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Induced pluripotent stem cells (iPSCs) can be used to generate different somatic cell types in vitro, including insulin-producing pancreatic β-cells. Here, we have generated iPSCs from a healthy male individual using an episomal reprogramming method. The resulting iPSCs are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Furthermore, we show that this iPSC line can be differentiated into pancreatic lineage cells. Taken together, this iPSC line will be useful to test differentiation protocols towards β-cell as well as other cell types and will also serve as a control for drug development and disease modelling studies., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Epidemiological studies suggest that an increased red meat intake is associated with a higher risk of type 2 diabetes, whereas an increased fiber intake is associated with a lower risk., Objectives: We conducted an intervention study to investigate the effects of these nutritional factors on glucose and lipid metabolism, body-fat distribution, and liver fat content in subjects at increased risk of type 2 diabetes., Methods: This prospective, randomized, and controlled dietary intervention study was performed over 6 mo. All groups decreased their daily caloric intake by 400 kcal. The "control" group (N = 40) only had this requirement. The "no red meat" group (N = 48) in addition aimed to avoid the intake of red meat, and the "fiber" group (N = 44) increased intake of fibers to 40 g/d. Anthropometric parameters and frequently sampled oral glucose tolerance tests were performed before and after intervention. Body-fat mass and distribution, liver fat, and liver iron content were assessed by MRI and single voxel proton magnetic resonance spectroscopy., Results: Participants in all groups lost weight (mean 3.3 ± 0.5 kg, P < 0.0001). Glucose tolerance and insulin sensitivity improved (P < 0.001), and body and visceral fat mass decreased in all groups (P < 0.001). These changes did not differ between groups. Liver fat content decreased significantly (P < 0.001) with no differences between the groups. The decrease in liver fat correlated with the decrease in ferritin during intervention (r2 = 0.08, P = 0.0021). This association was confirmed in an independent lifestyle intervention study (Tuebingen Lifestyle Intervention Program, N = 229, P = 0.0084)., Conclusions: Our data indicate that caloric restriction leads to a marked improvement in glucose metabolism and body-fat composition, including liver-fat content. The marked reduction in liver fat might be mediated via changes in ferritin levels. In the context of caloric restriction, there seems to be no additional beneficial impact of reduced red meat intake and increased fiber intake on the improvement in cardiometabolic risk parameters. This trial was registered at clinicaltrials.gov as NCT03231839.

In analytical chemistry serum as well as plasma are recommended as sample material of choice. However, blood processing for the generation of serum or plasma is rather different. Whether plasma or serum is the preferable sample material is still controversial discussed. We performed in paired samples three UHPLC-mass spectrometry-driven metabolomics studies. In study 1 metabolite profiles of serum vs plasma were compared. 46% out of 216 identified metabolites showed significant different levels (paired Wilcoxon signed-rank test, p < 0.05, FDR <0.01) with only three metabolites (methionine, C2:0- and C3:0-carnitine) showing lower levels in serum. In study 2 comparison of three different serum blood collection tubes revealed that coagulation and associated processes distinctly alter metabolite levels depending on the tube-specific clotting process. Most pronounced differences were found for the dipeptide phenylalanine-phenylalanine (highest levels in silicate containing serum blood collection tubes). In study 3 possible adverse effects of platelets, which still remain in standard plasma even after correct processing, were investigated. No differences in a pattern of 216 metabolites were detected in the comparison of standard and platelet-free plasma (PFP). Our results give novel insights in fundamental differences between serum and plasma, thereby providing valuable information for analytical chemists for decision making to either use serum or plasma before starting complex and time-consuming analytical investigations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Volume retention in nephrotic syndrome has been linked to activation of the epithelial sodium channel (ENaC) by proteolysis of its γ-subunit following urinary excretion of serine proteases such as plasmin. Here we tested whether pharmacological inhibition of urinary serine protease activity might protect from ENaC activation and volume retention in nephrotic syndrome. Urine from both nephrotic mice (induced by doxorubicin injection) and nephrotic patients exhibited high aprotinin-sensitive serine protease activity. Treatment of nephrotic mice with the serine protease inhibitor aprotinin by means of subcutaneous sustained-release pellets normalized urinary serine protease activity and prevented sodium retention, as did treatment with the ENaC inhibitor amiloride. In the kidney cortex from nephrotic mice, immunofluorescence revealed increased apical γ-ENaC staining, normalized by aprotinin treatment. In Xenopus laevis oocytes heterologously expressing murine ENaC, aprotinin had no direct inhibitory effect on channel activity but prevented proteolytic channel activation. Thus, our study shows that volume retention in experimental nephrotic syndrome is related to proteolytic ENaC activation by proteasuria and can be prevented by treatment with aprotinin. Hence, inhibition of urinary serine protease activity might become a therapeutic approach to treat patients with nephrotic-range proteinuria., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4) and Type 2 Diabetes Mellitus (T2DM). To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a C18R heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 C18R iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms., (Copyright © 2016 Helmholtz Zentrum München GmbH. Published by Elsevier B.V. All rights reserved.)

Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4) and Type 2 Diabetes Mellitus (T2DM). To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a P33T heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 P33T iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms., (Copyright © 2016 Helmholtz Zentrum München. Published by Elsevier B.V. All rights reserved.)

