Circulating exosomes deliver free fatty acids from the bloodstream to cardiac cells: Possible role of CD36.

Regulation of circulating free fatty acid (FFA) levels and delivery is crucial to maintain tissue homeostasis. Exosomes are nanomembranous vesicles that are released from diverse cell types and mediate intercellular communication by delivering bioactive molecules. Here, we sought to investigate the uptake of FFAs by circulating exosomes, the delivery of FFA-loaded exosomes to cardiac cells and the possible role of the FFA transporter CD36 in these processes. Circulating exosomes were purified from the serum of healthy donors after an overnight fast (F) or 20 minutes after a high caloric breakfast (postprandial, PP). Western blotting, Immunogold Electron Microscopy and FACS analysis of circulating exosomes showed that CD36 was expressed under both states, but was higher in postprandial-derived exosomes. Flow cytometry analysis showed that circulating exosomes were able to take-up FFA directly from serum. Importantly, preincubation of exosomes with a blocking CD36 antibody significantly impeded uptake of the FFA analogue BODIPY, pointing to the role of CD36 in FFA exosomal uptake. Finally, we found that circulating exosomes could delivery FFA analogue BODIPY into cardiac cells ex vivo and in vivo in a mice model. Overall, our results suggest a novel mechanism in which circulating exosomes can delivery FFAs from the bloodstream to cardiac tissue. Further studies will be necessary to understand this mechanism and, in particular, its potential involvement in metabolic pathologies such as obesity, diabetes and atherosclerosis.

Circulating sCD36 levels in patients with non-alcoholic fatty liver disease and controls.

BACKGROUND AND OBJECTIVE: CD36 is implicated in fatty-acid uptake in multiple tissues, including hepatocytes and adipocytes. Circulating CD36 (sCD36) is increased in non-alcoholic fatty liver disease (NAFLD). We explored this association further by investigating correlations between sCD36 levels, intrahepatic lipid content and markers of obesity in NAFLD patients and controls. METHODS: In total, 111 NAFLD patients and 33 normal/overweight controls were included. Intrahepatic lipid content was measured by magnetic resonance spectroscopy; and subgroups of participants had a dual-energy X-ray absorptiometry (n=99), magnetic resonance imaging (n=94, subcutaneous and visceral adipose tissue) and liver biopsy (n=28 NAFLD patients) performed. Plasma sCD36 was assessed by enzyme-linked immunosorbent assay. RESULTS: NAFLD patients had elevated sCD36 levels compared with controls (0.68 (0.12-2.27) versus 0.43 (0.10-1.18), P<0.01). sCD36 correlated with intrahepatic lipid (rs=0.30), alanine transaminase (ALT) (r=0.31), homeostasis model assessment index-insulin resistance (r=0.24), high-density lipoprotein (r=-0.32) and triglyceride (r=0.44, all P<0.01). Intrahepatic lipid and plasma triglyceride were independent predictors of sCD36 levels in a multiple regression analysis. Further, sCD36 and body mass index were weakly correlated (r=0.17, P=0.04); yet, we found no correlations between sCD36 and other measures of fat distribution except an inverse relation to visceral adipose tissue (rs=-0.21, P<0.05). We observed a trend for correlation between sCD36 and hepatic CD36 mRNA expression (r=0.37, P=0.07). CONCLUSIONS: sCD36 levels increased with the level of intrahepatic lipid, insulin resistance and dyslipidemia. The weak association with markers of obesity and the association with hepatic CD36 mRNA expression suggest that excess sCD36 in NAFLD patients is derived from the hepatocytes, which may support that CD36 is involved in NAFLD development. An unhealthy and unbalanced CD36 expression in adipose and hepatic tissue may shift the fatty-acid load to the liver.

Reduced sCD36 following weight loss corresponds to improved insulin sensitivity, dyslipidemia and liver fat in obese children.

