Overexpression of the obese (ob) gene in adipose tissue of human obese subjects.

Obesity is accompanied by complications such as hypertension, non-insulin-dependent diabetes mellitus and atherosclerosis, which in turn cause ischaemic heart disease, stroke and premature death. The underlying mechanisms behind imbalance in energy intake and energy expenditure that lead to obesity are still controversial. In most populations, obesity is more common among women than men and is a multifactorial phenotype, which may result from a complex network of genetic and nongenetic factors. The relative importance of genetic factors for obesity is under debate. Genome searches using polymorphic markers in inbred mice with phenotypes that result in extreme obesity and studies of human candidate genes are being performed in an attempt to identify genes that contribute to obesity. There is evidence that body weight is physiologically regulated and it has been postulated that the storage of fat may provide signals to the brain that the body is obese, which in turn may make the subject eat less and burn more fuel. One of the molecules that may be involved in such signalling is the obese (ob) gene product. Mutations in ob result in profound obesity and type II diabetes in mice. The mouse ob gene and its human homologue have been cloned and sequenced. The gene is expressed in adipose tissue and the product has features of a secreted protein. We have investigated human ob expression in subcutaneous and omental adipose tissue obtained from non-obese and massively obese subjects using in situ hybridization histochemistry and report on overexpression in obese people.

Mechanisms underlying regional differences in lipolysis in human adipose tissue.

Catecholamine-induced lipolysis was investigated in nonobese females and males. Isolated subcutaneous adipocytes were obtained from the abdominal and gluteal regions. The lipolytic effect of noradrenaline was four to fivefold more marked in abdominal adipocytes than in gluteal fat cells. This regional difference was more apparent in females than in males. No site differences were observed when lipolysis was stimulated with agents acting at different postreceptor levels. The beta-adrenergic lipolytic sensitivity was 10-20 times greater in abdominal adipocytes from both sexes than in gluteal adipocytes. Abdominal adipocytes from females showed a 40 times lower alpha 2-adrenergic antilipolytic sensitivity than did gluteal adipocytes, but the adenosine receptor sensitivity was similar in both sites. Beta-receptor affinity for agonists displayed no site or sex variation. Abdominal adipocytes showed a twofold increased beta-adrenoceptor density than did gluteal cells from both sexes. The alpha 2-adrenoceptor density was similar in all regions, but in females the affinity of clonidine for these sites was 10-15 times lower in the abdominal fat cells compared with gluteal cells. In conclusion, regional differences in catecholamine-induced lipolysis are regulated at the adrenoceptor level, chiefly because of site variations in beta-adrenoceptor density. Further variations in the affinity properties of alpha 2-adrenergic receptor in females may explain why the regional differences in catecholamine-induced lipolysis are more pronounced in women than in men.

Catecholamine-induced lipolysis in adipose tissue of the elderly.

Age-dependent alterations in the effects of catecholamines on lipolysis were investigated in 25 young (21-35 yr) and 10 elderly (58-72 yr) healthy, nonobese subjects using isolated adipocytes obtained from abdominal subcutaneous tissue. Basal lipolysis was not affected by aging, while the rate of catecholamine-stimulated lipolysis was reduced by 50% in the elderly subjects (P less than 0.005). To elucidate the mechanisms behind this phenomenon lipolysis was stimulated with agents that act at well-defined steps in the lipolytic cascade, from the receptor down to the final step: the activation of the protein kinase/hormone-sensitive lipase complex. All agents stimulated lipolysis at a 50% lower rate in elderly as compared with young subjects (P less than 0.05 or less). However, half-maximum effective concentrations of the lipolytic agents were similar in both groups. The antilipolytic effects of alpha 2-adrenoceptor agonists were also the same in young and old subjects. Moreover, the stoichiometric properties of the beta- and alpha 2-receptors did not change with increasing age. In vivo studies performed on the same individuals likewise demonstrated an impaired lipolytic responsiveness, with 50% lower plasma glycerol concentrations during exercise in the elderly subjects (P less than 0.05), in spite of a normal rise in plasma norepinephrine. The plasma glycerol levels correlated strongly to the glycerol release caused by catecholamine-stimulated lipolysis in vitro in both young and elderly subjects (r = 0.8-0.9, P less than 0.001). In conclusion, a decreased activation of the hormone-sensitive lipase complex appears to be the mechanism underlying a blunted lipolytic response of fat cells to catecholamine stimulation in elderly subjects. This finding may, explain the age-dependent decreased lipolytic response to exercise in vivo.