Structural brain imaging studies have shown that obesity is associated with widespread reductions in gray matter (GM) volume. Although the body mass index (BMI) is an easily accessible anthropometric measure, substantial health problems are more related to specific body fat compartments, like visceral adipose tissue (VAT). We investigated cortical thickness measures in a group of 72 healthy subjects (BMI range 20-35 kg/m(2), age range 19-50 years). Multiple regression analyses were performed using VAT and BMI as predictors and age, gender, total surface area and education as confounds. BMI and VAT were independently associated with reductions in cortical thickness in clusters comprising the left lateral occipital area, the left inferior temporal cortex, and the left precentral and inferior parietal area, while the right insula, the left fusiform gyrus and the right inferior temporal area showed a negative correlation with VAT only. In addition, we could show significant reductions in cortical thickness with increasing VAT adjusted for BMI in the left temporal cortex. We were able to detect widespread cortical thinning in a young to middle-aged population related to BMI and VAT; these findings show close resemblance to studies focusing on GM volume differences in diabetic patients. This may point to the influence of VAT related adverse effects, like low-grade inflammation, as a potentially harmful factor on brain integrity already in individuals at risk of developing diabetes, metabolic syndromes and arteriosclerosis.

In systems biology, the combination of multiple types of omics data, such as metabolomics, proteomics, transcriptomics, and genomics, yields more information on a biological process than the analysis of a single type of data. Thus, data from different omics platforms is usually combined in one experimental setup to obtain insight into a biological process or a disease state. Particularly high accuracy metabolomics data from modern mass spectrometry instruments is currently more and more integrated into biological studies. Reflecting this trend, we extended InCroMAP, a data integration, analysis and visualization tool for genomics, transcriptomics, and proteomics data. Now, the tool is able to perform an integrated enrichment analysis and pathway-based visualization of multi-omics data and thus, it is suitable for the evaluation of comprehensive systems biology studies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A common challenge for scientists working with animal tissue or human biopsy samples is the limitation of material and consequently, the difficulty to perform comprehensive metabolic profiling within one experiment. Here, we present a novel approach to simultaneously perform targeted and non-targeted metabolomics as well as lipidomics from one small piece of liver or muscle tissue by ultra-high performance liquid chromatography/mass spectrometry (UHPLC/MS) following a methyl tert-butyl ether (MTBE)-based extraction. Equal relative amounts of the resulting polar and non-polar fractions were pooled, evaporated and reconstituted in the appropriate solvent for UHPLC/MS analysis. This mix was comparable or superior in yield and reproducibility to a standard 80% methanol extraction for the profiling of polar and lipophilic metabolites (free carnitine, acylcarnitines and FFA). The mix was also suitable for non-targeted metabolomics, an easy measure to increase the metabolite coverage by 30% relative to using the polar fraction alone. Lipidomics was performed from an aliquot of the non-polar fraction. This novel strategy could successfully be applied to one mouse soleus muscle with a dry weight of merely 2.5 mg. By enabling a simultaneous profiling of lipids and metabolites with mixed polarity while saving material for molecular, biochemical or histological analyses, our approach may open up new perspectives toward a comprehensive investigation of small, valuable tissue samples., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hashimoto's thyroiditis (HT) can casually co-occur with an encephalopathy associated with autoimmune thyroid disease. Recently we found an increased occurrence of weaknesses in sustained attention and response inhibition in a subgroup of euthyroid patients with HT as obtained by the d2 attention test. Previous studies in healthy subjects and patients with brain lesions demonstrated a pivotal role for the left inferior frontal gyrus (LIFG) in these skills. Therefore, we studied the association between the performance in the d2 test and grey matter (GM) density of the LIFG in 13 euthyroid patients with HT compared to a control group of 12 euthyroid patients with other thyroid diseases. A significant correlation between GM density and d2 test total score was detected for the opercular part of the LIFG in patients with HT (p<0.001), but not in the control group (p=0.94). Regression in patients with HT was significantly stronger than in the control group (p=0.02). Moreover, GM density was significantly reduced when comparing HT patients with control patients that scored in the lower third during d2 attention testing (p<0.05). It can be concluded that in HT performance in the d2 test correlated with GM density of the LIFG. Particularly low achievement was associated with reduced GM density of this brain region suggesting an influence of autoimmune processes on the frontal cortex in this disease. This could be due to not yet known antibodies affecting brain morphology or an influence of thyroid antibodies themselves., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In a non-hypothesis driven metabolomics approach plasma samples collected at six different time points (before, during and after an exercise bout) were analyzed by gas chromatography-time of flight mass spectrometry (GC-TOF MS). Since independent component analysis (ICA) does not need a priori information on the investigated process and moreover can separate statistically independent source signals with non-Gaussian distribution, we aimed to elucidate the analytical power of ICA for the metabolic pattern analysis and the identification of key metabolites in this exercise study. A novel approach based on descriptive statistics was established to optimize ICA model. In the GC-TOF MS data set the number of principal components after whitening and the number of independent components of ICA were optimized and systematically selected by descriptive statistics. The elucidated dominating independent components were involved in fuel metabolism, representing one of the most affected metabolic changes occurring in exercising humans. Conclusive time dependent physiological changes of the metabolic pattern under exercise conditions were detected. We conclude that after optimization ICA can successfully elucidate key metabolite pattern as well as characteristic metabolites in metabolic processes thereby simplifying the explanation of complex biological processes. Moreover, ICA is capable to study time series in complex experiments with multi-levels and multi-factors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: Sensitization to human leukocyte antigen (HLA) prolongs waiting list time and reduces allograft survival in solid organ transplantation. Current strategies for pretransplant desensitization are based on B-cell depletion and extracorporeal treatment. The proteasome inhibitor bortezomib allows direct targeting of the antibody-producing plasma cell and has been used in antibody-mediated rejection (AMR) and recipient desensitization with varying results. Here, we report the effect of bortezomib preconditioning on HLA antibody titers and specificity in highly sensitized patients awaiting renal allograft transplantation., Patients and Methods: Two highly sensitized patients awaiting third kidney transplantation were given one cycle of bortezomib (1.3 mg/m², days 1, 4, 8, 11), as part of recipient desensitization. Time-course and levels of anti-HLA antibodies, as well as specificity to previous transplant antigens were monitored by luminex technology. In addition, measles and tetanus toxoid immunoglobulin G (IgG) was measured., Results: Following bortezomib, overall changes in IgG levels were small and no sustained reduction in anti-HLA class I or II antibody levels was observed over more than 100 days of follow-up to both, donor specific and non-donor specific antigens. Moreover, anti-measles and -tetanus toxoid IgG levels remained unchanged., Conclusions: Bortezomib preconditioning alone does not result in sustained reduction of HLA antibody levels or alter protective immunity in sensitized patients. This supports the notion, that bortezomib requires activation of plasma cells, as in AMR, to effectively reduce HLA antibody production. Hence, in a pretransplant setting, combination strategies may be required to derive benefit from proteasome inhibition., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The hypothalamus is the central homeostatic control region of the brain and, therefore, highly influenced by nutrients such as glucose and fat. Immediate and prolonged homeostatic effects of glucose ingestion have been well characterized. However, studies that used stimulation with fat have mainly investigated immediate perceptional processes. Besides homeostatic processes, the gustatory cortex, including parts of the insular cortex, is crucial for the processing of food items., Objective: The aim of this study was to investigate the effect of high- compared with low-fat meals on the hypothalamus and the insular cortex., Design: Eleven healthy men participated in a single-blinded, functional MRI study of high- and low-fat meals on 2 measurement days. Cerebral blood flow (CBF) was measured before and 30 and 120 min after intake of high- and low-fat yogurts. Hunger was rated and blood samples were taken before each CBF measurement., Results: High-fat yogurt induced a pronounced decrease in CBF in the hypothalamus, and the corresponding CBF change correlated positively with the insulin change. Furthermore, insular activity increased after 120 min in the low-fat condition only. The CBF change in both regions correlated positively in the high-fat condition., Conclusions: The decrease in hypothalamic activity and the interaction with the insular cortex elicited by fat may contribute to an efficient energy homeostasis. Therefore, fat might be a modulator of homeostatic and gustatory brain regions and their interaction. This trial was registered at clinicaltrials.gov as NCT01516021.

