Effects of four-week high-fructose diet on gene expression in skeletal muscle of healthy men

AIMS: A high-fructose diet (HFrD) may play a role in the obesity and metabolic disorders epidemic. In rodents, HFrD leads to insulin resistance and ectopic lipid deposition. In healthy humans, a four-week HFrD alters lipid homoeostasis, but does not affect insulin sensitivity or intramyocellular lipids (IMCL). The aim of this study was to investigate whether fructose may induce early molecular changes in skeletal muscle prior to the development of whole-body insulin resistance. METHODS: Muscle biopsies were taken from five healthy men who had participated in a previous four-week HFrD study, during which insulin sensitivity (hyperinsulinaemic euglycaemic clamp), and intrahepatocellular lipids and IMCL were assessed before and after HFrD. The mRNA concentrations of 16 genes involved in lipid and carbohydrate metabolism were quantified before and after HFrD by real-time quantitative PCR. RESULTS: HFrD significantly (P<0.05) increased stearoyl-CoA desaturase-1 (SCD-1) (+50%). Glucose transporter-4 (GLUT-4) decreased by 27% and acetyl-CoA carboxylase-2 decreased by 48%. A trend toward decreased peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was observed (-26%, P=0.06). All other genes showed no significant changes. CONCLUSION: HFrD led to alterations of SCD-1, GLUT-4 and PGC-1alpha, which may be early markers of insulin resistance.

Skeletal muscle cells from insulin-resistant (non-diabetic) individuals are susceptible to insulin desensitization by palmitate

We recently demonstrated that in vivo insulin resistance is not retained in cultured skeletal muscle cells. In the present study, we tested the hypothesis that treating cultured skeletal muscle cells with fatty acids has an effect on insulin action which differs between insulin-sensitive and insulin-resistant subjects. Insulin effects were examined in myotubes from 8 normoglycemic non-obese insulin-resistant and 8 carefully matched insulin-sensitive subjects after preincubation with or without palmitate, linoleate, and 2-bromo-palmitate. Insulin-stimulated glycogen synthesis decreased by 27 +/- 5 % after palmitate treatment in myotubes from insulin-resistant, but not from insulin-sensitive subjects (1.50 +/- 0.08-fold over basal vs. 1.81 +/- 0.09-fold, p = 0.042). Despite this observation, we did not find any impairment in the PI 3-kinase/PKB/GSK-3 pathway. Furthermore, insulin action was not affected by linoleate and 2-bromo-palmitate. In conclusion, our data provide preliminary evidence that insulin resistance of skeletal muscle does not necessarily involve primary defects in insulin action, but could represent susceptibility to the desensitizing effect of fatty acids and possibly other environmental or adipose tissue-derived factors.

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.

Ramipril increases the protein level of skeletal muscle IRS-1 and alters protein tyrosine phosphatase activity in spontaneously hypertensive rats

To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.

Insulin signaling and action in cultured skeletal muscle cells from lean healthy humans with high and low insulin sensitivity

The aim of these studies was to investigate whether insulin resistance is primary to skeletal muscle. Myoblasts were isolated from muscle biopsies of 8 lean insulin-resistant and 8 carefully matched insulin-sensitive subjects (metabolic clearance rates as determined by euglycemic-hyperinsulinemic clamp: 5.8 +/- 0.5 vs. 12.3 +/- 1.7 ml x kg(-1) x min(-1), respectively; P < or = 0.05) and differentiated to myotubes. In these cells, insulin stimulation of glucose uptake, glycogen synthesis, insulin receptor (IR) kinase activity, and insulin receptor substrate 1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity were measured. Furthermore, insulin activation of protein kinase B (PKB) was compared with immunoblotting of serine residues at position 473. Basal glucose uptake (1.05 +/- 0.07 vs. 0.95 +/- 0.07 relative units, respectively; P = 0.49) and basal glycogen synthesis (1.02 +/- 0.11 vs. 0.98 +/- 0.11 relative units, respectively; P = 0.89) were not different in myotubes from insulin-resistant and insulin-sensitive subjects. Maximal insulin responsiveness of glucose uptake (1.35 +/- 0.03-fold vs. 1.41 +/- 0.05-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.43) and glycogen synthesis (2.00 +/- 0.13-fold vs. 2.10 +/- 0.16-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.66) were also not different. Insulin stimulation (1 nmol/l) of IR kinase and PI 3-kinase were maximal within 5 min (approximately 8- and 5-fold over basal, respectively), and insulin activation of PKB was maximal within 15 min (approximately 3.5-fold over basal). These time kinetics were not significantly different between groups. In summary, our data show that insulin action and signaling in cultured skeletal muscle cells from normoglycemic lean insulin-resistant subjects is not different from that in cells from insulin-sensitive subjects. This suggests an important role of environmental factors in the development of insulin resistance in skeletal muscle.

Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase

Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

Skeletal benefits after long-term retirement in former elite female gymnasts

Bone strength benefits after long-term retirement from elite gymnastics in terms of bone geometry and volumetric BMD were studied by comparing retired female gymnasts to moderately active age-matched women. In a cross-sectional study, 30 retired female gymnasts were compared with 30 age-matched moderately active controls. Bone geometric and densitometric parameters were measured by pQCT at the distal epiphyses and shafts of the tibia, femur, radius, and humerus. Muscle cross-sectional areas were assessed from the shaft scans. Independent t-tests were conducted on bone and muscle variables to detect differences between the two groups. The gymnasts had retired for a mean of 6.1 +/- 0.4 yr and were engaged in

Prenatal sonographic diagnosis of skeletal dysplasias

OBJECTIVE: To assess the types and numbers of cases, gestational age at specific prenatal diagnosis and diagnostic accuracy of the diagnosis of skeletal dysplasias in a prenatal population from a single tertiary center. METHODS: This was a retrospective database review of type, prenatal and definitive postnatal diagnoses and gestational age at specific prenatal diagnosis of all cases of skeletal dysplasias from a mixed referral and screening population between 1985 and 2007. Prenatal diagnoses were grouped into 'correct ultrasound diagnosis' (complete concordance with postnatal pediatric or pathological findings) or 'partially correct ultrasound diagnosis' (skeletal dysplasias found postnatally to be a different one from that diagnosed prenatally). RESULTS: We included 178 fetuses in this study, of which 176 had a prenatal ultrasound diagnosis of 'skeletal dysplasia'. In 160 cases the prenatal diagnosis of a skeletal dysplasia was confirmed; two cases with skeletal dysplasias identified postnatally had not been diagnosed prenatally, giving 162 fetuses with skeletal dysplasias in total. There were 23 different classifiable types of skeletal dysplasia. The specific diagnoses based on prenatal ultrasound examination alone were correct in 110/162 (67.9%) cases and partially correct in 50/162 (30.9%) cases, (160/162 overall, 98.8%). In 16 cases, skeletal dysplasia was diagnosed prenatally, but was not confirmed postnatally (n = 12 false positives) or the case was lost to follow-up (n = 4). The following skeletal dysplasias were recorded: thanatophoric dysplasia (35 diagnosed correctly prenatally of 40 overall), osteogenesis imperfecta (lethal and non-lethal, 31/35), short-rib dysplasias (5/10), chondroectodermal dysplasia Ellis-van Creveld (4/9), achondroplasia (7/9), achondrogenesis (7/8), campomelic dysplasia (6/8), asphyxiating thoracic dysplasia Jeune (3/7), hypochondrogenesis (1/6), diastrophic dysplasia (2/5), chondrodysplasia punctata (2/2), hypophosphatasia (0/2) as well as a further 7/21 cases with rare or unclassifiable skeletal dysplasias. CONCLUSION: Prenatal diagnosis of skeletal dysplasias can present a considerable diagnostic challenge. However, a meticulous sonographic examination yields high overall detection. In the two most common disorders, thanatophoric dysplasia and osteogenesis imperfecta (25% and 22% of all cases, respectively), typical sonomorphology accounts for the high rates of completely correct prenatal diagnosis (88% and 89%, respectively) at the first diagnostic examination.

Association between statin-associated myopathy and skeletal muscle damage

BACKGROUND: Many patients taking statins often complain of muscle pain and weakness. The extent to which muscle pain reflects muscle injury is unknown. METHODS: We obtained biopsy samples from the vastus lateralis muscle of 83 patients. Of the 44 patients with clinically diagnosed statin-associated myopathy, 29 were currently taking a statin, and 15 had discontinued statin therapy before the biopsy (minimal duration of discontinuation 3 weeks). We also included 19 patients who were taking statins and had no myopathy, and 20 patients who had never taken statins and had no myopathy. We classified the muscles as injured if 2% or more of the muscle fibres in a biopsy sample showed damage. Using reverse transcriptase polymerase chain reaction, we evaluated the expression levels of candidate genes potentially related to myocyte injury. RESULTS: Muscle injury was observed in 25 (of 44) patients with myopathy and in 1 patient without myopathy. Only 1 patient with structural injury had a circulating level of creatine phosphokinase that was elevated more than 1950 U/L (10x the upper limit of normal). Expression of ryanodine receptor 3 was significantly upregulated in patients with biopsy evidence of structural damage (1.7, standard error of the mean 0.3). INTERPRETATION: Persistent myopathy in patients taking statins reflects structural muscle damage. A lack of elevated levels of circulating creatine phosphokinase does not rule out structural muscle injury. Upregulation of the expression of ryanodine receptor 3 is suggestive of an intracellular calcium leak.

Graded doses of recombinant interleukin-1beta induce generalized osteopenia in rats without altering skeletal growth and joint integrity

Whereas a primary role of interleukin-1beta (IL-1beta) in local bone remodelling and articular inflammation has been well established, the effect of prolonged systemic administration of this cytokine on total skeletal Ca, somatic growth and joint tissue has not yet been investigated.