Lipolytic catecholamine resistance due to decreased beta 2-adrenoceptor expression in fat cells.

The existence of lipolytic beta-adrenoceptor (BAR) resistance was investigated in vivo and in isolated abdominal subcutaneous adipocytes in 65 healthy and drug-free subjects. The concentration of isoprenaline (nonselective BAR agonist) causing half-maximum lipolysis effect (ED50) varied bimodally and 10(6)-fold between individuals but was almost constant in the same subject when measured two times at rest or before and 30 min after exercise. The subjects were categorized as having either high or low isoprenaline sensitivity. The former group had a 50% reduced in vivo lipolytic response to exercise and mental stress, despite a 50% increased plasma noradrenaline response (P < 0.01) and a 350% increased plasma adrenaline response (P < 0.02). In fat cells the lipolytic ED50 values for noradrenaline and terbutaline (BAR2 agonist) were 10 times lower (P < 0.001) in low-sensitive subjects, but the maximum lipolytic actions of these agents (and of isoprenaline) were similar in both groups. The action on lipolysis of dobutamine (BAR1 agonist), forskolin (stimulating adenylate cyclase), dibutyryl cyclic AMP (activating protein kinase), clonidine (alpha 2-adrenergic agonist), or phenyl isopropyladenosine (adenosine receptor agonist) were almost identical in high- and low-sensitivity subjects. ED50 for isoprenaline correlated with ED50 for terbutaline (r = 0.75), but not with ED50 for dobutamine. In high-sensitivity subjects the number of BAR2 was almost three-fold increased (P < 0.002) and the steady-state adipocyte mRNA level for BAR2 was sixfold increased (P < 0.005). BAR2 affinity as well as BAR1 number, affinity and mRNA expression were similar in both groups. In 11 cholecystectomy patients (otherwise healthy) lipolytic ED50 for beta agonists correlated in omental and subcutaneous fat cells (r = 0.85 for isoprenaline; r = 0.95 for terbutaline). In conclusion, lipolytic resistance to catecholamines is present in vivo in apparently healthy subjects due to reduced expression of BAR2 in adipocytes.

Leptin secretion from adipose tissue in women. Relationship to plasma levels and gene expression.

The role of expression and secretion of the ob gene product, leptin, for the regulation of plasma leptin levels has been investigated in vitro using abdominal subcutaneous adipose tissue of 20 obese, otherwise healthy, and 11 nonobese women. Body mass index (BMI, mean+/-SEM; kg/m2) in the two groups was 41+/-2 and 23+/-1, respectively. Fat cell volume was 815+/-55 pl in the obese and 320+/-46 pl in the nonobese group. In the obese group, plasma leptin concentrations and adipose leptin mRNA (relative to gamma actin) were increased five and two times, respectively. Moreover, adipose tissue secretion rates per gram lipid weight or per fat cell number were also increased two and seven times, respectively, in the obese group. There were strong linear correlations (r = 0.6-0.8) between plasma leptin, leptin secretion, and leptin mRNA. All of these leptin measurements correlated strongly with BMI and fat cell volume (r = 0.7- 0.9). About 60% of the variation in plasma leptin could be attributed to variations in leptin secretion rate, BMI, or fat cell volume. We conclude that elevated circulating levels of leptin in obese women above all result from accelerated secretion rates of the peptide from adipose tissue because of increased ob gene expression. However, leptin mRNA, leptin secretion, and circulating leptin levels are all more closely related to the stored amount of lipids in the fat cells of adipose tissue than they are to an arbitrary division into obese versus nonobese.

Demonstration of an in vivo functional beta 3-adrenoceptor in man.