Previous studies suggest impairments of physical, mental, and psychic well-being in patients with Hashimoto's thyroiditis (HT), but these impairments have been shown to be independent of thyroid dysfunction. In 64 euthyroid patients with HT, symptomatic distress was assessed with the Symptom Checklist-90-Revised (SCL-90-R), a 90-item multidimensional self-report symptom inventory using a 5-point rating scale. In a subgroup of patients, endocrine testing 3 years prior to the current investigation was available. Anti-thyroid peroxidase antibodies (TPO-Abs) were associated with the three SCL-90-R global indices Global Severity Index (GSI), Positive Symptom Distress Index (PSDI), and Positive Symptom Total (PST) as well as with somatization and obsessive-compulsive symptoms after adjustment for age, gender, and thyroid function as assessed by TSH levels (all p<0.05). HT patients positive for TPO-Abs showed poorer results in the three SCL-90-R global indices as well as in the three domains: somatization, obsessive-compulsive symptoms, and depression (all p≤0.02), though the aforementioned associations did not withstand sequential Bonferroni correction for multiple testing. In contrast, TPO-Abs positivity, defined as TPO-Abs >100 IU/l, significantly predicted poorer psychosocial well-being in all of the three SCL-90-R global indices after three years, even after correction (all p≤0.02). In conclusion, high TPO-Abs are associated with poor physical and psychological well-being and appear to predict future health perception in HT patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: In obesity, particularly increased visceral- (VAT), but not total (TAT) adipose tissue mass is a major source of proinflammatory cytokine expression and secretion. VAT, more than TAT, is associated with endothelial dysfunction (ED), which is an accepted risk factor for atherosclerosis. Consequently, we hypothesized that during a lifestyle intervention specifically a decrease in VAT, rather than TAT, is associated with improved ED and vascular adhesion molecules in type 2 diabetes prone subjects., Methods: Analyses were done in 189 individuals (age: 45.4±0.8 years) at increased risk of type 2 diabetes, who underwent a 9-month lifestyle intervention. ED expressed as flow mediated dilation (FMD) of the brachial artery, sE-selectin, sV-CAM, sI-CAM, TAT and VAT (measured by magnetic resonance tomography) was determined., Results: There was a mean decrease in body weight (-3%, p<0.0001), TAT (-7.6%, p<0.0001) and VAT (-12.5%, p<0.0001), while FMD increased (+9.1%, p=0.04). The change in FMD was not associated with change in body weight (p=0.35) or TAT (p=0.21) but with a decrease in VAT (r=-0.19, p=0.009). In a post hoc analysis, the subjects were divided by the median change in VAT into responders and non-responders. FMD increased only in the responders (from 6.2±0.4% to 8.0±0.5%, p=0.0005) but not in the non-responders (p=0.15). Also sE-selectin significantly decreased only in the responders (from 54±4 ng/ml to 47±3 ng/ml; p=0.03)., Conclusion: During a lifestyle intervention, not weight loss or decrease in TAT, but decrease in VAT is associated with improved ED in individuals prone to type 2 diabetes. Therefore, primary cardiovascular prevention should focus specifically on reducing VAT rather than body weight alone., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: High urine volume enhances urinary free cortisol (UFF) and cortisone (UFE) excretion rates in normal-weight adults and children. Renal excretion rates of glucocorticoids (GC) and their metabolites are frequently altered in obesity. The aim of the present study was to investigate whether UFF and UFE excretion is also affected by urine volume in severely obese subjects., Experimental: In 24-h urine samples of 59 extremely obese subjects (mean BMI 45.3+/-8.9 kg/m(2)) and 20 healthy lean subjects (BMI 22.1+/-1.8 kg/m(2)), UFF and UFE, tetrahydrocortisol (THF), 5alpha-tetrahydrocortisol (5alpha-THF), and tetrahydrocortisone (THE) were quantified by RIA. The sum of THF, 5alpha-THF, and THE (GC3), the three major GC metabolites, reflects daily cortisol secretion. 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity was assessed by the ratio UFE/UFF. Daily GC excretion rates were corrected for urine creatinine and adjusted for gender and body weight., Results: In extremely obese subjects, urine volume was significantly associated with creatinine-corrected UFE and 11beta-HSD2 activity after adjustment for gender and BMI (r=0.47, p=0.0002 and r=0.31, p=0.02, respectively). However, urine volume was not associated with creatinine-corrected UFF and GC3 (p=0.4 and p=0.6, respectively). In lean controls, urine volume was significantly associated with creatinine-corrected UFE and UFF (r=0.58, p=0.01 and r=0.55, p=0.02, respectively), whereas urine volume was not associated with 11beta-HSD2 activity after appropriate adjustment (p=0.3)., Conclusions: In severe obesity, in contrast to normal weight, renal excretion of UFE, but not of UFF is affected by fluid intake. This discrepancy may be due to the increased renal 11beta-HSD2 activity in obesity.