BACKGROUND/OBJECTIVES: Childhood obesity is a major health problem with serious long-term metabolic consequences. CD36 is important for the development of obesity-related complications among adults. We aimed to investigate circulating sCD36 during weight loss in childhood obesity and its associations with insulin resistance, dyslipidemia, hepatic fat accumulation and low-grade inflammation. SUBJECTS/METHODS: The impact of a 10-week weight loss camp for obese children (N=113) on plasma sCD36 and further after a 12-month follow-up (N=68) was investigated. Clinical and biochemical data were collected, and sCD36 was measured by an in-house assay. Liver fat was estimated by ultrasonography and insulin resistance by the homeostasis model assessment (HOMA-IR). RESULTS: Along with marked weight loss, sCD36 was reduced by 21% (P=0.0013) following lifestyle intervention, and individual sCD36 reductions were significantly associated with the corresponding decreases in HOMA-IR, triglycerides and total cholesterol. The largest sCD36 decrease occurred among children who reduced HOMA-IR and liver fat. After 12 months of follow-up, sCD36 was increased (P=0.014) and the metabolic improvements were largely lost. CONCLUSIONS: Weight-loss-induced sCD36 reduction, coincident with improved insulin resistance, circulating lipids and hepatic fat accumulation, proposes that sCD36 may be an early marker of long-term health risk associated with obesity-related complications.

The Impact of Lipoprotein-Associated Oxidative Stress on Cell-Specific Microvesicle Release in Patients with Familial Hypercholesterolemia.

Objective. Microvesicles (MVs) are small cell-derived particles shed upon activation. Familial hypercholesterolemia (FH) particularly when associated with Achilles tendon xanthomas (ATX) predisposes to atherosclerosis, possibly through oxLDL-C interaction with the CD36 receptor. To investigate the hypothesis that MVs derived from cells involved in atherosclerosis are increased in FH and that CD36 expressing MVs (CD36+ MVs) may be markers of oxLDL-C-induced cell activation, cell-specific MVs were measured in FH patients with and without ATX and their association with atherogenic lipid profile was studied. Approach and Results. Thirty FH patients with and without ATX and twenty-three controls were included. Plasma concentrations of MVs and CD36+ MVs derived from platelets (PMVs), erythrocytes (ErytMVs), monocytes (MMVs), and endothelial cells (EMVs), as well as tissue factor-positive cells (TF+ MVs), were measured by flow cytometry. Total MVs, MMVs, EMVs, ErytMVs, and TF+ MVs were significantly increased in FH patients, compared to controls. CD36+ MVs derived from endothelial cells and monocytes were significantly higher in FH patients and oxLDL-C predicted all the investigated cell-specific CD36+ MVs in FH patients with ATX. Conclusions. MVs derived from cells involved in atherosclerosis were increased in FH and may contribute to elevated atherothrombosis risk. The increased cell-specific CD36+ MVs observed in FH may represent markers of oxLDL-C-induced cell activation.

The macrophage activation marker sCD163 is associated with changes in NAFLD and metabolic profile during lifestyle intervention in obese children.

BACKGROUND: Obesity is associated with metabolic derangement and non-alcoholic fatty liver disease (NAFLD). Macrophages are involved in liver inflammation and fibrosis, and soluble (s)CD163 is a macrophage activation marker. OBJECTIVES: To associate sCD163 with parameters of paediatric obesity and NAFLD, as well as changes in these parameters during lifestyle intervention. METHODS: We studied 117 obese children during a 10-week lifestyle intervention; 71 completed the 12-month follow-up. We recorded clinical and biochemical data, and performed liver ultrasonography. RESULTS: Baseline sCD163 was higher in children with elevated alanine transaminase (ALT) (2.3 ± 0.7 vs. 2.0 ± 0.6 mg L(-1), P = 0.03), steatosis (2.3 ± 0.7 vs. 2.0 ± 0.6 mg L(-1), P = 0.01) and high paediatric NAFLD fibrosis index (2.3 ± 0.7 vs. 1.9 ± 0.6 mg L(-1) , P = 0.03). Baseline sCD163 was independently associated with ALT, cholesterol and high-sensitivity C-reactive protein (hs-CRP). The change in sCD163 during lifestyle intervention was associated with changes in ALT, homeostatic model assessment of insulin resistance (HOMA-IR), hs-CRP and cholesterol, and inversely associated with the change in high-density lipoprotein cholesterol. CONCLUSION: sCD163 was associated with markers of liver injury and metabolic parameters in obese children, and changes in these parameters during lifestyle intervention. This may suggest that activated macrophages play a role in NAFLD and sCD163 may serve as a marker of liver disease severity and treatment effect.

Circulating sCD36 is associated with unhealthy fat distribution and elevated circulating triglycerides in morbidly obese individuals.

