Maria K. Svensson

mTOR inhibition with rapamycin causes impaired insulin signalling and glucose uptake in human subcutaneous and omental adipocytes

Rapamycin is an immunosuppressive agent used after organ transplantation, but its molecular effects on glucose metabolism needs further evaluation. We explored rapamycin effects on glucose uptake and insulin signalling proteins in adipocytes obtained via subcutaneous (n=62) and omental (n=10) fat biopsies in human donors. At therapeutic concentration (0.01 μM) rapamycin reduced basal and insulin-stimulated glucose uptake by 20-30%, after short-term (15 min) or long-term (20 h) culture of subcutaneous (n=23 and n=10) and omental adipocytes (n=6 and n=7). Rapamycin reduced PKB Ser473 and AS160 Thr642 phosphorylation, and IRS2 protein levels in subcutaneous adipocytes. Additionally, it reduced mTOR-raptor, mTOR-rictor and mTOR-Sin1 interactions, suggesting decreased mTORC1 and mTORC2 formation. Rapamycin also reduced IR Tyr1146 and IRS1 Ser307/Ser616/Ser636 phosphorylation, whereas no effects were observed on the insulin stimulated IRS1-Tyr and TSC2 Thr1462 phosphorylation. This is the first study to show that rapamycin reduces glucose uptake in human adipocytes through impaired insulin signalling and this may contribute to the development of insulin resistance associated with rapamycin therapy.

Acute hyperinsulinemia raises plasma interleukin-6 in both nondiabetic and type 2 diabetes mellitus subjects, and this effect is inversely associated with body mass index.

Hyperinsulinemia is a characteristic of type 2 diabetes mellitus (T2DM) and is believed to play a role in the low-grade inflammation seen in T2DM. The main aim was to study the effect of hyperinsulinemia on adipokines in individuals with different levels of insulin resistance, glycemia, and obesity. Three groups of sex-matched subjects were studied: young healthy subjects (YS; n = 10; mean age, 26 years; body mass index [BMI], 22 kg/m(2)), patients with T2DM (DS; n = 10; 61 years; BMI, 27 kg/m(2)), and age- and BMI-matched controls to DS (CS; n = 10; 60 years; BMI, 27 kg/m(2)). Plasma concentrations of adipokines were measured during a hyperinsulinemic euglycemic clamp lasting 4 hours. Moreover, insulin-stimulated glucose uptake in isolated adipocytes was analyzed to address adipose tissue insulin sensitivity. Plasma interleukin (IL)-6 increased significantly (P < or = .01) in all 3 groups during hyperinsulinemia. However, the increase was smaller in both DS (P = .06) and CS (P < .05) compared with YS (approximately 2.5-fold vs approximately 4-fold). A significant increase of plasma tumor necrosis factor (TNF) alpha was observed only in YS. There were only minor or inconsistent effects on adiponectin, leptin, and high-sensitivity C-reactive protein levels during hyperinsulinemia. Insulin-induced rise in IL-6 correlated negatively to BMI (P = .001), waist to hip ratio (P = .05), and baseline (fasting) insulin (P = .03) and IL-6 (P = .02) levels and positively to insulin-stimulated glucose uptake in isolated adipocytes (P = .07). There was no association with age or insulin sensitivity. In a multivariate analysis, also including T2DM/no T2DM, an independent correlation (inverse) was found only between BMI and fold change of IL-6 (r(2) = 0.41 for model, P < .005). Hyperinsulinemia per se can produce an increase in plasma IL-6 and TNFalpha, and this can potentially contribute to the low-grade inflammation seen in obesity and T2DM. However, obesity seems to attenuate the ability of an acute increase in insulin to further raise circulating levels of IL-6 and possibly TNFalpha.