Monocyte-chemoattractant-protein (MCP)-1 and its receptor CCR2 have been shown to play a pivotal role in vascular inflammation and atherosclerotic plaque formation. However, it is currently unclear whether MCP-1/CCR2 triggered inflammation affects nitric oxide (NO)-bioavailability, hence influencing vascular function, a sign of early atherosclerosis. Therefore, we sought to investigate the association between serum levels of MCP-1 and NO-bioavailability, expressed as flow mediated dilation (FMD) in vivo, and the impact of CCR2 gene variations on FMD. We studied a German population of 242 prediabetic individuals (144 women, 98 men; mean age 45+/-0.8 years) via FMD by high-resolution ultrasound (13MHz). In order to replicate our findings, a second, independent population (n=115; 44 women, 77 men; mean age 48+/-1.0 years) (total=357 individuals) from Italy was studied. Vascular function in the Italian population was studied via intra-arterial application of acetylcholine. MCP-1 serum-levels were assessed by ELISA and CCR2 polymorphisms were determined by sequencing. MCP-1 serum levels showed no association with FMD (p=0.90), whereas the CCR2 promoter polymorphism was associated with elevated FMD (T/T: 5.6+/-0.3%; T/A: 6.7+/-0.4%; A/A: 8.3+/-0.8%; p=0.01) after adjusting for possible confounders. These results were confirmed in the independent Italian population (A/A: 97.1+/-20.3 vs. T/T: 60.5+/-5.6% forearm blood-flow increase; p<0.05). When testing for the functional relevance of the T-960A (rs3918359) polymorphism, we found that the A/A-genotype was associated with moderately increased protein binding in EMSA, increased promoter activity in luciferase assays and reduced transendothelial monocyte migration. In conclusion, MCP-1 serum levels do not reflect endothelial function in vivo in prediabetic individuals. However, the functionally relevant CCR2 promoter polymorphism T-960A (rs3918359) is associated with elevated vascular function. This might be due to reduced subendothelial inflammation, mediated by reduced transendothelial monocyte-migration ability.

Glucocorticoids blunt insulin release, an effect partially due to activation of Kv channels. Similar to those channels Na+/K+ ATPase activity repolarizes the plasma membrane. The present study explored whether glucocorticoids increase the Na+/K+ ATPase activity in pancreatic beta-cells. The glucocorticoid dexamethasone (100 nmol/l for 1 day) significantly increased Na+/K+ ATPase alpha1/beta1-subunit transcript levels and ouabain-sensitive outward current reflecting Na+/K+ ATPase activity in INS-1 cells, effects blunted by glucocorticoid-receptor-blocker RU487 (1 micromol/l). Dexamethasone (100 nmol/l) increased K+ current in beta-cells from wild type mice but not from knockout mice lacking functional serum and glucocorticoid inducible kinase SGK1. Thus, glucocorticoids indeed up-regulate Na+/K+ ATPase activity, an effect requiring SGK1.

Chronic microinflammation increases cardiovascular morbidity in chronic hemodialysis (HD) patients. Previously published studies are controversial with respect to acute effects of HD treatment on up- or downregulation of cytokine protein levels. Twenty-nine chronic HD patients were hemodialysed for 4 h with a 4008 dialyser using high-flux membranes. Patients were separated into a low (up to 1 mg/dl) and a high (1.1 to 5.5 mg/dl) C-reactive protein (CRP) group. Blood was drawn before HD and 240 min after initiation of HD. Acute changes of transcript levels encoding pro- and anti-inflammatory mediators were analyzed in RNA stabilized immediately from blood leukocytes using microarray analysis (n=1) and quantitative real-time polymerase chain reaction (PCR) (Light Cycler) (n=29). In both patient groups, HD treatment significantly increased the transcript levels of several pro-inflammatory cytokines, such as tumor necrosis factor alpha and interleukin-8 (IL-8), and chemokine receptors such as C-X-C chemokine receptor type 4, C-C chemokine receptor type 7, and the fractakine receptor CX3C chemokine receptor 1. In the low CRP group, the increase of transcript levels for anti-inflammatory IL-1-receptor antagonist and of the receptor for the anti-inflammatory cytokines IL-10 and interferon gamma was significantly more pronounced than in the high CRP group. Subgroup analysis revealed no difference between diabetic vs non-diabetic patients. These observations point towards a marked influence of a routine hemodialysis treatment on transcription in leukocytes of pro- and anti-inflammatory cytokines and receptors relevant for microinflammation. Diminished upregulation of receptors for anti-inflammatory factors in HD patients with high CRP levels could contribute to enhanced microinflammation in those patients.