Although it is well established in several mammalian species that beta 3-adrenoceptors play a major role in regulating lipolysis and thermogenesis in adipose tissue, the functional existence and role of this receptor subtype in man has been controversial. We investigated whether the beta 3-adrenoceptor functionally co-exists with beta 1- and beta 2-adrenoceptors in vivo in human adipose tissue. Subcutaneous abdominal adipose tissue of healthy non-obese subjects was microdialyzed with equimolar concentrations of dobutamine (selective beta 1-adrenoceptor agonist), terbutaline (selective beta 2-adrenoceptor agonist), or CGP 12177 (selective beta 3-adrenoceptor agonist). All three agents caused a rapid, sustained, concentration-dependent and significant elevation of the glycerol level in the microdialysate (lipolysis index). However, only terbutaline stimulated the nutritive blood flow in adipose tissue, as measured by an ethanol escape technique. Dobutamine and CGP 12177 was equally effective in elevating the glycerol level (maximum effect 150% above baseline). Terbutaline was significantly more effective than the other two beta-agonists (maximum effect 200% above baseline). When adipose tissue was pretreated with the beta 1/beta 2-selective adrenoceptor blocker propranolol the glycerol increasing effect of dobutamine or terbutaline was inhibited by 80-85% but the glycerol response to CGP 12177 was not influenced. It is concluded that a functional beta 3-adrenoceptor is present in vivo in man. It co-exists with beta 1- and beta 2-adrenoceptors in adipose tissue and may therefore play a role in lipolysis regulation. It appears, however, that the beta 2-adrenoceptor is the most important beta-adrenoceptor subtype for the mobilization of lipids from abdominal subcutaneous adipose tissue because of its concomitant stimulatory effect on lipolysis and blood flow.

A pathogenic role of visceral fat beta 3-adrenoceptors in obesity.

Increased release of free fatty acids (FFA) from visceral fat cells to the portal venous system may cause several metabolic disturbances in obesity. However, this hypothesis and the underlying mechanism remain to be demonstrated. In this study catecholamine-induced lipid mobilization through lipolysis in omental adipose tissue was investigated in vitro in 25 markedly obese subjects (body mass index range 35-56 kg/m2) undergoing weight reduction surgery and in 19 nonobese subjects (body mass index range 20-28 kg/m2) undergoing cholecystectomy. Release of FFA and glycerol, induced by norepinephrine or adrenergic receptor subtype-specific agonists, were determined in isolated omental fat cells. The obese subjects had higher fat cell volume, blood pressure, plasma insulin levels, blood glucose, plasma triglycerides, and plasma cholesterol than the controls. There was evidence of upper-body fat distribution in the obese group. The rate of FFA and glycerol response to norepinephrine was increased twofold in the cells of obese subjects; no significant reutilization of FFA during catecholamine-induced lipolysis was observed in any of the groups (glycerol/FFA ratio near 1:3). There were no differences in the lipolytic sensitivity to beta 3- or beta 2-adrenoceptor specific agonists between the two groups. However, beta 3-adrenoceptor sensitivity was approximately 50 times enhanced (P = 0.0001), and the coupling efficiency of these receptors was increased from 37 to 56% (P = 0.01) in obesity. Furthermore, the obese subjects demonstrated a sixfold lower alpha 2-adrenoceptor sensitivity (P = 0.04). beta 3-Adrenoceptor sensitivity, but not alpha 2-, beta 1-, or beta 2-adrenoceptor sensitivity, correlated with norepinephrine-induced lipolysis (r = -0.67, P = 0.0001) and fat cell volume (r = -0.71, P = 0.0001). In conclusion, catecholamine-induced rate of FFA mobilization from omental fat cells is accelerated due to elevated rate of lipolysis in obesity, mainly because of an increased beta 3-adrenoceptor function, but partly also because of a decreased alpha 2-adrenoceptor function. This promotes an increased release of FFA to the portal system, which may contribute to the parallel metabolic disturbances observed in upper-body obesity.

Human beta-2 adrenoceptor gene polymorphisms are highly frequent in obesity and associate with altered adipocyte beta-2 adrenoceptor function.

