Hu Häring

Leptin stimulates glucose transport and glycogen synthesis in C2C12 myotubes : evidence for a PI3-kinase mediated effect

Summary It was recently shown that leptin impairs insulin signalling, i. e. insulin receptor autophosphorylation and insulin-receptor substrate (IRS)-1 phosphorylation in rat-1 fibroblasts, NIH3T3 cells and HepG2 cells. To evaluate whether leptin might impair the effects of insulin in muscle tissue we studied the interaction of insulin and leptin in a muscle cell system, i. e. C2C12 myotubes. Preincubation of C2C12 cells with leptin (1–500 ng/ml) did not significantly affect insulin stimulated glucose transport and glycogen synthesis (1.8 to 2 fold stimulation); however, leptin by itself (1 ng/ml) was able to mimic approximately 80–90 % of the insulin effect on glucose transport and glycogen synthesis. Both glucose transport as well as glycogen synthesis were inhibited by the phosphatidylinositol-3 (PI3)-kinase inhibitor wortmannin and the protein kinase C inhibitor H7 while no effect was observed with the S6-kinase inhibitor rapamycin. We determined whether the effect of leptin occurs through activation of IRS-1 and PI3-kinase. Leptin did not stimulate PI3-kinase activity in IRS-1 immunoprecipitates; however, PI3-kinase activation could be demonstrated in p85α immunoprecipitates (3.04 ± 1.5 fold of basal). In summary the data provide the first evidence for a positive crosstalk between the signalling chain of the insulin receptor and the leptin receptor. Leptin mimics in C2C12 myotubes insulin effects on glucose transport and glycogen synthesis most likely through activation of PI3-kinase. This effect of leptin occurs independently of IRS-1 activation in C2C12 cells. [Diabetologia (1997) 40: 606–609]

Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways

SummaryWe have recently shown that leptin mimicks insulin effects on glucose transport and glycogen synthesis through a phosphatidylinositol-3 (PI) kinase dependent pathway in C2C12 myotubes. The aim of the present study was to identify the signalling path from the leptin receptor to the PI-3 kinase. We stimulated C2C12 myotubes with insulin (100 nmol/1, 5 min) or leptin (0.62 nmol/1,10 min) and determined PI-3 kinase activity in immunoprecipitates with specific non-crossreacting antibodies against insulinreceptor substrate (IRS 1/IRS 2) and against janus kinase (JAK 1 and JAK 2). While insulin-stimulated PI-3 kinase activity is detected in IRS-1 and IRS-2 immunoprecipitates, leptin-stimulated PI-3 kinase activity is found only in IRS-2 immunoprecipitates, suggesting that the leptin signal to PI-3 kinase occurs via IRS-2 and not IRS-1. Leptin-, but not insulin-stimulated PI-3 kinase activity is also detected in immunoprecipitates with antibodies against JAK-2, but not JAK-1. The data suggest that JAK-2 and IRS-2 couple the leptin signalling pathway to the insulin signalling chain. Since we have also detected leptin-stimulated tyrosine phosphorylation of JAK-2 and IRS-2 in C2C12 myotubes it can be assumed that leptin activates JAK-2 which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase. As we could not detect the long leptin receptor isoform in C2C12 myotubes we conclude that this signalling pathway is activated by a short leptin receptor isoform.

Variation in the FTO gene influences food intake but not energy expenditure.

Polymorphisms in the FTO (fat mass- and obesity-associated) gene are associated with obesity. The mechanisms how genetic variation in this gene influences body weight are unknown. Body weight is determined by energy intake/storage and energy expenditure. In this study, we investigated whether genetic variation in FTO influences energy expenditure or food intake in carefully phenotyped subjects. In 380 German subjects, insulin sensitivity was measured by a hyperinsulinemic euglycemic clamp. Lean body mass and body fat were quantified using the bioimpedance method. Indirect calorimetry was used to estimate the metabolic rate. Food intake was assessed using food diaries (mean 11+/-1 d) in 151 subjects participating in a lifestyle intervention program to prevent diabetes. All subjects were genotyped for the FTO single nucleotide polymorphism (SNP) rs8050136. The risk allele of SNP rs8050136 was associated with higher body fat-related parameters (all p< or =0.04, additive inheritance model). Energy expenditure was not affected by the SNP. However, the risk allele of rs8050136 was significantly associated with higher energy intake (p=0.01, dominant inheritance model) during dietary restriction. Our data suggest that the increased body weight in carriers of the risk allele of FTO SNP rs8050136 is a consequence of increased food intake, but not of impaired energy expenditure.