Increased serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is associated with cellular insulin resistance. We have recently identified serine 318 (Ser318) as a novel protein kinase C-zeta (PKC-zeta)-dependent phosphorylation site within IRS-1. As other kinases may phosphorylate at this serine residue as well, we aimed to identify such kinases in the present study. In C2C12 myotubes, exposure to insulin or phorbol ester markedly increased Ser318 phosphorylation. In contrast, high glucose, tumor necrosis factor-alpha, and free fatty acids did not provoke Ser318 phosphorylation. JNK and the PI 3-kinase/mTOR pathway were found to be implicated in insulin-induced Ser318 phosphorylation, but not in TPA-stimulated phosphorylation that was, at least partly, mediated by classical or novel PKC. In conclusion, with JNK and the PI 3-kinase/mTOR pathway as mediators of insulin-induced Ser318 phosphorylation, we have identified kinases that have previously been reported to play key roles in phosphorylation of other serine residues in IRS-1.

Reduced bio-availability of nitric oxide leading to disturbed flow mediated (endothelial dependent) vasodilation (FMD) has been shown to be an early functional abnormality of the vascular system in insulin resistant individuals and other subjects at high risk for accelerated atherosclerosis. In addition, an increase of the intima-media thickness (IMT) is regarded as an early marker of morphological alterations of the vessel wall. Whether endothelial dysfunction (ED) is evident already at an early stage when morphological changes of the vessel wall are not apparent is still an open question. We, therefore, examined IMT and peripheral endothelial function in a group of young insulin resistant subjects in a cross-sectional study and compared these results with a metabolically healthy (insulin sensitive) control group. We measured IMT (distal common carotid arteries), endothelium-dependent and endothelium-independent vasodilation (flow mediated and glyceroltrinitrate induced vasodilation of the brachial artery) non-invasively with high resolution ultrasound (13 MHz) in 91 young normoglycemic subjects (40/51 M/F, median: 31 years, range 18-50 years). Insulin sensitivity was measured with a euglycemic, hyperinsulinemic glucose clamp. Despite a marked reduction in flow-mediated vasodilation in insulin resistant (IR) subjects (FMD: median 3.4%, range -4.0 to 12.5 in IR versus 6.6%, range -1.2 to 20.1% in insulin sensitive subjects; P = 0.017), there was no difference in endothelial independent vasodilation (16.3%, range 5.7-41.0% versus 16.1%, range 0.5-39.2%) and in IMT (0.50 mm, range 0.39-0.66 and 0.51, 0.40-0.70 mm, respectively). These data suggest that ED can be detected very early in the life of insulin resistant subjects whereas no significant structural changes, indicated by a thickening of the intima-media layer, could be found. We therefore conclude that for identification of subjects with a high risk for accelerated atherosclerosis at an early stage, measurement of flow mediated vasodilation of the brachial artery may be more helpful than measuring thickness of the vascular wall., (Copyright 2003 Elsevier Ireland Ltd.)

Increasing evidence indicates a role of insulin signalling for insulin secretion from the pancreatic beta-cells. Therefore, regulators of insulin signalling, like protein tyrosine phosphatases, could also have an impact on insulin secretion. Here, we investigated a possible role of the negative regulator protein tyrosine phosphatase alpha (PTP alpha) for insulin secretion. RT-PCR analysis confirmed that both splice variants of the extracellular domain of PTP alpha that vary by an insert of 9 amino acids are expressed in human islets and insulinoma cells (INS-1E, RIN1046-38). Overexpression of the wild type PTP alpha splice variant containing the 9 amino acids reduced insulin secretion, as did a mutant form unable to bind Grb2 (Tyr798Phe). By contrast, overexpression of a phosphatase inactive mutant improved insulin secretion. These data reveal a functional relevance of PTP alpha for insulin secretion.

The peroxisome proliferator-activated receptor gamma activating compounds thiazolidinedione (TZD) have been shown to inhibit diabetes-induced glomerular transforming growth factor-beta1 (TGF-beta1) expression, thereby ameliorating diabetic nephropathy. Here we examined the hypothesis that TZDs block high glucose-induced TGF-beta1 gene activation by interaction with the activated protein kinase C-c-Fos-TGF-beta1 promoter cascade in mesangial cells. The TZD compounds troglitazone and rosiglitazone completely prevented the high glucose induction of both TGF-beta1 promoter activity and elevation in nuclear c-Fos protein levels. The scavenging properties of troglitazone were shown not to be responsible for this inhibitory action, because hydrogen peroxide-mediated stimulation of TGF-beta1 promoter activity was not blocked. TZD-treatment did not interfere with the transcriptional activity of c-Fos responsible for stimulation of the TGF-beta1 promoter. The findings suggest a molecular mechanism by which TZD-treatment reduces specifically high glucose-induced, c-Fos-mediated gene activation, since phorbol ester-induced c-Fos mRNA and protein expression and subsequent elevation of TGF-beta1 mRNA expression were not prevented by TZDs.

It is believed that free fatty acids contribute to the pathogenesis of type 2 diabetes in humans. We have recently shown that lipoapoptosis of human beta-cells is specifically induced by saturated fatty acids while unsaturated had no effect. In the present study we tested the effect of co-incubation of different saturated and unsaturated free fatty acids on lipoapoptosis in beta-cells. RIN1046-38 cells and isolated human beta-cells were incubated with combinations of saturated fatty acids (palmitate, stearate) and mono- or polyunsaturated fatty acids (palmitoleate, oleate, and linoleate). Cells were incubated for 24-72 h with 1mM fatty acids. All unsaturated fatty acids tested completely prevented palmitate- or stearate-induced apoptosis of rat and human beta-cells as assessed by flow cytometric cell cycle analysis and TUNEL assay. This might suggest that apoptosis in vivo is predominantly determined by the content of unsaturated fatty acids in a mixed fatty acid pool.