BACKGROUND: The recently identified circulating sCD36 has been proposed to reflect tissue CD36 expression, and is upregulated in case of obesity, insulin resistance and hepatic steatosis. The aim of this study was to explore the effect of weight loss secondary to bariatric surgery in relation to sCD36 among morbidly obese individuals. Furthermore, we investigated the levels of sCD36 in relation to obesity-related metabolic complications, low-grade inflammation and fat distribution. METHODS: Twenty morbidly obese individuals (body mass index (BMI) 43.0±5.4 kg m(-2)) with a referral to Roux-en-Y gastric bypass were included. Anthropometric measurements and fasting blood samples were collected at a preoperative baseline visit and 3 months after surgery. sCD36 was measured by an in-house assay, whereas insulin sensitivity and the hepatic fat accumulation were estimated by the homeostasis model assessment (HOMA-%S) and liver fat percentage (LF%), respectively. RESULTS: Postoperatively, BMI was reduced by 20% to 34.3±5.2 kg m(-2) (P<0.001). sCD36 was reduced by 31% (P=0.001) and improvements were observed in the amount of fat mass (P<0.001), truncal fat mass (P<0.001), circulating triglycerides (P=0.001), HOMA-%S (P=0.007), LF% (P=0.001) and the inflammatory marker high-sensitive C-reactive protein (P=0.005). sCD36 correlated with triglycerides (ρ=0.523, P=0.001) and truncal fat mass (ρ=0.357, P=0.026), and triglycerides were found to be an independent predictor of sCD36. At baseline, participants with the metabolic syndrome had a higher LF% and higher levels of the inflammatory biomarker YKL-40 (P=0.003 and P=0.014) as well as a tendency towards higher levels of sCD36. CONCLUSION: sCD36 was reduced by weight loss and associated with an unhealthy fat accumulation and circulating triglycerides, which support the proposed role of sCD36 as a biochemical marker of obesity-related metabolic complications and risks.

Biochemical markers of bone turnover in diabetes patients--a meta-analysis, and a methodological study on the effects of glucose on bone markers.

UNLABELLED: This study examined whether markers of bone turnover differ between individuals with and without diabetes. Bone markers showed heterogeneity between studies and were discrepant for markers of bone creation and markers of bone degradation. Bone markers may be of lesser value in diabetes due to heterogeneity. INTRODUCTION: The aim of this meta-analysis was to compare existing literature regarding changes in bone markers among diabetics compared to healthy controls. To exclude that blood glucose levels among diabetes patients could influence the assays used for determining bone turnover markers, a methodological study was performed. METHODS: Medline at Pubmed Embase, Cinahl, Svemed+, Cochrane library, and Bibliotek.dk was searched in August 2012. The studies should examine biochemical bone turnover among diabetes patients in comparison to controls in an observational design. In the methodological study, fasting blood samples were drawn from two individuals. Glucose was added to the blood samples in different concentrations and OC, CTX, and procollagen type 1 amino terminal propeptide were measured after 0, 1, 2, and 3 h. RESULTS: Twenty-two papers fulfilled the criteria for the meta-analysis. From the pooled data in the meta-analysis, the bone markers osteocalcin (OC) (-1.15 ng/ml [-1.78,-0.52]) and C-terminal cross-linked telopeptide (CTX) (-0.14 ng/ml [-0.22, -0.05]) were significantly lower among diabetes patients than non-diabetes patients, however other markers did not differ. All markers displayed very high heterogeneity by I2 statistics. In the methodological study, the addition of glucose did not significantly change the bone markers neither by level of glucose nor with increasing incubation time. CONCLUSION: The dissociative pattern of biochemical bone markers of bone formation and bone resorption present in diabetes patients is thus not caused by glucose per se but may be modulated by unknown factors associated with diabetes mellitus.

High sCD36 plasma level is associated with steatosis and its severity in patients with genotype 1 chronic hepatitis C.