The PPARγ2 amino acid polymorphism Pro 12 Ala is prevalent in offspring of Type II diabetic patients and is associated to increased insulin sensitivity in a subgroup of obese subjects

Aims/hypothesis. Recently a mutation in the coding sequence of the adipocyte specific isoform peroxisome proliferator-activated receptor γ2 (PPARγ2) was described, leading to the substitution of Proline to Alanine at codon 12. Mutations in PPARγ2 could have a role in people who are at increased risk for the development of obesity and Type II (non-insulin-dependent) diabetes mellitus. Methods. Non-diabetic first-degree relatives (n = 108) of subjects with Type II diabetes were characterized by oral glucose tolerance tests and euglycaemic hyperinsulinaemic glucose clamp to determine insulin sensitivity. Results. We found 75 (69 %) probands without the PPARγ ProAla12 substitution, 28 heterozygotes (26 %) and 5 (4 %) homozygotes. When the whole group was analysed for an association between the mutation and insulin sensitivity, no statistical significance could be shown. Only in the group with severe obesity more than 30 kg/m2, an association (p = 0.016) of the polymorphism with an increase in insulin sensitivity was found. Conclusion/interpretation. These observations suggest that the mutation in the PPARγ2 molecule may have a role in subgroups prone to the development of obesity and Type II diabetes. [Diabetologia (1999) 42: 758–762]

Protein kinase C isoforms α, δ and θ require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells)

Summary Protein kinase C (PKC) isoforms are potentially important as modulators of the insulin signalling chain and could be involved in the pathogenesis of cellular insulin resistance. We have previously shown that phorbol ester stimulated PKC β1 and β2 as well as tumor necrosis factor-α (TNFα) stimulated PKC ɛ inhibit human insulin receptor (HIR) signalling. There is increasing evidence that the insulin receptor substrate-1 (IRS-1) is involved in inhibitory signals in insulin receptor function. The aim of the present study was to elucidate the role of IRS-1 in the inhibitory effects of protein kinase C on human insulin receptor function. HIR, PKC isoforms (α, β1, β2, γ, δ, ɛ, η, θ and ζ) and IRS-1 were coexpressed in human embryonic kidney (HEK) 293 cells. PKCs were activated by preincubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (CTPA) (10––7 mol/l) following insulin stimulation. While PKCs α, δ and θ were not inhibitory in HEK 293 cells which were transfected only with HIR and PKC, additional transfection of IRS-1 induced a strong inhibitory effect of these PKC isoforms being maximal for PKC θ (99 ± 1.8 % inhibition of insulin stimulated receptor autophosphorylation, n = 7, p < 0.001). No effect was seen with PKC γ, ɛ, ζ and η while the earlier observed insulin receptor kinase inhibition of PKC β2 was further augmented (91 ± 13 %, n = 7, p < 0.001 instead of 45 % without IRS-1). The strong inhibitory effect of PKC θ is accompanied by a molecular weight shift of IRS-1 (183 kDa vs 180 kDa) in the sodium dodecyl sulphate polyacrylamide gel. This can be reversed by alkaline phosphatase treatment of IRS-1 suggesting that this molecular weight shift is due to an increased phosphorylation of IRS-1 on serine or threonine residues. In summary, these data show that IRS-1 is involved in the inhibitory effect of the PKC isoforms α, β2, δ and θ and it is likely that this involves serine/threonine phosphorylation of IRS-1. [Diabetologia (1998) 41: 833–838]

Lifestyle intervention in individuals with normal versus impaired glucose tolerance

Background  Lifestyle intervention is effective in the prevention of type 2 diabetes in individuals with impaired glucose tolerance (IGT). It is currently unknown whether it has beneficial effects on metabolism to a similar extent, in individuals with normal glucose tolerance (NGT) compared to individuals with IGT.