Catecholamines play a central role in the regulation of energy expenditure, in part by stimulating lipid mobilization through lipolysis in fat cells. The beta-2 adrenoceptor (BAR-2) is a major lipolytic receptor in human fat cells. To determine whether known polymorphisms in codons 16, 27, and 164 of this receptor play a role in obesity and subcutaneous adipocyte BAR-2 lipolytic function, we investigated a group of 140 women with a large variation in body fat mass. Only the polymorphisms in codons 16 and 27 were common in the study population. The Gln27Glu polymorphism was markedly associated with obesity with a relative risk for obesity of approximately 7 and an odds ratio of approximately 10. Homozygotes for Glu27 had an average fat mass excess of 20 kg and approximately 50% larger fat cells than controls. However, no significant association with changes in BAR-2 function was observed. The Arg16Gly polymorphism was associated with altered BAR-2 function with Gly16 carriers showing a fivefold increased agonist sensitivity and without any change in BAR-2 expression. However, it was not significantly linked with obesity. These findings suggest that genetic variability in the human BAR-2 gene could be of major importance for obesity, energy expenditure, and lipolytic BAR-2 function in adipose tissue, at least in women.

Tissue distribution of beta 3-adrenergic receptor mRNA in man.

Expression of mRNA for beta 1-, beta 2-, and beta 3-adrenergic receptors (beta 1-, beta 2-, and beta 3-AR) was investigated in human tissues. beta 1- and beta 2-AR mRNA distribution correlated with that of the cognate receptors established by pharmacological studies. beta 3-AR transcripts were abundant in infant perirenal brown adipose tissue, characterized by the presence of uncoupling protein (UCP) mRNA. In adult whole adipose tissues, beta 3-AR mRNA levels were high in deep deposits such as perirenal and omental, and lower in subcutaneous. In these deposits, UCP mRNA levels paralleled those of beta 3-AR. However, isolated omental and subcutaneous adipose cells, enriched in white adipocytes, expressed beta 3-AR but no UCP transcripts. beta 3-AR mRNA was highly expressed in gallbladder, and to a much lower extent in colon, independently of UCP mRNA. Quadriceps or abdominal muscles, heart, liver, lung, kidney, thyroid, and lymphocytes did not express intrinsic beta 3-AR mRNA. This study demonstrates that substantial amounts of brown adipocytes exist throughout life in adipose deposits, which are generally classified as white. These deposits are the main sites of beta 3-AR expression, which also occurs in gallbladder and colon. beta 3-AR may thus be involved in the control of lipid metabolism, possibly from fat assimilation in the digestive tract, to triglyceride storage and mobilization in adipose tissues.

Abnormal action of catecholamines on lipolysis in adipocytes of type I diabetic patients treated with insulin.

Catecholamine-induced lipolysis was investigated in adipocytes obtained before and after 30 min of exercise from 10 insulin-treated type I (insulin-dependent) diabetic men and 10 male matched control subjects. The alpha 2-adrenoceptor-mediated antilipolytic effect of catecholamines was normal, but the beta-adrenoceptor-mediated lipolytic sensitivity was increased 10-fold (P less than .01) in diabetic subjects before and after exercise. The latter correlated inversely (r greater than .7) with the circulating norepinephrine level, which was significantly reduced in diabetic subjects. Basal lipolysis and lipolysis activated at different steps distal to the beta-receptor were similar in the two groups. There was no major change in the total number of beta- and alpha-adrenoceptors in the diabetic patients. However, the proportion of high-affinity beta-adrenoceptors was significantly increased in these patients compared with control subjects. In the diabetic patients, approximately 50% of the beta-adrenoceptors were in a high-affinity state, compared to approximately 30% in the control subjects (P less than .025). In diabetic subjects there was an enhanced plasma glycerol response to exercise, despite a blunted plasma norepinephrine response. The data suggest enhanced sensitivity of catecholamine-induced lipolysis in type I diabetes due to an increase in the number of high-affinity (i.e., coupled) beta-adrenoceptors in fat cells. This mechanism may be due to low levels of circulating norepinephrine and may also explain the exaggerated lipolytic response to exercise in the diabetic state.

