown. Here, we established a mouse model of pre-diabetes, in which 6-week-old male C57BL6/J mice were fed either normal diet or high-fat diet (HFD) for 8 or 16 weeks. Both non-fasting and fasting glucose levels and the results of glucose and insulin tolerance tests showed that mice fed an 8-week HFD developed pre-diabetes with IGT; whereas mice fed a 16-week HFD presented with impaired fasting glucose and impaired glucose tolerance (IFG-IGT). Mice at both stages of pre-diabetes displayed decreased numbers of mitochondria in skeletal muscle. Moreover, IFG-IGT mice exhibited decreased mitochondrial membrane potential and ATP production in skeletal muscle and muscle degeneration characterized by a shift in muscle fibers from predominantly oxidative type I to glycolytic type II. Western blotting and histological analysis confirmed that myoblast differentiation was only inhibited in IFG-IGT mice. For primary skeletal muscle satellite cells, inhibition of differentiation was observed in palmitic acid-induced insulin resistance model. Moreover, enhanced myoblast differentiation increased glucose uptake and insulin sensitivity. These findings indicate that pre-diabetes result in mitochondrial dysfunction and inhibition of myoblast differentiation in skeletal muscle. Therefore, interventions that enhance myoblast differentiation may improve insulin resistance of diabetes at the earlier stage.     

Increased thermoregulation in cold-exposed transgenic mice overexpressing lipoprotein lipase in skeletal muscle: an avian phenotype?

LPL is an enzyme involved in the breakdown and uptake of lipoprotein triglycerides. In the present study, we examined how the transgenic (Tg) overexpression of human LPL in mouse skeletal muscle affected tolerance to cold temperatures, cold-induced thermogenesis, and fuel utilization during this response. Tg mice and their nontransgenic controls were placed in an environmental chamber and housed in metabolic chambers that monitored oxygen consumption and carbon dioxide production with calorimetry. When exposed to 4 degrees C, an attenuation in the decline in body temperature in Tg mice was accompanied by an increased metabolic rate (15%; P < 0.001) and a reduction in respiratory quotient (P < 0.05). Activity levels, the expression of uncoupling proteins in brown fat and muscle, and lean mass failed to explain the enhanced cold tolerance and thermogenesis in Tg mice. The more oxidative type IIa fibers were favored over the more glycolytic type IIb fibers (P < 0.001) in the gastrocnemius and quadriceps muscles of Tg mice. These data suggest that Tg overexpression of LPL in skeletal muscle increases cold tolerance by enhancing the capacity for fat oxidation, producing an avian-like phenotype in which skeletal muscle contributes significantly to the thermogenic response to cold temperatures.     

Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity

According to the concept of lipotoxicity, ectopic accumulation of lipids in non-adipose tissue induces pathological changes. The most prominent effects are seen in fatty liver disease, lipid cardiomyopathy, non-insulin-dependent diabetes mellitus, insulin resistance and skeletal muscle myopathy. We used the MCK(m)-hLPL mouse distinguished by skeletal and cardiac muscle-specific human lipoprotein lipase (hLPL) overexpression to investigate effects of lipid overload in skeletal muscle. We were intrigued to find that ectopic lipid accumulation induced proteasomal activity, apoptosis and skeletal muscle damage. In line with these findings we observed reduced Musculus gastrocnemius and Musculus quadriceps mass in transgenic animals, accompanied by severely impaired physical endurance. We suggest that muscle loss was aggravated by impaired muscle regeneration as evidenced by reduced cross-sectional area of regenerating myofibers after cardiotoxin-induced injury in MCK(m)-hLPL mice. Similarly, an almost complete loss of myogenic potential was observed in C2C12 murine myoblasts upon overexpression of LPL. Our findings directly link lipid overload to muscle damage, impaired regeneration and loss of performance. These findings support the concept of lipotoxicity and are a further step to explain pathological effects seen in muscle of obese patients, patients with the metabolic syndrome and patients with cancer-associated cachexia.     

Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.     

Dexamethasone facilitates lipid accumulation and mild feed restriction improves fatty acids oxidation in skeletal muscle of broiler chicks (Gallus gallus domesticus)

Effects of dexamethasone (DEX) and mild feed restriction on the uptake and utilization of fatty acids in skeletal muscle of broiler chicks (Gallus gallus domesticus) were investigated. Male Arbor Acres chicks (7-days old, n = 30) were injected with DEX or saline for 3 days, and a feed restriction group was included. DEX enhanced circulating very low density lipoprotein (VLDL) level and the lipid accumulation in both adipose and skeletal muscle tissues. Compared with the control, liver-carnitine palmitoyltransferase 1 (L-CPT1) and AMP-activated protein kinase (AMPK) α2 mRNA level of M. biceps femoris (BF) were down-regulated significantly by DEX, while mRNA expression of lipoprotein lipase (LPL), fatty acid transport protein 1 (FATP1), heart-fatty acid binding protein (H-FABP), long-chain acyl-CoA dehydrogenase (LCAD), activities of LPL and AMPK in both skeletal muscles were not obviously affected. Feed restriction increased the mRNA expression of LPL, L-CPT1 and LCAD of M. pectoralis major (PM), and FATP1, H-FABP, L-CPT1 and LCAD of BF. In conclusion, DEX retards the growth of body mass but facilitates lipid accumulation in both adipose and skeletal muscle tissues. In contrast to the favorable effect of mild feed restriction, DEX did not alter the uptake of fatty acids in the skeletal muscle. The result suggests that DEX may promote intramyocellular lipid accumulation by suppressed fatty acid oxidation while mild feed restriction improved fatty acid oxidation in skeletal muscle, especially in red muscle. Glucocorticoids (GCs) regulated muscle fatty acid metabolism in a different way from energy deficit caused by mild feed restriction.     