The immunosuppressive agents rapamycin, cyclosporin A and tacrolimus increase lipolysis, inhibit lipid storage and alter expression of genes involved in lipid metabolism in human adipose tissue

Cyclosporin A (CsA), tacrolimus and rapamycin are immunosuppressive agents (IAs) associated with insulin resistance and dyslipidemia, although their molecular effects on lipid metabolism in adipose tissue are unknown. We explored IAs effects on lipolysis, lipid storage and expression of genes involved on lipid metabolism in isolated human adipocytes and/or adipose tissue obtained via subcutaneous and omental fat biopsies. CsA, tacrolimus and rapamycin increased isoproterenol-stimulated lipolysis and inhibited lipid storage by 20-35% and enhanced isoproterenol-stimulated hormone-sensitive lipase Ser552 phosphorylation. Rapamycin also increased basal lipolysis (~20%) and impaired insulins antilipolytic effect. Rapamycin, down-regulated the gene expression of perilipin, sterol regulatory element-binding protein 1 (SREBP1) and lipin 1, while tacrolimus down-regulated CD36 and aP2 gene expression. All three IAs increased IL-6 gene expression and secretion, but not expression and secretion of TNF-α or adiponectin. These findings suggest that CsA, tacrolimus and rapamycin enhance lipolysis, inhibit lipid storage and expression of lipogenic genes in adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance associated with immunosuppressive therapy.

The SNARE protein SNAP23 and the SNARE-interacting protein Munc18c in human skeletal muscle are implicated in insulin resistance/type 2 diabetes

OBJECTIVE Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp. Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6. RESULTS We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects. Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23.

FKBP5 expression in human adipose tissue increases following dexamethasone exposure and is associated with insulin resistance

OBJECTIVE To study effects of dexamethasone on gene expression in human adipose tissue aiming to identify potential novel mechanisms for glucocorticoid-induced insulin resistance. MATERIALS/METHODS Subcutaneous and omental adipose tissue, obtained from non-diabetic donors (10 M/15 F; age: 28-60 years; BMI: 20.7-30.6 kg/m²), was incubated with or without dexamethasone (0.003-3 μmol/L) for 24 h. Gene expression was assessed by microarray and real time-PCR and protein expression by immunoblotting. RESULTS FKBP5 (FK506-binding protein 5) and CNR1 (cannabinoid receptor 1) were the most responsive genes to dexamethasone in both subcutaneous and omental adipose tissue (~7-fold). Dexamethasone increased FKBP5 gene and protein expression in a dose-dependent manner in both depots. The gene product, FKBP51 protein, was 10-fold higher in the omental than in the subcutaneous depot, whereas the mRNA levels were similar. Higher FKBP5 gene expression in omental adipose tissue was associated with reduced insulin effects on glucose uptake in both depots. Furthermore, FKBP5 gene expression in subcutaneous adipose tissue was positively correlated with serum insulin, HOMA-IR and subcutaneous adipocyte diameter and negatively with plasma HDL-cholesterol. FKBP5 SNPs were found to be associated with type 2 diabetes and diabetes-related phenotypes in large population-based samples. CONCLUSIONS Dexamethasone exposure promotes expression of FKBP5 in adipose tissue, a gene that may be implicated in glucocorticoid-induced insulin resistance.

Albuminuria and renal function as predictors of cardiovascular events and mortality in a general population of patients with type 2 diabetes: a nationwide observational study from the Swedish National Diabetes Register.

Objective: Reduced renal function and albuminuria predict cardiovascular (CV) events and mortality in type 2 diabetes (T2D). In addition, we evaluated the role of co-existing congestive heart failure (CHF) and other CV risk factors on CV events in a large observational population-based cohort of T2D patients. Research design and methods: We included 66,065 patients with T2D who were reported to the National Diabetes Register (NDR) in Sweden between 2003–2006 with a follow-up of 5.7 years. Data on outcomes were collected from the cause of death and hospital discharge registers. Results: A total of 10% of patients experienced a CV event and 3.7% of these were fatal. Increasing levels of albuminuria and renal impairment were independently associated with increasing risk of CV events and all-cause mortality also when adjusting for CHF. In normoalbuminuric patients, a reduction in renal function is an important predictor of CV events and all-cause mortality. Glycaemic control (high HbA1c), smoking and hyperlipidaemia had important effects on risk for CV events in patients with albuminuria, while high blood pressure, but not glycaemic control, had an effect in patients with normoalbuminuric renal impairment. Conclusion: Albuminuria and renal impairment are independent risk factors for CV outcomes and mortality in T2D, albuminuria being the strongest risk factor and relevant at all levels of renal function. In normoalbuminuric patients, a reduction in renal function is an important predictor of CV events and all-cause mortality.