The adipogenic transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a pivotal role in the regulation of whole body insulin sensitivity. Little, however, is known about hormonal and molecular modulation of PPARgamma gene expression. Therefore, we investigated the temporal and conditional expression of PPARgamma in a serum-free model of 3T3-L1 adipocyte differentiation. We show here that expression of PPARgamma depends on the full set of known adipogenic stimuli and not on a single hormone/agent. Unexpectedly, an indeed marked but only transient peak of PPARgamma expression (39+/-5-fold increase over basal on day 3 after hormonal stimulation) occurs during serum-free adipose conversion. To our knowledge, this finding is novel and probably remained hidden until now because of the common use of serum-containing preadipocyte culture systems. We conclude that maintenance, but not induction, of PPARgamma gene expression in vitro must be achieved by one or more still unknown serum component(s).

Protein kinase C seems to be linked to the regulation of insulin secretion as well as mitogenic signaling in pancreatic beta cells. To study the impact of different PKC isoforms on insulin secretion and mitogenic activity we stably overexpressed the PKC isoforms alpha, beta2, epsilon, and zeta in the rat clonal beta cell line RIN 1046-38. Under basal conditions PKC alpha, beta2, epsilon, and zeta were identified mainly in the cytosol. Treatment with the phorbol ester TPA caused translocation of PKC alpha, beta2, and epsilon to the plasma membrane. Glucose- and TPA-dependent increases in insulin release were comparable in all cell lines regardless of whether PKC was overexpressed or not. While PKC isoforms alpha, beta2, and epsilon had no effect on the [(3)H]thymidine incorporation rate, overexpression of PKC zeta specifically increased basal as well as IGF-1-dependent [(3)H]thymidine incorporation. Incubation with the MAP-kinase inhibitor PD98056 abolished this effect. Furthermore, treatment with IGF-1 led to activation of the beta cell-specific transcription factor PDX-1 in RIN 1046-38 cells overexpressing PKC zeta. Our data suggest that PKC zeta is involved in basal as well as IGF-1-dependent mitogenesis in RIN 1046-38 cells, while none of the PKC isoforms tested seem to be related to glucose-stimulated insulin release., ((c)2002 Elsevier Science.)

Background: Hyperglycemia-induced overexpression of prosclerotic transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of diabetic nephropathy. Since previous in vivo studies demonstrated a renoprotective effect of low-molecular-weight (LMW) heparin in experimental animals, and recent in vitro data showed an interaction of this drug with the overactivated TGF-beta 1 cascade in high glucose- and phorbol ester-stimulated mesangial cells, we studied the molecular mechanism of these effects on TGF-beta 1 gene expression., Methods: Mesangial cells were stimulated with 30 mmol/L glucose or with 0.5 micromol/L phorbol ester [phorbol myristate acetate (PMA)] in the absence or presence of LMW heparin. TGF-beta 1 promoter activity was determined in promoter-reporter luciferase assays. The effect of LMW heparin on the binding of nuclear proteins to a regulatory activator protein-1 (AP-1) site, which mediates the high glucose and PMA responsiveness of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays., Results: The presence of LMW heparin completely prevented TGF-beta 1 gene activation in both high glucose- and PMA-stimulated cells. Preincubation of the cells with LMW heparin and subsequent stimulation of the cells with both stimuli yielded the same result. Furthermore, treatment with LMW heparin prevented the enhanced binding of nuclear proteins to the regulatory AP-1 site, while binding to a Sp1 site was unaffected. Basal promoter activity and basal AP-1 binding also was reduced by LMW heparin. The LMW heparin effect on basal promoter activity was abolished by mutation of the regulatory AP-1 box B and by deletion of this AP-1 binding site., Conclusions: LMW heparin prevents high glucose- and PMA-mediated TGF-beta 1 expression by inhibiting the activation of the TGF-beta 1 promoter and by preventing the enhanced binding of nuclear proteins to the regulatory AP-1 site.

A gas chromatographic-mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC-MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1,000 microg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1 +/- 13.5 microg/l for 2-ethylhexanoic acid, 104.8 +/- 80.6 microg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2 +/- 389.5 microg/l for 2-ethyl-3-oxohexanoic acid.

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., (Copyright 2001 Academic Press.)

Urinary modified nucleosides were determined by capillary electrophoresis using a 300 mM SDS-25 mM sodium tetraborate-50 mM sodium dihydrogenphosphate buffer. The nucleosides were extracted from urine by phenylboronate affinity gel chromatography. In cancer patients the levels of the modified nucleosides are generally elevated. By an artificial neural network method breast cancer patients were differentiated from normal individuals, which indicates that the modified nucleosides could be of clinical value as tumor markers.

The combination of a new thermodesorption module with a cooled injection system now provides a powerful system for direct analysis of volatile trace compounds in gaseous, liquid and solid samples by gas chromatography-mass spectrometry (GC-MS). As a cooled injection system is used for the cryofocusing of the desorbed volatiles the GC-MC system still can be used for the regular analysis of liquid samples. Although plasticizers usually are analyzed by GC-MS after solvent extraction, contaminated solvents and glassware are very well known problems. Analysis of plasticizers in plastic materials by direct thermodesorption instead saves time and avoids cross contaminations. Many medical products are made of plasticized polyvinyl chloride. Extraction of the common plasticizer di(2-ethylhexyl) phthalate (DEHP) into blood will occur, and harmful effects of DEHP in the human body have been suggested. We therefore analyzed 21 different plastic devices which are used for various invasive techniques in medicine by direct thermodesorption GC-MS. In some of the plastics up to 30 different components were identified. By far the most common plasticizer found was DEHP, followed by diethyl and dibutyl phthalates.