Soluble CD36 (sCD36) plasma levels, a known marker of cardiometabolic disorders, are associated with surrogate markers of steatosis, while experimental and human studies show a link between CD36 expression in the liver and steatosis. In a cohort of patients with genotype 1 chronic hepatitis C (G1 CHC), we tested the association of sCD36 plasma levels with host and viral factors and sustained virological response (SVR). One hundred and seventy-five consecutive biopsy-proven patients were studied. sCD36 plasma levels were assessed by an in-house ELISA. All biopsies were scored by one pathologist for staging and grading (Scheuer) and graded for steatosis, which was considered moderate-severe if ≥20%. Patients underwent standard of care therapy with pegylated interferon and ribavirin. The severity of steatosis progressively increased according to sCD36 quartiles (P = 0.02); total and low-density lipoprotein (LDL) cholesterol levels were significantly higher in patients in the lower quartile compared to all the others. Gamma-glutamyl transferase (P = 0.02), homoeostasis model assessment (HOMA) score (P = 0.002) and sCD36 (P = 0.04) were independently associated with the severity of steatosis as continuous variable. Multivariate logistic regression analysis showed that HOMA (OR 1.243, 95% CI 1.04-1.484, P = 0.01) and sCD36 (OR 1.445, 95%CI 1.135-1.839, P = 0.003) were independently linked to steatosis ≥20%. No association was found between sCD36 and SVR. CD36 is linked to steatosis and insulin resistance in patients with G1 CHC, but does not predict response to treatment. The potential of sCD36 as a surrogate marker of steatosis should be further investigated.

Plasma sCD36 is associated with markers of atherosclerosis, insulin resistance and fatty liver in a nondiabetic healthy population.

OBJECTIVES: Insulin resistance is associated with increased CD36 expression in a number of tissues. Moreover, excess macrophage CD36 may initiate atherosclerotic lesions. The aim of this study was to determine whether plasma soluble CD36 (sCD36) was associated with insulin resistance, fatty liver and carotid atherosclerosis in nondiabetic subjects. METHODS: In 1296 healthy subjects without diabetes or hypertension recruited from 19 centres in 14 European countries (RISC study), we determined the levels of sCD36, adiponectin, lipids and liver enzymes, insulin sensitivity (M/I) by euglycaemic-hyperinsulinaemic clamp, carotid atherosclerosis as intima-media thickness (IMT) and two estimates of fatty liver, the fatty liver index (FLI) and liver fat percentage (LF%). RESULTS: IMT, FLI, LF%, presence of the metabolic syndrome, impaired glucose regulation, insulin and triglycerides increased across sCD36 quartiles (Q2-Q4), whereas adiponectin and M/I decreased (P ≤ 0.01). sCD36 was lower in women than in men (P = 0.045). Log sCD36 showed a bimodal distribution, and amongst subjects with sCD36 within the log-normal distribution (log-normal population, n = 1029), sCD36 was increased in subjects with impaired glucose regulation (P = 0.045), metabolic syndrome (P = 0.006) or increased likelihood of fatty liver (P < 0.001). sCD36 correlated significantly with insulin, triglycerides, M/I and FLI (P < 0.05) after adjustment for study centre, gender, age, glucose tolerance status, smoking habits and alcohol consumption. In the log-normal population, these relationships were stronger than in the total study population and, additionally, sCD36 was significantly associated with LF% and IMT (P < 0.05). CONCLUSIONS: In this cross-sectional study of nondiabetic subjects, sCD36 was significantly associated with indices of insulin resistance, carotid atherosclerosis and fatty liver. Prospective studies are needed to further evaluate the role of sCD36 in the inter-relationship between atherosclerosis, fatty liver and insulin resistance.

Surveillance for avian influenza viruses in wild birds in Denmark and Greenland, 2007-10.

In Denmark and Greenland, extensive surveillance of avian influenza (AI) viruses in wild bird populations has been conducted from 2007 through 2010. In Denmark, the surveillance consisted of passive surveillance of wild birds found dead or sick across Denmark and active surveillance of apparently healthy live birds in waterfowl reservoirs and along migratory flyways, birds living in proximity to domestic poultry, and hunted game birds. Dead birds were sampled by oropharyngeal swabbing. Healthy live wild birds were captured with nets, traps, or by hand and were sampled by swabbing of the oropharyngeal and cloacal tracts, or swabs were collected from fresh fecal droppings. Hunted game birds were delivered to game-handling establishments, where each bird was sampled by oropharyngeal and cloacal swabbing. During the 2007-10 period, a total of 11,055 wild birds were sampled in Denmark, of which 396 were birds that were found dead. In Greenland, samples were collected mainly from fecal droppings in breeding areas. Samples from 3555 live and apparently healthy wild birds were tested. All swab samples were tested by pan-influenza reverse transcriptase-PCR (RT-PCR), and the positive samples were further tested by H5/H7 specific RT-PCRs. H5/H7-positive samples were subjected to hemagglutination cleavage site sequencing for pathotyping. In addition, all RT-PCR-positive samples were subjected to virus isolation, and the virus isolates were subsequently subtyped. In Denmark, low pathogenic (LP) H5 viruses were detected throughout the period, in addition to a few LPAI H7 and several other subtypes. In Greenland, very few samples were positive for AI. None of them were found to be of the H5 or H7 subtypes by RT-PCR. Isolation of these viruses in eggs was unsuccessful; thus, they were not subtyped further. The findings did, however, demonstrate the presence of LPAI viruses in Greenland. For several water bird species overwintering in North America and northwest Europe, respectively, Greenland constitutes a common breeding area. This raises the possibility that viruses could be transmitted to North America via Greenland and vice versa. In Denmark, the screenings for AI showed LPAI viruses to be naturally occurring in the wild bird population, particularly in waterfowl. The occurrence of AI viruses in the wild bird population may pose a risk for AI infections in Danish