Reduction of plasma triglycerides in apolipoprotein C-II transgenic mice overexpressing lipoprotein lipase in muscle

LPL and its specific physiological activator, apolipoprotein C-II (apoC-II), regulate the hydrolysis of triglycerides (TGs) from circulating TG-rich lipoproteins. Previously, we developed a skeletal muscle-specific LPL transgenic mouse that had lower plasma TG levels. ApoC-II transgenic mice develop hypertriglyceridemia attributed to delayed clearance. To investigate whether overexpression of LPL could correct this apoC-II-induced hypertriglyceridemia, mice with overexpression of human apoC-II (CII) were cross-bred with mice with two levels of muscle-specific human LPL overexpression (LPL-L or LPL-H). Plasma TG levels were 319 +/- 39 mg/dl in CII mice and 39 +/- 5 mg/dl in wild-type mice. Compared with CII mice, apoC-II transgenic mice with the higher level of LPL overexpression (CIILPL-H) had a 50% reduction in plasma TG levels (P = 0.013). Heart LPL activity was reduced by approximately 30% in mice with the human apoC-II transgene, which accompanied a more modest 10% decrease in total LPL protein. Overexpression of human LPL in skeletal muscle resulted in dose-dependent reduction of plasma TGs in apoC-II transgenic mice. Along with plasma apoC-II concentrations, heart and skeletal muscle LPL activities were predictors of plasma TGs. These data suggest that mice with the human apoC-II transgene may have alterations in the expression/activity of endogenous LPL in the heart. Furthermore, the decrease of LPL activity in the heart, along with the inhibitory effects of excess apoC-II, may contribute to the hypertriglyceridemia observed in apoC-II transgenic mice.     

Nutritional regulation and role of peroxisome proliferator-activated receptor delta in fatty acid catabolism in skeletal muscle

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARdelta displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h starvation period, PPARdelta mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARdelta is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while refeeding promotes down-regulation of both genes. To directly access the role of PPARdelta in muscle cells, we forced its expression and that of a dominant-negative PPARdelta mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARdelta agonist by induction of genes involved in lipid metabolism and increment of fatty acid oxidation. Overexpression of PPARdelta enhanced these cellular responses, whereas expression of the dominant-negative mutant exerts opposite effects. These data strongly support a role for PPARdelta in the regulation of fatty acid oxidation in skeletal muscle and in adaptive response of this tissue to lipid catabolism.     

UCP-mediated energy depletion in skeletal muscle increases glucose transport despite lipid accumulation and mitochondrial dysfunction

To address the potential role of lipotoxicity and mitochondrial function in insulin resistance, we studied mice with high-level expression of uncoupling protein-1 in skeletal muscle (UCP-H mice). Body weight, body length, and bone mineral density were decreased in UCP-H mice compared with wild-type littermates. Forelimb grip strength and muscle mass were strikingly decreased, whereas muscle triglyceride content was increased fivefold in UCP-H mice. Electron microscopy demonstrated lipid accumulation and large mitochondria with abnormal architecture in UCP-H skeletal muscle. ATP content and key mitochondrial proteins were decreased in UCP-H muscle. Despite mitochondrial dysfunction and increased intramyocellular fat, fasting serum glucose was 22% lower and insulin-stimulated glucose transport 80% higher in UCP-H animals. These beneficial effects on glucose metabolism were associated with increased AMP kinase and hexokinase activities, as well as elevated levels of GLUT4 and myocyte enhancer factor-2 proteins A and D in skeletal muscle. These results suggest that UCP-H mice have a mitochondrial myopathy due to depleted energy stores sufficient to compromise growth and impair muscle function. Enhanced skeletal muscle glucose transport in this setting suggests that excess intramyocellular lipid and mitochondrial dysfunction are not sufficient to cause insulin resistance in mice.     

Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a > 80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.     

Catalytically inactive lipoprotein lipase overexpression increases insulin sensitivity in mice.