Acute myocardial infarction is associated with profound alterations in the plasma lipoprotein profile. The mechanism of these alterations is not clear, and both cholesterol biosynthesis up- and downregulation could possibly be a consequence of acute myocardial infarction. We determined plasma lipids, lipoproteins, apolipoproteins, and lathosterol-which is regarded as an estimate of whole body cholesterol biosynthesis in humans-concentrations in 34 patients (age 68+/-10 years, 24 male, 10 female) admitted to our hospital with acute MI and with onset of symptoms within the last 12 h. Samples were taken immediately after admission to the hospital, and 1, 2, and 10 days after admission. On the first day after admission there was a decrease in total cholesterol (C) by 14.1%, (P = 0.01), in LDL-C by 14.4% (P = 0.03), in HDL-C by 9.3% (NS), and in triglycerides by 19.5% (NS). Apolipoprotein B100 was reduced by 18.3% (P = 0.008), and apolipoprotein AI by 12.3% (NS). The lathosterol/cholesterol ratio was increased by 23.1% after 1 day, and by 28.7% after 2 days (P = 0.05). After 10 days, all variables except the apolipoproteins had essentially returned to baseline values. In conclusion, the changes in the plasma lipid profile after acute myocardial infarction are associated with a profound increase of whole body cholesterol biosynthesis as judged by the lathosterol/cholesterol ratio. These changes may possibly enhance the delivery of cholesterol to cells involved in tissue repair mechanisms after acute myocardial infarction.

The insertion(I)/deletion(D) polymorphism of the angiotensin-converting-enzyme (ACE) gene has been associated with an increased risk of myocardial infarction, lacunar stroke, and with an increased intimal-medial thickness in several populations. The aim of this study was to evaluate whether the ACE I/D genotype is associated with stenosis of extracranial arteries and stroke in middle-aged and aged men and women. We studied 388 patients (247 male, 141 female) using Doppler and Duplex ultrasound of the extracranial arteries. Patients' history was obtained by standard questionnaire and by the hospital case records. Genomic DNA was analyzed by polymerase chain reaction (PCR) to identify the I/D polymorphism, with a second insertion specific PCR in samples classified as homozygous DD genotypes to prevent mistyping. The ACE genotype groups (DD 132, ID 164, II 92) were well matched for the basic characteristics. The DD genotype was more common in patients with extracranial artery stenosis > or = 50%, compared with patients without stenosis (59/147 versus 73/241, odds ratio 1.54, 95%-CI 1.01-2.37), but was not associated with a history of stroke (30/91 versus 102/297, odds ratio 0.94, 95%-CI 0.57-1.54). The association of the DD genotype with extracranial artery stenosis was also present in hypertensive subjects (n = 206, odds ratio 1.76, 95%-CI 0.99-3.17). In the whole group multiple logistic regression analysis revealed that the association of the DD genotype with extracranial artery stenosis was independent of age, gender, hypertension, hyperlipidemia, and diabetes. In conclusion, the ACE DD genotype is a weak risk factor for hemodynamically relevant stenosis of extracranial arteries, but not for stroke.

The results reported in this communication demonstrate that capillary zone electrophoresis (CZE) can be used easily for the quantitative determination of the potential anticancer drugs berberine and isoguanosine in the extract of the traditional Chinese medicinal herb. Isoguanosine and berberine can be monitored selectively and sensitively at 254 nm within 14 min in the plant extract using a 100-mM sodium citrate running buffer (adjusted to pH 2.7; applied voltage 12 kV). The concentration range of 0.1-50 micrograms ml-1 proved to be sufficient for exact quantification and the peak profile showed good reproducibility [relative standard deviations (n = 32); for the migration time 0.22% (isoguanosine) and 1.32% (berberine); for the peak area 2.8% (isoguanosine) and 3.2% (berberine)]. The measured concentrations in the crude extract were 1.3 micrograms ml-1 for isoguanosine and 8.7 micrograms ml-1 for berberine. In addition to the better separation performance, CZE shows several other remarkable advantages over high-performance liquid chromatography (HPLC), such as rapidity of analysis, small sample volume, no requirement of organic solvent in the running buffer and low cost of reagents.

Insulin binding to its receptor activates a cascade of signaling events which are initiated by tyrosine autophosphorylation of the receptor and activation of the tyrosine kinase activity towards the insulin receptor substrates. In addition to phosphorylation at tyrosine residues a serine phosphorylation of the insulin receptor is observed. Neither the functional significance of serine phosphorylation of the receptor nor the location of relevant regulatory sites has been determined exactly so far. We studied potential functions of serine residues in human insulin receptor (HIR) with respect to its ability to undergo insulin stimulated autophosphorylation. Using site directed mutagenesis of HIR we exchanged serine to alanine at 13 different positions in the HIR beta-subunit. Sites were chosen according to the criteria of known serine phosphorylation sites (1023/25, 1293/94, 1308/09), conserved positions in hIR, hIGF-1 receptor, hIRR, and dIR (962, 994, 1037, 1055, 1074/78, 1168, 1177/78/82, 1202, 1263, 1267). All HIR mutants were expressed in HEK 293 cells and basal and insulin stimulated autophosphorylation were determined. We found that the exchange of serine to alanine at position 994 and at position 1023/25 increased insulin stimulated receptor autophosphorylation significantly (147% +/- 12% and 129% +/- 6% of control, p < 0.01, n = 7), while all other exchanges did not significantly alter insulin stimulated HIR autophosphorylation. The data suggest that the serine residues at position 994 as well as 1023/25 might be part of inhibitory domains of the insulin receptor.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder. It is characterized by a decreased affinity of low density lipoprotein (LDL) for the LDL receptor, as a consequence of a substitution of adenine by guanine in exon 26 of the apolipoprotein B-100 gene, coding for the putative LDL receptor-binding domain of the mature protein. This disorder is associated with a strikingly high incidence of arteriosclerosis and tends to cause disease and premature death. In this communication we describe a rapid capillary gel electrophoretic method in combination with molecular biology techniques to facilitate the diagnosis of FDB. Mutation screening for FDB is performed by an allele-specific amplification followed by capillary gel electrophoresis (CGE). For the combined polymerase chain reaction (PCR)-CGE method, a total analysis time of only 3 h is needed, a period that is normally necessary for the run and for staining of the gel only, not including the time for PCR, gel casting, etc. In our pilot study 4 of 43 hypercholesterolemic patients were found to have the predominant apoB 3500 codon mutation. The verification is demonstrated by DNA-sequencing. This pilot study will be followed by a large cohort analysis of the south-west German population to determine the frequency of FDB in this area. The PCR-CGE method on the Dionex capillary electrophoresis system (CES I) allows rapid, fully automated detection of the mutation resulting in the unequivocal diagnosis of FDB.