Effect of weight reduction on insulin sensitivity, sex hormone-binding globulin, sex hormones and gonadotrophins in obese children.

OBJECTIVE: Obesity in men is associated with reduced insulin sensitivity and hypoandrogenism, while obesity in women is associated with reduced insulin sensitivity and hyperandrogenism. In children, the effect of obesity and weight reduction on the hypothalamo-pituitary-gonadal axis is rarely investigated. The aim of the present study was to investigate the effect of weight reduction in obese Caucasian children on insulin sensitivity, sex hormone-binding globulin (SHBG), DHEAS and the hypothalamo-pituitary-gonadal axis. METHODS: One hundred and sixteen (65 females) obese children with a median age of 12.3 (7-15) years were examined before and after a 10-week stay at a weight loss camp. Examination included anthropometry and fasting blood samples measuring plasma glucose, serum insulin, SHBG, DHEAS, testosterone, 17ß-oestradiol, FSH and LH. RESULTS: Body mass index (BMI) decreased (P<0.01), insulin sensitivity and SHBG increased (P<0.01), independent of gender and puberty. The changes in insulin sensitivity and the changes in SHBG correlated significantly (P<0.01) independent of gender, puberty and the changes in BMI. Testosterone increased in boys (P<0.01) and tended to decrease in girls (P=0.05, in girls after menarche (P=0.03)). FSH increased in boys and girls. LH increased in boys and was unchanged in girls. CONCLUSIONS: During weight loss, insulin sensitivity and SHBG increased significantly in obese children, and the changes in insulin sensitivity and the changes in SHBG correlated significantly independent of gender, puberty and the changes in BMI. There was sexual dimorphism in the changes of testosterone, with the changes in boys towards increased virilisation and the changes in girls towards less virilisation.

Soluble CD36 (sCD36) clusters with markers of insulin resistance, and high sCD36 is associated with increased type 2 diabetes risk.

CONTEXT AND OBJECTIVE: Soluble CD36 (sCD36) may be an early marker of insulin resistance and atherosclerosis. The objective of this prospective study was to evaluate sCD36 as a predictor of type 2 diabetes and to study its relationship with components of the metabolic syndrome (MetSy). DESIGN, SETTING, PARTICIPANTS, AND OUTCOME MEASURES: We conducted a case-referent study nested within a population-based health survey. Baseline variables included sCD36, body mass index, blood pressure, blood lipids, adipokines, inflammatory markers, and beta-cell function. A total of 173 initially nondiabetic cohort members who developed type 2 diabetes during 10 yr of follow-up were matched (1:2) with referents. Exploratory factor analysis was applied to hypothesize affiliation of sCD36 to the MetSy components. RESULTS: Doubling of baseline sCD36 increases the odds ratio for diabetes development by 1.24 in the general study population and by 1.45 in the female population (P < 0.025). Comparing upper sCD36 quartiles with lower, odds ratio for diabetes was 4.6 in women (P = 0.001), 3.15 in men (P = 0.011), and 2.6 in obese individuals (P < 0.025). Multivariate analysis shows that sCD36 does not predict diabetes independent of fasting plasma glucose and insulin. Factor analysis of 15 variables generates a six-factor model explaining 66-69% of total variance, where sCD36, body mass index, insulin, proinsulin, and leptin were assigned to the obesity/insulin resistance cluster. CONCLUSIONS: Upper quartile sCD36 is associated with elevated diabetes risk independent of age, gender, and obesity. Baseline sCD36 does not, however, predict diabetes independent of fasting glucose and insulin. sCD36 clusters with important markers of insulin resistance and MetSy that are key predictors of type 2 diabetes.