Leptin secretion from subcutaneous and visceral adipose tissue in women.

Upper body obesity is a risk factor for type 2 diabetes. Little is known about the regulation of body fat distribution, but leptin may be involved. This study examined the secretion of leptin in subcutaneous and omental fat tissue in 15 obese and 8 nonobese women. Leptin secretion rates were two to three times higher in subcutaneous than in omental fat tissue in both obese and nonobese women (P < 0.0001 and P < 0.001, respectively). There was a positive correlation between BMI and leptin secretion rates in both subcutaneous (r = 0.87, P < 0.0001) and omental (r = 0.74, P < 0.0001) fat tissue. Furthermore, leptin secretion rates in subcutaneous and omental fat tissue correlated well with serum leptin levels (r = 0.84, P < 0.0001 and r = 0.73, P = 0.001, respectively), although in multivariate analysis, the subcutaneous leptin secretion rate was the major regressor for serum leptin (F = 42). Subcutaneous fat cells were approximately 50% larger than omental fat cells, and there was a positive correlation between fat cell size and leptin secretion rate in both fat depots (r = 0.8, P < 0.01). Leptin (but not gamma-actin) mRNA levels were twofold higher in subcutaneous than in omental fat tissue (P < 0.05). Thus the subcutaneous fat depot is the major source of leptin in women owing to the combination of a mass effect (subcutaneous fat being the major depot) and a higher secretion rate in the subcutaneous than in the visceral region, which in turn could be due to increased cell size and leptin gene expression.

Peroxisome proliferator-activated receptor gamma polymorphisms affect systemic inflammation and survival in end-stage renal disease patients starting renal replacement therapy.

BACKGROUND: Inflammation may contribute to the markedly increased cardiovascular morbidity and mortality in end-stage renal disease (ESRD). However, the prevalence of inflammation varies in different ESRD populations. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is an important nuclear signaling protein that may regulate inflammatory response, and recent studies have revealed genetic polymorphisms that have significant effect on PPAR-gamma signaling. The aim of this study was to clarify whether the PPAR-gamma 161C/T and PPAR-gamma2 Pro12Ala single-nucleotide polymorphisms (SNPs) influence the inter-individual variance of inflammation and mortality in ESRD patients. METHODS: The present prospective study included 229 incident Caucasian ESRD patients (62% males) just prior to starting renal replacement therapy and 207 healthy controls (62% males). Blood samples were taken for measuring systemic inflammatory (CRP, TNF-alpha, IL-6) and nutritional (S-albumin) parameters. The presence of diabetes mellitus, malnutrition (subjective global assessment (SGA)) and cardiovascular disease (CVD) were also assessed. Genotyping of the two PPAR-gamma SNPs was performed using Pyrosequencing. During follow-up (1621+/-63 days), both all-cause and CVD-mortality were investigated. RESULTS: ESRD patients had a higher prevalence of both the PPAR-gamma 161 CC and PPAR-gamma2 Pro12Pro genotypes than the general population (p<0.01). Whereas the Pro12Pro genotype was associated with higher median serum levels of both hs-CRP (p<0.05) and TNF-alpha (p<0.01) the 161CC genotype was associated with a significantly higher (6.6 mg/L versus 3.3 mg/L; p<0.01) median hs-CRP level. Following adjustment for age, gender, SGA and CVD a significantly higher mortality rate was observed in patients with the Pro12Pro genotype. CONCLUSION: This study demonstrates significant differences in PPAR-gamma genotype distribution between ESRD patients and healthy controls. Furthermore, as the PPAR-gamma2 Pro12Pro genotype was associated with both higher levels of biomarkers of inflammation as well as shorter survival, genetic polymorphisms seem to play a role in determining systemic inflammatory status and outcome in this patient group.

Beta-adrenoreceptor subtype expression in human liver.