Abnormalities in lipoprotein lipase (LPL) function contribute to the development of hypertriglyceridemia, one of the characteristic disorders observed in the metabolic syndrome. In addition to the hydrolyzing activity of triglycerides, LPL modulates various cellular functions via its binding ability to the cell surface. Here we show the effects of catalytically inactive LPL overexpression on high-fat diet (HFD)-induced decreased systemic insulin sensitivity in mice. The binding capacity of catalytically inactive G188E-LPL to C2C12 skeletal muscle cells was not significantly different from that of wild type LPL. Insulin-stimulated IRS-1 phosphorylation and glucose uptake were increased by addition of wild type or mutant LPL in C2C12 cells. After 10 weeks' of HFD feeding, mice had significantly higher blood glucose levels than chow-fed mice in insulin tolerance tests. The blood glucose levels after insulin injection was significantly decreased in mutated LPL-overexpressing mice (G188E mice), as well as in wild type LPL-overexpressing mice (WT mice). Overexpression of catalytically inactive LPL, as well as wild type LPL, improved impaired insulin sensitivity in mice. These results show that decreased expression of LPL possibly causes the insulin resistance, in addition to hypertriglyceridemia, in metabolic syndrome.     

PPARα-LXR as a novel metabolostatic signalling axis in skeletal muscle that acts to optimize substrate selection in response to nutrient status

LXR (liver X receptor) and PPARα (peroxisome-proliferator-activated receptor α) are nuclear receptors that control the expression of genes involved in glucose and lipid homoeostasis. Using wild-type and PPARα-null mice fed on an LXR-agonist-supplemented diet, the present study analysed the impact of pharmacological LXR activation on the expression of metabolically important genes in skeletal muscle, testing the hypothesis that LXR activation can modulate PPAR action in skeletal muscle in a manner dependent on nutritional status. In the fed state, LXR activation promoted a gene profile favouring lipid storage and glucose oxidation, increasing SCD1 (stearoyl-CoA desaturase 1) expression and down-regulating PGC-1α (PPARγ co-activator-1α) and PDK4 (pyruvate dehydrogenase kinase 4) expression. PPARα deficiency enhanced LXR stimulation of SCD1 expression, and facilitated elevated SREBP-1 (sterol-regulatory-element-binding protein-1) expression. However, LXR-mediated down-regulation of PGC-1α and PDK4 was opposed and reversed by PPARα deficiency. During fasting, prior LXR activation augmented PPARα signalling to heighten FA (fatty acid) oxidation and decrease glucose oxidation by augmenting fasting-induced up-regulation of PGC-1α and PDK4 expression, effects opposed by PPARα deficiency. Starvation-induced down-regulation of SCD1 expression was opposed by antecedent LXR activation in wild-type mice, an effect enhanced further by PPARα deficiency, which may elicit increased channelling of FA into triacylglycerol to limit lipotoxicity. Our results also identified potential regulatory links between the protein deacetylases SIRT1 (sirtuin 1) and SIRT3 and PDK4 expression in muscle from fasted mice, with a requirement for PPARα. In summary, we therefore propose that a LXR-PPARα signalling axis acts as a metabolostatic regulatory mechanism to optimize substrate selection and disposition in skeletal muscle according to metabolic requirement.     

Nutritional regulation and role of peroxisome proliferator-activated receptor δ in fatty acid catabolism in skeletal muscle

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARδ displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h starvation period, PPARδ mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARδ is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while refeeding promotes down-regulation of both genes. To directly access the role of PPARδ in muscle cells, we forced its expression and that of a dominant-negative PPARδ mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARδ agonist by induction of genes involved in lipid metabolism and increment of fatty acid oxidation. Overexpression of PPARδ enhanced these cellular responses, whereas expression of the dominant-negative mutant exerts opposite effects. These data strongly support a role for PPARδ in the regulation of fatty acid oxidation in skeletal muscle and in adaptive response of this tissue to lipid catabolism.     

Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice

Bioactive components from bitter melon (BM) have been reported to improve glucose metabolism in vivo, but definitive studies on efficacy and mechanism of action are lacking. We sought to investigate the effects of BM bioactives on body weight, muscle lipid content and insulin signaling in mice fed a high-fat diet and on insulin signaling in L6 myotubes. Male C57BL/6J mice were randomly divided into low-fat diet control (LFD), high-fat diet (HFD) and HFD plus BM (BM) groups. Body weight, body composition, plasma glucose, leptin, insulin and muscle lipid profile were determined over 12 weeks. Insulin signaling was determined in the mouse muscle taken at end of study and in L6 myotubes exposed to the extract. Body weight, plasma glucose, insulin, leptin levels and HOMA-IR values were significantly lower in the BM-fed HFD group when compared to the HFD group. BM supplementation significantly increased IRS-2, IR β, PI 3K and GLUT4 protein abundance in skeletal muscle, as well as phosphorylation of IRS-1, Akt1 and Akt2 when compared with HFD (P<.05 and P<.01). BM also significantly reduced muscle lipid content in the HFD mice. BM extract greatly increased glucose uptake and enhanced insulin signaling in L6 myotubes. This study shows that BM bioactives reduced body weight, improved glucose metabolism and enhanced skeletal muscle insulin signaling. A contributing mechanism to the enhanced insulin signaling may be associated with the reduction in skeletal muscle lipid content. Nutritional supplementation with this extract, if validated for human studies, may offer an adjunctive therapy for diabetes.     

Skeletal muscle reprogramming by activation of calcineurin improves insulin action on metabolic pathways

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow- versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-gamma co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.