Molecular analysis of the muscle protein projectin in Lepidoptera.

Striated muscles of both vertebrates and insects contain a third filament composed of the giant proteins, namely kettin and projectin (insects) and titin (vertebrates). All three proteins have been shown to contain several domains implicated in conferring elasticity, in particular a PEVK segment. In this study, the characterization of the projectin protein in the silkmoth, Bombyx mori L. (Lepidoptera: Bombycidae), and the monarch butterfly, Danaus plexippus L. (Lepidoptera: Nymphalidae), as well as a partial characterization in the Carolina sphinx, Manduca sexta L. (Lepidoptera: Sphingidae), are presented. This study showed that, similar to other insects, projectin's overall modular organization was conserved, but in contrast, the PEVK region had a highly divergent sequence. The analysis of alternative splicing in the PEVK region revealed a small number of possible isoforms and the lack of a flight-muscle specific variant, both characteristics being in sharp contrast with findings from other insects. The possible correlation with difference in flight muscle stiffness and physiology between Lepidoptera and other insect orders is discussed.

Projectin PEVK domain, splicing variants and domain structure in basal and derived insects.

The third elastic filament of striated muscles consists of giant proteins: titin (in vertebrates) and kettin/projectin (in insects). In all three proteins, elasticity is at least partly associated with the so-called PEVK domain. The projectin PEVK domains of diverse insects are highly divergent compared with an otherwise conserved protein organization. We present the characterization of the PEVK domain in two dragonflies and in human lice. A conserved segment at the end of the PEVK, the NH(2)-terminal conserved segment-1 (NTCS-1), may serve as an anchor point for projectin to either myosin or actin, providing a mechanical link. The analysis of alternative splicing variants identifies the shortest PEVK isoform as the predominant form in the flight muscles of several insects, possibly contributing to myofibrillar stiffness.

Stearoyl CoA desaturase 1 is elevated in obesity but protects against fatty acid-induced skeletal muscle insulin resistance in vitro.

AIMS/HYPOTHESIS: Stearoyl CoA desaturase 1 (SCD1) is implicated in mediating obesity and insulin resistance. Paradoxically, SCD1 converts saturated fatty acids, the lipid species implicated in mediating insulin resistance, to monounsaturated fatty acids. The aim of the present study was to assess the molecular mechanisms that implicate SCD1 in the aetiology of fatty acid-induced insulin resistance. METHODS: SCD1 protein was transiently decreased or increased in rat L6 skeletal muscle myotubes using SCD1 short interfering RNA (siRNA) or liposome-mediated transfection of pcDNA3.1/Hygro-mSCD1, respectively. RESULTS: Reducing SCD1 protein resulted in marked esterification of exogenous fatty acids into diacylglycerol (DAG) and ceramide. Insulin-stimulated Akt activity and phosphorylation and 2-deoxyglucose uptake were reduced with SCD1 siRNA. Exposure of L6 myotubes to palmitate abolished insulin-stimulated glucose uptake in both control and SCD1 siRNA myotubes. Overexpression of SCD1 resulted in triacylglycerol esterification but attenuated ceramide and DAG accumulation and protected myotubes from fatty acid-induced insulin resistance. CONCLUSIONS/INTERPRETATION: SCD1 protects from cellular toxicity in L6 myotubes by preventing excessive accumulation of bioactive lipid metabolites.

The effect of exercise and training status on platelet activation: do cocoa polyphenols play a role?

Sedentary and trained men respond differently to the same intensity of exercise, this is probably related to their platelet reactivity and antioxidant capacity. There is growing interest in the utilization of antioxidant-rich plant extracts as dietary food supplements. The aim of this study was to investigate the effect of an acute bout of sub maximal exercise on platelet count and differential response of platelet activation in trained and sedentary subjects and to observe if cocoa polyphenols reverse the effect of exercise on platelet function. The practical significance of this study was that many sedentary people engage in occasional strenuous exercise that may predispose them to risk of heart disease. Fasting blood samples were collected from 16 male subjects, pre and post 1-h cycling exercise at 70% of maximal aerobic power (VO2max) before and after consumption of cocoa or placebo. Agonist stimulated citrated whole blood was utilized for measuring platelet aggregation, adenosine triphosphate (ATP) release and platelet activation. Baseline platelet count (221 +/- 33 x 10(9)/L) and ATP release (1.4 +/- 0.6 nmol) increased significantly (P < 0.05) after exercise in all subjects. Baseline platelet numbers in the trained were higher (P < 0.05) than in the sedentary (235 +/- 37 vs. 208 +/- 34 x 10(9)/L), where as platelet activation in trained was lower (P < 0.05) than sedentary (51 +/- 6 vs. 59 +/- 5%). Seven days of cocoa polyphenol supplementation had little effect on any of the parameters measured. We conclude that trained subjects show decreased activation of stimulated platelets when compared to the sedentary subjects and short-term cocoa polyphenol supplementation did not decrease platelet activity in response to exercise independent of prior training status.