The human insulin receptor exists in two isoforms, HIR-A and HIR-B. We studied whether both insulin receptor isotypes are able to mediate an insulin signal to phospholipase C. Plasma membranes were prepared from rat-1 fibroblasts transfected either with HIR-A or HIR-B and insulin stimulated PIP-hydrolysis was determined. We found that insulin stimulates PIP-hydrolysis in a similar dose dependent manner and to a similar extent in plasma membranes expressing HIR-A and HIR-B. These data suggest that both receptor isoforms are equally able to activate phospholipase-C.

Human insulin receptor (HIR) is expressed in two isoforms which differ in the C-terminal end of the alpha-subunit (HIR-A = -12 aa, HIR-B = +12 aa). We studied internalization kinetics of HIR-A and HIR-B in Rat1 fibroblasts. Internalized receptors were quantified by 125I-insulin binding after cell trypsinisation and solubilization, surface receptors were determined by 125I-insulin binding to intact cells and by chemical crosslinking with B26-125I-insulin. HIR-A and HIR-B show different kinetics of receptor internalization. While in HIR-A cells the maximum of internalization (approx. 65% of total) is reached after 10 min followed by a high recycling rate (approx. 80% of internalized receptors after 20 min), the internalization in HIR-B cells reaches a maximum (approx. 60% of total) after 15 min without detectable recycling within 30 min. The data show that the different alpha-subunits of both receptor types determine different velocities of internalization and determine whether a fast recycling occurs.

Activation of a phosphatidylinositol-3-kinase (PI-3-kinase) is one of the earliest consequences of insulin binding to the receptor. The human insulin receptor exists in two isoforms which differ in the length of the alpha-subunit (HIR-A = 719 aa, HIR-B = 731 aa). To test whether both isoforms transduce an insulin signal on PI-3-kinase we used rat-1-fibroblasts expressing HIR-A or HIR-B. We found that insulin stimulates 32P incorporation into PIP through both HIR-A and HIR-B to a similar extent (approx. 8-10 fold).

Proteinkinase-C (PKC) stimulating phorbolesters induce in vitro insulin resistance of isolated adipocytes. This effect might be explained by an inhibition of insulin signal transduction at the level of the insulin receptor kinase. There is now some evidence that a phospholipase C is a potential candidate as a signal transducer at the postreceptor level. In order to determine whether phorbol esters might inhibit insulin signalling also at the level of a phospholipase C, we studied the insulin dependent [3H] phosphatidylinositol 4-monophosphate (PIP) hydrolysis of fat cell membranes. PIP hydrolysis was measured after in vitro stimulation with and without insulin. Insulin stimulated PIP hydrolysis in a dose dependent way. When plasma membranes from phorbolester (TPA) treated fat cells were used, this insulin stimulated phospholipase C activity was suppressed, provided, membranes have been prepared in a buffer containing serine phosphatase inhibitors. These data suggest that fat cell membranes contain an insulin dependent phospholipase C which is inhibited by TPA most likely via serine phosphorylation through proteinkinase C.

Insulin activates the glucose transport in isolated fat cells through a translocation of the insulin sensitive glucose carrier subtype (GLUT4) and by activation of glucose carriers in the plasma membrane. Protein kinase C stimulating phorbol esters are able to mimick partially the insulin effect on glucose transport. In order to determine whether this phorbol ester effect occurs through a translocation of the insulin sensitive glucose carrier (GLUT4) we used a monoclonal antibody against GLUT4 to determine its distribution in subcellular fractions of rat adipocytes. We found that the phorbol ester TPA is able to increase the amount of GLUT4 in the plasma membrane fraction about two-fold.

The effect of cAMP on insulin binding and insulin stimulation of glucose transport was investigated in isolated rat adipocytes. Preincubation for 30 min in medium containing 16 mmol/l glucose and either db-cAMP or bromo-cAMP in concentrations of 10(-4)-10(-3) M inhibited high affinity binding of insulin by 15 to 30% and glucose transport by 30 to 50%. Preincubation with IBMX (10(-4)-10(-3) M) reduced insulin binding by 25% and glucose transport by 70%. Closer analysis of these data indicated that preincubation with these compounds caused not only a decrease in insulin binding but also a post-receptor resistance. High intracellular cyclic AMP-levels seem therefore to induce insulin resistance at both receptor and post-receptor levels.

The C-kinase activating phorbolester TPA (12-O-Tetradecanoyl-beta-phorbol-13-acetate) and PdBu (4-beta-Phorbol-12,13,dibutyrate) stimulated D-glucose transport twofold in isolated rat adipocytes but inhibited high affinity insulin binding and the responsiveness of D-glucose transport to insulin stimulation by about 30%. Phorbolesters have therefore insulin-like effects but antagonize insulin on receptor and postreceptor level.