GLUT11, but not GLUT8 or GLUT12, is expressed in human skeletal muscle in a fibre type-specific pattern.

Nine novel sugar transporter-like proteins have been discovered in the past 5 years. The mRNA for three of these, the glucose transporters (GLUT) GLUT8, GLUT11 and GLUT12, have been detected in human skeletal muscle. In the present study, we examined the pattern of expression and localization of the GLUT isoforms 8, 11 and 12 in human skeletal muscle using an immunohistochemical approach. Biopsies of human skeletal muscle from sedentary or trained healthy adults, from fetal muscle (24 weeks of gestation), from obese type-2 diabetic subjects, and from patients suffering from polymyositis or amyotrophic lateral sclerosis (ALS) were studied. GLUT8 and 12 immunoreactivity was below detection level in both developing and adult muscle fibres. GLUT11 immunoreactivity, however, was present in slow-twitch muscle fibres, but not in fast twitch fibres. Since, in contrast, GLUT4 was expressed in all investigated muscle fibres, the pattern of expression of GLUT11 differs from that of GLUT4, suggesting a specialized function for GLUT11 with a regulation independent from that of GLUT4. Obesity, type-2 diabetes, training, conditions of de- and reinnervation (ALS) and regeneration (polymyositis) failed to induce GLUT8 or -12 expression. Likewise, the fibre type-dependent pattern of GLUT11 immunoreactivity was unaltered. However, some slow muscle fibres lose their GLUT11 immunoreactivity under regeneration. Our results indicate that GLUT11 immunoreactivity, in contrast to that of GLUT4, is expressed exclusively in slow-twitch muscle fibres and is unaffected by physiological and pathophysiological conditions except in primary myopathy. GLUT8 and GLUT12 do not appear to be of importance in human muscle under physiological and pathophysiological conditions.

The basal kinetic parameters of glycogen synthase in human myotube cultures are not affected by chronic high insulin exposure.

There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV(0.1)) of GS, increased the sensitivity of GS to the allosteric activator glucose 6-phosphate (A(0.5)) and increased the sensitivity of GS to its substrate UDPG (K(m(0.1))) when myotubes were precultured at low insulin with/without high glucose conditions. However, this effect of acute insulin stimulation was abolished in myotubes precultured at high insulin with or without high glucose. Furthermore, we found significant correlations between the fractional velocities FV(0.1) of GS and K(m(0.1)) (rho=-0.72, P<0.0001), between FV(0.1) and A(0.5) (rho=-0.82, P<0.0001) and between K(m(0.1)) and A(0.5) values (rho=0.71, P<0.0001). Our results show that chronic exposure of human myotubes to high insulin with or without high glucose did not affect the basal kinetic parameters but abolished the reactivity of GS to acute insulin stimulation. We suggest that insulin induced insulin resistance of GS is caused by a failure of acute insulin stimulation to decrease A(0.5) and K(m(0.1)) in human skeletal muscle.

Insulin action and long-term electrically induced training in individuals with spinal cord injuries.

PURPOSE: Individuals with spinal cord injuries (SCI) have an increased prevalence of insulin resistance and type 2 diabetes mellitus. In able-bodied individuals, training with large muscle groups increases insulin sensitivity and may prevent type 2 diabetes mellitus. However, individuals with SCI cannot voluntarily recruit major muscle groups, but by functional electrical stimulation (FES) they can now perform ergometer bicycle training. METHODS: Ten subjects with SCI (35 +/- 2 yr (mean +/- SE), 73 +/- 5 kg, level of lesion C6--Th4, time since injury: 12 +/- 2 yr) performed 1 yr of FES cycling (30 min x d(-1), 3 d x wk(-1) (intensive training)). Seven subjects continued 6 months with reduced training (1 d x wk(-1) (reduced training)). A sequential, hyperinsulinemic (50 mU x min(-1) x m(-2) (step 1) and 480 mU x min(-1) x m(-2) (step 2)), euglycemic clamp, an oral glucose tolerance test (OGTT), and determination of GLUT 4 transporter protein in muscle biopsies were performed before and after training. RESULTS: Insulin-stimulated glucose uptake rates increased after intensive training (from 4.9 +/- 0.5 mg x min(-1) x kg(-1) to 6.2 +/- 0.6 mg x min(-1) x kg(-1) (P < 0.008) (step 1) and from 9.0 +/- 0.8 mg x min(-1) x kg(-1) to 10.6 +/- 0.8 mg x min(-1) x kg(-1) (P = 0.103) (step 2)). With the reduction in training, insulin sensitivity decreased to a similar level as before training (P > 0.05). GLUT 4 increased by 105% after intense training and decreased again with the training reduction. The subjects had impaired glucose tolerance before and after training, and neither glucose tolerance nor insulin responses to OGTT were significantly altered by training. CONCLUSIONS: Electrically induced bicycle training, performed three times per week increases insulin sensitivity and GLUT 4 content in skeletal muscle in subjects with SCI. A reduction in training to once per week is not sufficient to maintain these effects. FES training may have a role in the prevention of the insulin resistance syndrome in persons with SCI.