The pharmacological and gene expressions of beta 1- and beta 2-adrenoceptor subtypes (BAR1 and BAR2) were investigated in human liver by radioligand binding assays, adenylate cyclase experiments, and RNA excess solution hybridization. [125I]Cyanopinodolol, nonlabeled adrenergic agents, and BAR1/BAR2 cRNA were used as probes. The relationship between binding sites for BAR1 and BAR2 was markedly different from that between the basal mRNA expression for the two receptor subtypes. Plasma membranes as well as a microsomel-enriched fraction contained binding sites only for BAR2. The potency of BAR agonists and antagonists in stimulating adenylate cyclase activity of plasma membranes was typical of a BAR2 response. Northern blot analysis of total cellular RNA isolated from liver tissue showed hybridization of the BAR1 probe to a mRNA species of 2.5-2.6 kilobases and of the BAR2 probe to a mRNA species of 2.2-2.3 kilobases. The basal level of BAR1 mRNA was 5-fold higher than of BAR2 mRNA, as assayed by solution hybridization. No difference in BAR subtype mRNA stability was observed, as indicated by a mRNA half-life of approximately 5.5 h for both receptor subtypes. It is concluded that specific factors are involved in the steady state regulation of BAR subtype expression in human liver. This tissue contains solely BAR2 owing to a posttranscriptional block of basal BAR1 expression.

Evidence for a functional beta 3-adrenoceptor in man.

1. The existence of a functional beta 3-adrenoceptor in man was investigated by studying the lipolytic action of selective beta-adrenoceptor agents in isolated white omental and subcutaneous fat cells. 2. The non-selective beta 1/beta 2-adrenoceptor antagonist, CGP 12177 was lipolytic in both omental and subcutaneous fat cells. The intrinsic activity relative to isoprenaline was greater in omental than in subcutaneous cells. 3. Addition of the beta 2-adrenoceptor antagonist, ICI 118,551 and the beta 1-adrenoceptor antagonist CGP 20712A in combination or the non-selective beta-adrenoceptor antagonist propranolol alone (all 10(-7) M), induced a rightward shift of the dose-response curves for isoprenaline- and BRL37344-stimulated lipolysis of about 4 and 2 log-units, respectively. However, the antagonists did not alter lipolysis induced by CGP12177. 4. Several concentrations of beta-adrenoceptor antagonists were used to determine the pA2 values by Schild analysis. The values for CGP 20712A and ICI 118,551 (6.63 +/- 0.20 and 6.25 +/- 0.12) as antagonists of the lipolytic effects of CGP 12177 were over 2 units lower than the pA2 value for CGP 20712A against the response to the selective beta 1-agonist dobutamine (8.58 +/- 0.23) and the pA2 value for ICI 118,551 against the response to the selective beta 2-agonist terbutaline (9.15 +/- 0.26). 5. beta 3-Adrenoceptor mRNA expression, investigated with a polymerase chain reaction assay, was demonstrated in both types of adipocytes in the same cell preparations that had a lipolytic response to CGP 12177. 6. In conclusion, human white fat cells express an atypical beta-adrenoceptor in addition to beta 1- and beta 2-adrenoceptors. This receptor is stimulated more selectively by the beta1/beta2-antagonist CGP 12177 than by BRL 37344 and is poorly sensitive to blockade by selective beta1- and beta2-antagonists. On the basis of the pharmacological properties and the mRNA analyses, we suggest that this atypical receptor corresponds to the beta 3-adrenoceptor subtype.

Effects of mental stress on lipolysis in humans.