Discordant gene expression in skeletal muscle and adipose tissue of patients with type 2 diabetes: effect of interleukin-6 infusion.

AIMS/HYPOTHESIS: We compared metabolic gene expression in adipose tissue and skeletal muscle from patients with type 2 diabetes and from well-matched healthy control subjects. We hypothesised that gene expression would be discordantly regulated when comparing the two groups. Our secondary aim was to determine the effect of Interleukin-6 (IL6) infusion on circulating adipokines and on gene expression in human adipose tissue. To do this we used real-time RT-PCR. METHODS: Both diabetic and control subjects underwent basal skeletal muscle and subcutaneous adipose tissue biopsies. A subset of these individuals underwent a 3-h infusion of recombinant human IL6 and had adipose tissue samples taken before and after infusion. RESULTS: The mRNA gene expression of suppressor of cytokine signalling (SOCS) 3, peroxisome proliferative activated receptor (PPAR) alpha/delta, PPAR gamma, coactivator 1, alpha (PPARGC1A), carnitine palmitoyltransferase 1B and solute carrier family 2 (facilitated glucose transporter), member 4 (formerly known as glucose transporter 4/GLUT4), was higher in adipose tissue, but lower in skeletal muscle of diabetic patients than in that of control subjects. In addition, uncoupling protein 1 (UCP1) gene was detected in the adipose tissue of some of the diabetic patients, but not in the control subjects. The following genes were increased by infusion of recombinant human IL6 in both groups: SOCS1/3, resistin, adiponectin, AMP-activated protein kinase-alpha-1 and PPARA. Plasma tumour necrosis factor alpha, adiponectin and resistin were all unaffected by IL6 infusion, but plasma resistin was lower in the diabetic subjects than in control subjects. CONCLUSIONS/INTERPRETATION: The observation that PPARGC1A and the PPARs were upregulated in the adipose tissue of type 2 diabetic patients, along with the finding that adipose tissue from some patients with type 2 diabetes can express UCP1 mRNA, suggests that in these patients white adipose tissue may move towards a brown adipose tissue phenotype.

Suppression of plasma free fatty acids upregulates peroxisome proliferator-activated receptor (PPAR) alpha and delta and PPAR coactivator 1alpha in human skeletal muscle, but not lipid regulatory genes.

Fatty acids are an important ligand for peroxisome proliferator-activated receptor (PPAR) activation and transcriptional regulation of metabolic genes. To examine whether reduced plasma free fatty acid (FFA) availability affects the mRNA content of proteins involved in fuel metabolism in vivo, the skeletal muscle mRNA content of various transcription factors, transcriptional coactivators and genes encoding for lipid regulatory proteins were examined before and after 3 h of cycle exercise with (NA) and without (CON) pre-exercise ingestion of nicotinic acid (NA). NA resulted in a marked (3- to 6-fold) increase (P<0.05) in PPARalpha, PPARdelta and PPAR coactivator 1alpha (PGC1alpha) mRNA, but was without effect on nuclear respiratory factor-1 and Forkhead transcription factor, fatty acid transcolase/CD36, carnitine palmitoyl transferase 1, hormone sensitive lipase (HSL) and pyruvate dehydrogenase kinase 4. Exercise in CON was associated with increased (P<0.05) PPARalpha, PPARdelta and PGC1alpha mRNA, which was similar in magnitude to levels observed with NA at rest. Exercise was generally without effect on the mRNA content of lipid regulatory proteins in CON and did not affect the mRNA content of the measured subset of transcription factors, transcriptional co-activators and lipid regulatory proteins during NA. To determine the possible mechanisms by which NA might affect PGC1alpha expression, we measured p38 MAP kinase (MAPK) and plasma epinephrine. Phosphorylation of p38 MAPK was increased (P<0.05) by NA treatment at rest, and this correlated (r2=0.84, P<0.01) with increased PGC1alpha. Despite this close relationship, increasing p38 MAPK in human primary myotubes was without effect on PGC1alpha mRNA content. Plasma epinephrine was elevated (P<0.05) by NA at rest (CON: 0.27+/-0.06, NA: 0.72+/-0.11 nM) and throughout exercise. Incubating human primary myotubes with epinephrine increased PGC1alpha independently of changes in p38 MAPK phosphorylation. Hence, despite the fact that NA ingestion decreased FFA availability, it promoted the induction of PPARalpha/delta and PGC1alpha gene expression to a similar degree as prolonged exercise. We suggest that the increase in PGC1alpha may be due to the elevated plasma epinephrine levels. Despite these changes in transcription factors/coactivators, the mRNA content of lipid regulatory proteins was generally unaffected by plasma FFA availability.