GLUT4 is reduced in slow muscle fibers of type 2 diabetic patients: is insulin resistance in type 2 diabetes a slow, type 1 fiber disease?

To gain further insight into the mechanisms underlying muscle insulin resistance, the influence of obesity and type 2 diabetes on GLUT4 immunoreactivity in slow and fast skeletal muscle fibers was studied. Through a newly developed, very sensitive method using immunohistochemistry combined with morphometry, GLUT4 density was found to be significantly higher in slow compared with fast fibers in biopsy specimens from lean and obese subjects. In contrast, in type 2 diabetic subjects, GLUT4 density was significantly lower in slow compared with fast fibers. GLUT4 density in slow fibers from diabetic patients was reduced by 9% compared with the weight-matched obese subjects and by 18% compared with the lean control group. The slow-fiber fraction was reduced to 86% in the obese subjects and to 75% in the diabetic subjects compared with the control group. Estimated GLUT4 contribution from slow fibers was reduced to 77% in the obese subjects and to 61% in type 2 diabetic patients compared with the control subjects. We propose that a reduction in the fraction of slow-twitch fibers, combined with a reduction in GLUT4 expression in slow fibers, may reduce the insulin-sensitive GLUT4 pool in type 2 diabetes and thus contribute to skeletal muscle insulin resistance.

Glucose transporter expression in human skeletal muscle fibers.

The present study was initiated to investigate GLUT-1 through -5 expression in developing and mature human skeletal muscle. To bypass the problems inherent in techniques using tissue homogenates, we applied an immunocytochemical approach, employing the sensitive enhanced tyramide signal amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas after birth, the characteristic subcellular localization is as seen in adult muscle fibers. Our results show that GLUT-1, GLUT-3, and GLUT-4 seem to be of importance during muscle fiber growth and development. GLUT-5 protein was undetectable in fetal and adult skeletal muscle fibers. In adult muscle fibers, only GLUT-4 was expressed at significant levels. GLUT-1 immunoreactivity was below the detection limit in muscle fibers, indicating that this glucose transporter is of minor importance for muscle glucose supply. Thus we hypothesize that GLUT-4 also mediates basal glucose transport in muscle fibers, possibly through constant exposure to tonal contraction and basal insulin levels.

Direct evidence of fiber type-dependent GLUT-4 expression in human skeletal muscle.

GLUT-4 expression in individual fibers of human skeletal muscles in younger and older adults was studied. Furthermore, the dependency of insulin-stimulated glucose uptake on fiber type distribution was investigated. Fiber type distribution was determined in cryosections of muscle biopsies from 8 younger (29 yr) and 8 older (64 yr) healthy subjects, and estimates of GLUT-4 expression in individual fibers were obtained by combining immunohistochemistry and stereology. GLUT-4 was more abundantly expressed in slow compared with fast muscle fibers in both younger (P < 0.007) and older (P < 0. 001) subjects. A 25% reduction of GLUT-4 density in fast fibers (P < 0.001) and an unchanged GLUT-4 density in slow fibers were demonstrated in older compared with younger subjects. Insulin-stimulated glucose uptake rates measured by hyperinsulinemic, euglycemic clamp were not correlated with the fraction of slow fibers in the young (r = -0.45, P > 0.25) or in the elderly (r = 0. 11, P > 0.75) subjects. In conclusion, in human skeletal muscle, GLUT-4 expression is fiber type dependent and decreases with age, particularly in fast muscle fibers.

Intracellular skeletal muscle glucose metabolism is differentially altered by dexamethasone treatment of normoglycemic relatives of type 2 diabetic patients.