Characterization of brown adipose tissue in the human perirenal depot.

To characterize brown adipose tissue (BAT) in the human perirenal adipose tissue depot.

Pharmacokinetic-Pharmacodynamic Assessment of the Interrelationships Between Tesaglitazar Exposure and Renal Function in Patients With Type 2 Diabetes Mellitus

The effects of tesaglitazar on renal function (assessed as urinary clearance of 125I‐sodium iothalamate or estimated by the modification of diet in renal disease formula) were studied in a 24‐week open‐label trial in type 2 diabetes mellitus patients randomized to daily doses of either tesaglitazar 2 mg or pioglitazone 45 mg. The aim of the analysis was to develop a population pharmacokinetic‐pharmacodynamic model that could simultaneously describe the interrelationship between tesaglitazar exposure and reduction in renal function over time in patients with type 2 diabetes mellitus. The pharmacokinetic‐pharmacodynamic model could adequately describe the interplay between tesaglitazar and glomerular filtration rate. A one‐compartment model in which the apparent clearance was influenced by glomerular filtration rate characterized the pharmacokinetics of tesaglitazar. An indirect‐response model was used for the slow time course of change in glomerular filtration rate, which decreased from 100 to 78 mL/min/1.73m2 after 12 weeks of treatment. All tesaglitazar‐treated patients had a reduction in glomerular filtration rate, and available demographic variables could not explain differences in response. Patients treated with an angiotensin converting enzyme inhibitor were more sensitive to tesaglitazar and had larger glomerular filtration rate decrease compared to nontreated patients. Approximately 8 weeks after discontinuing treatment, mean glomerular filtration rate had returned towards baseline. The model and data give valuable insights into the dynamic changes in glomerular filtration rate over time.

Differences between men and women in the regulation of adipose 11β‐HSD1 and in its association with adiposity and insulin resistance

This study explored sex differences in 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) activity and gene expression in isolated adipocytes and adipose tissue (AT), obtained via subcutaneous biopsies from non‐diabetic subjects [58 M, 64 F; age 48.3 ± 15.3 years, body mass index (BMI) 27.2 ± 3.9 kg/m2]. Relationships with adiposity and insulin resistance (IR) were addressed. Males exhibited higher 11β‐HSD1 activity in adipocytes than females, but there was no such difference for AT. In both men and women, adipocyte 11β‐HSD1 activity correlated positively with BMI, waist circumference, % body fat, adipocyte size and with serum glucose, triglycerides and low‐density lipoprotein:high‐density lipoprotein (LDL:HDL) ratio. Positive correlations with insulin, HOMA‐IR and haemoglobin A1c (HbA1c) and a negative correlation with HDL‐cholesterol were significant only in males. Conversely, 11β‐HSD1 activity in AT correlated with several markers of IR and adiposity in females but not in males, but the opposite pattern was found with respect to 11β‐HSD1 mRNA expression. This study suggests that there are sex differences in 11β‐HSD1 regulation and in its associations with markers of obesity and IR.

Selection of uremic patients for kidney transplantation

Selection of patients for kidney transplantations is necessary due to the shortage of organs. The process has not been greatly studied. Twelve hypothetical cases were constructed, each with one or several relative contraindications, such as cardiovascular disease, diabetes, old age or a mental disorder. The cases were submitted to 40 nephrologists, chosen to represent the recruitment areas of the four Swedish transplant units. They were asked to declare whether the ‘patient’ was suitable for transplantation or not, and, independently, whether the patient would be referred to the transplant unit. The same cases were evaluated by 3–4 representatives of each transplant unit. The response rate was 100%. A median of 6 cases was considered suitable (range 3–11). The acceptance rate differed significantly between the four unit areas, from 4 cases (3–7) to 7 (4–11), p=0.014. Nephrologists would accept fewer patients than staff from the transplant units, 5 (3–10) vs. 7 (3–11), p=0.009. Most of the latter difference was compensated for by referral of borderline cases to the unit. Only 5 individual cases were equally judged by at least 75% of the respondents. Discrepancies in view were noted with respect to the significance of old or young age, the patients determination and severe obesity.