Anna Krook

Non-CpG Methylation of the PGC-1α Promoter through DNMT3B Controls Mitochondrial Density

Epigenetic modification through DNA methylation is implicated in metabolic disease. Using whole-genome promoter methylation analysis of skeletal muscle from normal glucose-tolerant and type 2 diabetic subjects, we identified cytosine hypermethylation of peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1 alpha (PGC-1alpha) in diabetic subjects. Methylation levels were negatively correlated with PGC-1alpha mRNA and mitochondrial DNA (mtDNA). Bisulfite sequencing revealed that the highest proportion of cytosine methylation within PGC-1alpha was found within non-CpG nucleotides. Non-CpG methylation was acutely increased in human myotubes by exposure to tumor necrosis factor-alpha (TNF-alpha) or free fatty acids, but not insulin or glucose. Selective silencing of the DNA methyltransferase 3B (DNMT3B), but not DNMT1 or DNMT3A, prevented palmitate-induced non-CpG methylation of PGC-1alpha and decreased mtDNA and PGC-1alpha mRNA. We provide evidence for PGC-1alpha hypermethylation, concomitant with reduced mitochondrial content in type 2 diabetic patients, and link DNMT3B to the acute fatty-acid-induced non-CpG methylation of PGC-1alpha promoter.

Insulin action and insulin resistance in human skeletal muscle.

The pivotal role in vivo of insulin is to control fuel homeostasis. Insulin exerts its effect on three main target tissues: liver, fat and skeletal muscle. Resistance to the normal action of insulin contributes to the pathogenesis of a number of common human disorders, including Type I (insulin-dependent) and Type II (non-insulin-dependent) diabetes mellitus, hypertension and the metabolic syndrome x, thus constituting a major public health problem. Environmental factors such as poor nutrition (high-fat diet) or inactivity (lower oxygen consumption i.e. VO2max) or both also seem to have a major effect on the development of peripheral insulin resistance [1±4]. Although the primary defect in the pathogenesis of Type II diabetes is not known, a combination of genetic and environmental factors probably contribute to the manifestation of this progressive metabolic disorder, which is usually not apparent clinically until mid-life. Type II diabetic patients are characterised by fasting hyperglycaemia and by increased, normal or low concentrations of insulin. Any one of these could be a cause or consequence of defects in insulin secretion from the beta cell or peripheral insulin resistance in skeletal muscle or adipose tissue. Defects in insulin secretion have been noted in lean or normal weight patients with Type II diabetes [5, 6] and insulin resistance in skeletal muscle has been reported in Type II diabetic patients regardless of weight [7]. Skeletal muscle is the principal tissue responsible for insulinstimulated glucose disposal [8] and the major site of peripheral insulin resistance [9]. Intense interest has been focused on explaining the molecular basis for the development of insulin resistance in skeletal muscle in an effort to control and improve whole-body glucose homeostasis. This review is focused on current understanding of the molecular mechanisms regulating insulin action and the factors contributing to insulin resistance in human skeletal muscle.

Insulin-Stimulated Akt Kinase Activity Is Reduced in Skeletal Muscle From NIDDM Subjects

The serine/threonine kinase Akt (PKB/Rac) has been implicated as playing a role in the insulin-signaling pathway to glucose transport. Little is known regarding the regulation of Akt kinase activity in insulin-sensitive tissues, such as skeletal muscle, or whether this regulation is altered in insulin-resistant states such as NIDDM. We examined the effect of insulin on Akt kinase activity in skeletal muscle from six NIDDM patients and six healthy subjects. Whole-body insulin sensitivity, assessed by the euglycemic-hyperinsulinemic clamp, was significantly lower in NIDDM subjects (P < 0.001), and this was accompanied by impaired in vitro insulin-stimulated glucose transport in skeletal muscle. In both groups, insulin induced a significant increase in Akt kinase activity, but the response to maximal insulin (60 nmol/1) was markedly reduced in skeletal muscle from NIDDM subjects (66% of control levels, P < 0.01). Impaired Akt kinase activity was not accompanied by decreased protein expression of Akt. Instead, a trend toward increased Akt expression was noted in skeletal muscle from NIDDM subjects (P < 0.1). These parallel defects in insulin-stimulated Akt kinase activity and glucose transport in diabetic skeletal muscle suggest that reduced Akt kinase activity may play a role in the development of insulin resistance in NIDDM.

Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle

The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one‐leg cycle ergometry at ∼70% VO2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction‐induced signaling in human skeletal. Activation of the different MAP kinase pathways byphysical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.—Widegren, U., Jiang, X.‐J., Krook, A., Chibalin, A. V., Bjo¨rnholm, M., Tally, M., Roth, R. A., Henriksson, J., Wallberg‐ Henriksson, H., Zierath, J. R. Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle. FASEB J. 12, 1379– 1389 (1998)

Skeletal Muscle PGC-1α1 Modulates Kynurenine Metabolism and Mediates Resilience to Stress-Induced Depression

Depression is a debilitating condition with a profound impact on quality of life for millions of people worldwide. Physical exercise is used as a treatment strategy for many patients, but the mechanisms that underlie its beneficial effects remain unknown. Here, we describe a mechanism by which skeletal muscle PGC-1α1 induced by exercise training changes kynurenine metabolism and protects from stress-induced depression. Activation of the PGC-1α1-PPARα/δ pathway increases skeletal muscle expression of kynurenine aminotransferases, thus enhancing the conversion of kynurenine into kynurenic acid, a metabolite unable to cross the blood-brain barrier. Reducing plasma kynurenine protects the brain from stress-induced changes associated with depression and renders skeletal muscle-specific PGC-1α1 transgenic mice resistant to depression induced by chronic mild stress or direct kynurenine administration. This study opens therapeutic avenues for the treatment of depression by targeting the PGC-1α1-PPAR axis in skeletal muscle, without the need to cross the blood-brain barrier.

Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.

Regulation of Skeletal Muscle Physiology and Metabolism by Peroxisome Proliferator-Activated Receptor δ

Agonists directed against the α and γ isoforms of the peroxisome proliferator-activated receptors (PPARs) have become important for the respective treatment of hypertriglyceridemia and insulin resistance associated with metabolic disease. PPARδ is the least well characterized of the three PPAR isoforms. Skeletal muscle insulin resistance is a primary risk factor for the development of type 2 diabetes. There is increasing evidence that PPARδ is an important regulator of skeletal muscle metabolism, in particular, muscle lipid oxidation, highlighting the potential utility of this isoform as a drug target. In addition, PPARδ seems to be a key regulator of skeletal muscle fiber type and a possible mediator of the adaptations noted in skeletal muscle in response to exercise. In this review we summarize the current status regarding the regulation, and the metabolic effects, of PPARδ in skeletal muscle.

Role of AMP kinase and PPAR delta in the regulation of lipid and glucose metabolism in human skeletal muscle

The peroxisome proliferator-activated receptor (PPAR)δ has been implicated in the regulation of lipid metabolism in skeletal muscle. Furthermore, activation of PPARδ has been proposed to improve insulin sensitivity and reduce glucose levels in animal models of type 2 diabetes. We recently demonstrated that the PPARδ agonist GW501516 activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake in skeletal muscle. However, the underlying mechanism remains to be clearly identified. In this study, we first confirmed that incubation of primary cultured human muscle cells with GW501516 induced AMPK phosphorylation and increased fatty acid transport and oxidation and glucose uptake. Using small interfering RNA, we have demonstrated that PPARδ expression is required for the effect of GW501516 on the intracellular accumulation of fatty acids. Furthermore, we have shown that the subsequent increase in fatty acid oxidation induced by GW501516 is dependent on both PPARδ and AMPK. Concomitant with these metabolic changes, we provide evidence that GW501516 increases the expression of key genes involved in lipid metabolism (FABP3, CPT1, and PDK4) by a PPARδ-dependent mechanism. Finally, we have also demonstrated that the GW501516-mediated increase in glucose uptake requires AMPK but not PPARδ. In conclusion, the PPARδ agonist GW501516 promotes changes in lipid/glucose metabolism and gene expression in human skeletal muscle cells by PPARδ- and AMPK-dependent and -independent mechanisms.