Young first-degree relatives of type 2 diabetic patients are insulin-resistant, with the insulin resistance mainly located in skeletal muscle due to decreased insulin-induced nonoxidative glucose metabolism and muscle glycogen synthase activation. We investigated whether the mechanism differs for dexamethasone (dex)-induced insulin resistance in first-degree relatives of type 2 diabetics versus healthy control subjects by quantifying intracellular glucose processing in muscle biopsies taken before and after 5 days of dex treatment (4 mg/d) in 20 normal glucose-tolerant relatives of type 2 diabetic patients and 20 matched controls (age, 29.4 +/- 1.7 v 29.4 +/- 1.6 years; body mass index, 25.1 +/- 1.0 v 25.1 +/- 0.9 kg/m2). In addition, an intravenous glucose tolerance test (IVGTT) combined with continuous indirect calorimetry was performed. Following 5 days of dex treatment, glucose tolerance deteriorated in both the relatives and the control subjects. Fasting dry-weight muscle glucose and fasting intracellular muscle glucose concentrations increased in response to dex only in the relatives (2.43 +/- 0.21 v 2.97 +/- 0.26 mmol/kg dry weight, P < .05; 0.28 +/- 0.07 v 0.45 +/- 0.08 mmol/L intracellular water, P < .05); no increases were observed in the control subjects. Fasting dry-weight muscle lactate also increased post-dex only in the relatives (7.37 +/- 0.40 v 10.77 +/- 1.22 mmol/kg dry weight, P < .001). Both basal muscle glucose and lactate concentrations from the IVGTT study correlated with the 2-hour post-dex glucose value obtained during the OGTT study in the relatives (R = .76 and R = .74, respectively, both P < .0001) but not in the control subjects. Basal intramuscular glycogen synthase activity decreased approximately 25% in both the relatives and control subjects post-dex; the decrement was significant (P < .01) only in control subjects. Indirect calorimetry during the post-dex IVGTT demonstrated increased glucose oxidation (P < .03) and reduced lipid oxidation (P < .03) in the relatives only. We postulate that the insulin resistance induced by dex in first-degree relatives of type 2 diabetic patients is associated with a preferential channeling of glucose into the glycolytic pathway (increased glucose oxidation and lactate production), probably associated with a preexisting downregulation of the glycosen synthase pathway.

Diurnal variation of the serum leptin concentration in patients with anorexia nervosa.

OBJECTIVE: In rodents, leptin is involved in regulating eating behaviour, fat storage, and reproductive function. In humans, the serum leptin concentration in obese and normal weight subjects correlates with body mass index, reflecting the body fat store. The serum leptin exhibit diurnal variation, however, this has been reported to be absent in normal weighted amenorrheic athletes. Anorexia nervosa is associated with multiple endocrine abnormalities. Hypothalamic amenorrhoea often precedes the weight loss and may persist after weight recovery. We hypothesized that leptin could be involved in the regulation of eating behaviour and gonadal function in anorexia nervosa. DESIGN: We measured the concentration of leptin in serum samples taken after an overnight fast in 18 female anorexia nervosa patients and 11 controls. To study diurnal variation, eight patients and 11 controls were hospitalized for 24 h and had a standardized diet at regular times. Seven blood samples were obtained at 4 h intervals from each subject. PATIENTS: The patients fulfilled the DSM-IV criteria for anorexia nervosa. The mean body mass index for the patients was 14.2 +/- 2.3 kg/m2 and for controls 20.3 +/- 1.7 kg/m2. RESULTS: The mean fasting leptin concentration as well as the 24 h mean concentration were significantly lower in the anorectic group than in the control group (2.5 +/- 0.9 vs 10.1 +/- 6.1 micrograms/l, P < 0.01 and 2.7 +/- 1.5 vs 10.6 +/- 7.1 micrograms/l, P < 0.01 respectively). In the whole group of subjects (n = 28) a significant positive correlation between the leptin level and body mass index was found (r = 0.63, P < 0.001). In the anorectic group it was found that the leptin level correlated better with body fat percentage than with body mass index. In normalized data the time course of the mean leptin levels showed a monophasic variation with nadir and zenith at about 0900 and 0100 h respectively. However, the individual coefficients of variance were significantly lower in the anorectic group compared to the group of healthy women. CONCLUSION: In patients with anorexia nervosa the leptin level is low, reflecting the low body fat mass, and the relative diurnal variation is strikingly reduced. The similarity to that of normal weighted women with hypothalamic amenorrhoea suggest that altered leptin oscillations may be of particular significance in the hypothalamic regulation of reproductive function.