Lipid mobilization was investigated in subcutaneous adipose tissue specimens obtained before and after a standardized mental-stress test in 14 non-obese healthy subjects. All participants responded with an increased heart rate and elevation of plasma glycerol levels. Plasma norepinephrine concentrations remained unchanged throughout the test. In six subjects, mental stress induced a significant increase in plasma epinephrine levels, to more than 0.26 nmol/L (responders), while the remaining eight individuals showed a response of less than 0.12 nmol/L (nonresponders). In the responders, a 30% increase in catecholamine-stimulated in vitro lipolysis was found after the mental-stress test, while the lipolytic response in isolated fat cells in vitro decreased slightly in the nonresponders after mental stress. A strong correlation (r = .84) was observed between the increased in vitro lipolytic responsiveness due to mental stress and circulating plasma epinephrine levels. In vitro data suggest that the augmentation in lipolytic activity induced by acute mental stress was caused by alterations between the beta-adrenoceptor and adenylate cyclase, ie, probably an increased coupling between beta-receptors and the stimulatory guanosine triphosphate [GTP]-binding protein (G2). This, in combination with elevated levels of circulating epinephrine, may explain the increased lipolysis during mental stress in some individuals (ie, responders). However, other parallel mechanisms for activation of lipolysis during mental stress must also exist in certain individuals (ie, nonresponders), and seem not to involve the adrenergic system.

Elevated resistin levels in chronic kidney disease are associated with decreased glomerular filtration rate and inflammation, but not with insulin resistance.

In the present study, we explore the role of decreased renal function and a genetic polymorphism on the recently discovered protein resistin, apparently able to inhibit hepatic insulin action in mice. We also investigate possible links with inflammation and the insulin resistance present in patients with chronic kidney disease (CKD). This is a post hoc, cross-sectional study comparing 239 prevalent CKD patients with varying degrees of renal function impairment with an age- and gender-matched randomly selected control group of 25 individuals. Glomerular filtration rate (GFR) was estimated by the mean of urea and creatinine clearance (24-h urine samples) (n=204) or by iohexol clearance (n=60). Plasma analysis of blood lipids, insulin, glucose, inflammatory markers (high-sensitivity C-reactive protein, interleukin-6, tumor necrosis factor-alpha, vascular cellular adhesion molecule, intercellular adhesion molecule) and resistin (kit from LINCO Research, St Charles, MS) was performed using commercially available assays or routine methods. Insulin resistance was estimated by quantitative insulin-sensitivity check index (QUICKI) and homeostasis model assessment for insulin resistance (HOMA-IR) and body composition by dual-energy X-ray absorptiometry. Genotyping of a C/G promoter single nucleotide polymorphism (n=168) at position -180 of the resistin gene was performed by PyroSequencing. Serum levels of resistin were markedly elevated in the CKD patients with both advanced (39.9+/-1.3 ng/ml) and mild to moderate (23.2+/-1.0 ng/ml) renal function impairment, as compared to controls (8.5+/-0.7 ng/ml; P<0.001). In a multiple linear regression model in patients (adjusted r(2)=0.60), only GFR (beta=3.4; P<0.0001), lean body mass (beta=2.2; P<0.001) and the inflammatory markers were independently associated with circulating resistin levels. There was a weak but significant impact of -180 C/G genotype on plasma levels of resistin (median 43.0+/-2.4 ng/ml in CC, 37.5+/-2.0 ng/ml in CG, and 41.1+/-4.9 ng/ml in GG; P<0.05). Univariate analysis of non-diabetic patients and controls showed that serum resistin was associated with markers of glucose metabolism. However, in a multiple regression model, resistin, as well as all the measured markers of inflammation, was only associated with insulin resistance if GFR was not taken into account. Circulating resistin levels are strongly associated with both GFR and inflammatory biomarkers in CKD. As the significant relationship between plasma resistin levels and insulin resistance was lost following the correction for GFR, resistin is not a likely mediator of insulin resistance in patients with CKD. Renal function is an important factor to take into account in clinical studies relating insulin sensitivity to inflammatory biomarkers in CKD as well as in patients with diabetes mellitus, who often have an impaired renal function.

Enhanced insulin-stimulated glycogen synthesis in response to insulin, metformin or rosiglitazone is associated with increased mRNA expression of GLUT4 and peroxisomal proliferator activator receptor gamma co-activator 1.