Metabolic and mitogenic signal transduction in human skeletal muscle after intense cycling exercise

We determined whether mitogen‐activated protein kinase (MAPK) and 5′‐AMP‐activated protein kinase (AMPK) signalling cascades are activated in response to intense exercise in skeletal muscle from six highly trained cyclists (peak O2 uptake (V̇O2,peak) 5.14 ± 0.1 l min−1) and four control subjects (V̇O2,peak 3.8 ± 0.1 l min−1) matched for age and body mass. Trained subjects completed eight 5 min bouts of cycling at ≈85% of V̇O2,peak with 60 s recovery between work bouts. Control subjects performed four 5 min work bouts commencing at the same relative, but a lower absolute intensity, with a comparable rest interval. Vastus lateralis muscle biopsies were taken at rest and immediately after exercise. Extracellular regulated kinase (ERK1/2), p38 MAPK, histone H3, AMPK and acetyl CoA‐carboxylase (ACC) phosphorylation was determined by immunoblot analysis using phosphospecific antibodies. Activity of mitogen and stress‐activated kinase 1 (MSK1; a substrate of ERK1/2 and p38 MAPK) and α1 and α2 subunits of AMPK were determined by immune complex assay. ERK1/2 and p38 MAPK phosphorylation and MSK1 activity increased (P < 0.05) after exercise 2.6‐, 2.1‐ and 2.0‐fold, respectively, in control subjects and 1.5‐, 1.6‐ and 1.4‐fold, respectively, in trained subjects. Phosphorylation of histone H3, a substrate of MSK1, increased (P < 0.05) ≈1.8‐fold in both control and trained subject. AMPKα2 activity increased (P < 0.05) after exercise 4.2‐ and 2.3‐fold in control and trained subjects, respectively, whereas AMPKα1 activity was not altered. Exercise increased ACC phosphorylation (P < 0.05) 1.9‐ and 2.8‐fold in control and trained subjects. In conclusion, intense cycling exercise in subjects with a prolonged history of endurance training increases MAPK signalling to the downstream targets MSK1 and histone H3 and isoform‐specific AMPK signalling to ACC. Importantly, exercise‐induced signalling responses were greater in untrained men, even at the same relative exercise intensity, suggesting muscle from previously well‐trained individuals requires a greater stimulus to activate signal transduction via these pathways.

Interleukin-6 Directly Increases Glucose Metabolism in Resting Human Skeletal Muscle

Interleukin (IL)-6 is a proinflammatory cytokine shown to modify insulin sensitivity. Elevated plasma levels of IL-6 are observed in insulin-resistant states. Interestingly, plasma IL-6 levels also increase during exercise, with skeletal muscle being the predominant source. Thus, IL-6 has also been suggested to promote insulin-mediated glucose utilization. In this study, we determined the direct effects of IL-6 on glucose transport and signal transduction in human skeletal muscle. Skeletal muscle strips were prepared from vastus lateralis biopsies obtained from 22 healthy men. Muscle strips were incubated with or without IL-6 (120 ng/ml). We found that IL-6 increased glucose transport in human skeletal muscle 1.3-fold (P < 0.05). A 30-min pre-exposure to IL-6 did not affect insulin-stimulated glucose transport. IL-6 also increased skeletal muscle glucose incorporation into glycogen, as well as glucose oxidation (1.5- and 1.3-fold, respectively; P < 0.05). IL-6 increased phosphorylation of STAT3 (signal transducer and activator of transcription 3; P < 0.05), AMP-activated protein kinase (P = 0.063), and p38 mitogen-activated protein kinase (P < 0.05) and reduced phosphorylation of S6 ribosomal protein (P < 0.05). In contrast, phosphorylation of protein kinase B/Akt, AS160 (Akt substrate of 160 kDa), and GSK3α/β (glycogen synthase kinase 3α/β) as well as insulin receptor substrate 1–associated phosphatidylinositol 3-kinase activity remained unaltered. In conclusion, acute IL-6 exposure increases glucose metabolism in resting human skeletal muscle. Insulin-stimulated glucose transport and insulin signaling were unchanged after IL-6 exposure.