AIMS/HYPOTHESIS: The aim of this study was to determine the effect of several antidiabetic agents on insulin-stimulated glycogen synthesis, as well as on mRNA expression. METHODS: Cultured primary human skeletal myotubes obtained from six healthy subjects were treated for 4 or 8 days without or with glucose (25 mmol/l), insulin (400 pmol/l), rosiglitazone (10 micromol/l), metformin (20 micromol/l) or the AMP-activated kinase activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (200 micromol/l). After this, insulin-stimulated glycogen synthesis was determined. mRNA levels of the glucose transporters GLUT1 and GLUT4, the peroxisomal proliferator activator receptor gamma (PPAR gamma) co-activator 1 (PGC1) and the myocyte-specific enhancer factors (MEF2), MEF2A, MEF2C and MEF2D were determined using real-time PCR analysis after 8 days exposure to the various antidiabetic agents. RESULTS: Insulin-stimulated glycogen synthesis was significantly increased in cultured human myotubes treated with insulin, rosiglitazone or metformin for 8 days, compared with non-treated cells. Furthermore, an 8-day exposure of myotubes to 25 mmol/l glucose impaired insulin-stimulated glycogen synthesis. In contrast, treatment with AICAR was without effect on insulin-mediated glycogen synthesis. Exposure to insulin, rosiglitazone or metformin increased mRNA expression of PGC1 and GLUT4, while AICAR or 25 mmol/l glucose treatment increased GLUT1 mRNA expression. Metformin also increased mRNA expression of the MEF2 isoforms. CONCLUSIONS/INTERPRETATION: Enhanced insulin-stimulated glycogen synthesis in human skeletal muscle cell culture coincides with increased GLUT4 and PGC1 mRNA expression following treatment with various antidiabetic agents. These data show that chronic treatment of human myotubes with insulin, metformin or rosiglitazone has a direct positive effect on insulin action and mRNA expression.

Interactions between adenylate cyclase inhibitors and beta-adrenoceptors in isolated human fat cells.

Interactions between adenylate cyclase inhibitors and beta-adrenoceptors were investigated in isolated human fat cells. Phenylisopropyl adenosine, nicotinic acid and prostaglandine E2 induced a dose-dependent decrease in beta-adrenoceptor sensitivity; the concentration of isoprenaline causing half-maximum lipolytic effect increased 100-fold. The affinity constants for the high and low affinity beta-adrenoceptor states were increased 3000 and 700 times, respectively, but the total number of binding sites was unchanged. Pertussis toxin caused a dose-dependent increase of beta-adrenoceptor sensitivity to isoprenaline. There was a 200-fold increase in isoprenaline sensitivity in the lipolysis experiments and corresponding increases in the receptor affinity in the binding experiments. It is concluded that the affinity of human fat cell beta-adrenoceptors is reduced by adenylate cyclase inhibitors. This seems to be mediated by the Gi-protein and represents a new potential mechanism by which lipolysis is regulated by inhibitors of adenylate cyclase in man.

Antilipolytic effects of insulin and adenylate cyclase inhibitors on isolated human fat cells.

Antilipolysis induced by insulin by adenylate cyclase inhibitors was compared in isolated human fat cells when lipolysis was activated at well-defined steps in the cyclic AMP system. The latter was achieved with isoprenaline (beta-adrenoreceptor agonist), cholera toxin and pertussis toxin (acting on the GTP-sensitive coupling proteins), forskolin (stimulating the catalytic component of adenylate cyclase), enprofylline (selective phosphodiesterase inhibitor) and N6-monobutyryl-cyclic-AMP or 8-bromo cyclic-AMP (cyclic AMP analogues which are resistant or sensitive to phosphodiesterase, respectively). Clonidine (alpha 2-adrenoreceptor agonist), prostaglandin E2 and N6-(phenylisopropyl) adenosine (adenosine analogue) failed to inhibit lipolysis stimulated by cholera toxin or pertussis toxin, but were effective under all other conditions. Insulin failed to inhibit lipolysis stimulated by enprofylline or N6-monobutyryl cyclic AMP, but was effective under all other circumstances. In conclusion, insulin and adenylate cyclase inhibitors are antilipolytic in human fat cells through different mechanisms. Adenylate cyclase inhibitors act predominantly on the GTP-sensitive coupling proteins and, to a minor extent, at some yet unidentified distal step in the lipolytic machinery. As regards insulin, the major site of the antilipolytic action is phosphodiesterase.