Protein kinase C isoforms β 1 and β 2 inhibit the tyrosine kinase activity of the insulin receptor

Summary Downregulation of insulin receptor tyrosine kinase (IRK) activity yields to impaired insulin signalling and contributes to the pathogenesis of cellular insulin resistance. Activation of protein kinase C (PKC) by different agents is associated with an inhibition of IRK activity in various cell types. There is evidence that this effect on IRK activity might be mediated through phosphorylation of specific serine residues of the insulin receptor β -subunit. Neither the domains of the IRK where inhibiting serine phosphorylation occurs nor the PKC isoform responsible for IRK inhibition have been identified. PKC consists of a family of at least 12 isoforms. The aim of the present study was to determine which PKC isoform might be capable of IRK inhibition. The human insulin receptor and the PKC isoforms α, β 1, β 2, γ,δ,ɛ,η,θ and ζ were overexpressed in human embryo kidney fibroblasts (HEK 293 cells) in order to answer this question. PKCs were activated by preincubation with the phorbolester (TPA) (10−7 mol/l) following insulin stimulation of the cells. When the IRK was coexpressed with the PKC isoforms β 1 and β 2, a 50 ± 15.7 and 45 ± 10.1 % inhibition of tyrosine autophosphorylation of IRK was observed while coexpression with the other isoforms did not significantly modify IRK autophosphorylation. The data suggest that the PKC isoforms β 1 and β 2 might be candidates for insulin receptor inhibition. [Diabetologia (1997) 40: 863–866]

Age and gender related effects on adipose tissue compartments of subjects with increased risk for type 2 diabetes: a whole body MRI / MRS study

Quantitative measurement of adipose tissue (AT) compartments in the entire body and of lipids in muscle and liver cells by means of MRI and MRS. Assessment of ageand gender related differences in AT compartments and determination of cross-correlations between AT compartments in a heterogeneous cohort at increased risk for metabolic diseases. One hundred and fifty healthy volunteers with increased risk to type 2 diabetes were examined. T1-weighted MRI was applied for whole-body adipose tissue quantification. Adipose tissue compartments were subdivided in lower extremities, trunk (abdominal subcutaneous (SCAT) and visceral (VAT) adipose tissue), and upper extremities. Intrahepatocellular lipids (IHCL) and intramyocellular lipids (IMCL) in tibialis anterior and soleus muscle were determined by volume selective MRS. Females are characterized by lower %VAT (2.8±1.3% vs. 4.6±1.4% ,p<0.001) and higher %SCAT (14.7±3.9% vs. 9.3±2.9% ,p<0.001). There is a strong correlation between %VAT and age (r=0.64/0.60 for females/males), whereas %SCAT remained virtually unchanged in males (r=-0.09) and was only slightly increaseding in females (r =0.30,p<0.01). For IHCL, age related differences were observed in females with significantly increased IHCL in the older women, but not in males. IMCL contents in both muscles were found almost independent of age in both, males and females. Furthermore, VAT and IHCL show significant correlations in both groups. Assessed age and gender related differences, especially the age related significant increase of VAT and IHCL, as well as cross-correlations between different lipid compartments might contribute to a better understanding of the lipid metabolism under normal and pathologic metabolic conditions in humans.

Tissue selectivity of insulin detemir action in vivo

Aims/hypothesisRecombinant DNA technology is a useful tool that can be used to create insulin analogues with modified absorption kinetics to improve glycaemic control in patients with type 1 and type 2 diabetes. Among conventional insulin analogues, which are usually created by amino acid exchange, insulin detemir is the first analogue to be acylated with a fatty acid to enable reversible albumin binding. In this study we determined activation of the insulin receptor (IR)-signalling cascade by insulin detemir at the level of IR and IR substrate (Irs) phosphorylation, as well as downstream signalling elements such as phosphatidylinositol 3-kinase and Akt, and performed epidural EEG in vivo.MethodsC57Bl/6 mice were injected i.v. with either insulin detemir or human insulin and Western blot analysis was performed on liver, muscle, hypothalamic and cerebrocortical tissues. Moreover, cerebrocortical activity was detected by EEG in awake mice and cerebral insulin concentrations were measured following human insulin and insulin detemir injection.ResultsThe time course and extent of IR phosphorylation in peripheral tissues were similar following insulin detemir treatment compared with human insulin, but insulin signalling in hypothalamic and cerebrocortical tissue determined by tyrosine-phosphorylation of the IR and Irs2 proteins occurred faster and was enhanced due to a higher insulin detemir concentration in the brain. Moreover, epidural EEG in mice displayed increased cortical activity using insulin detemir.Conclusions/interpretationTaken together, these data suggest that insulin detemir has a tissue-selective action, with a relative preference for brain compared with peripheral tissues.

Variation in the FTO gene locus is associated with cerebrocortical insulin resistance in humans

regulation. Based on this, we investigated the relationship between the FTO variant rs8050136 and BMI using data obtained from the Tubingen Family (TUF) Study [5]. BMI was higher in carriers of the risk allele than in wild-type individuals (AA [n=463] 27.2±0.3 kg/m 2 ,A C [n=732] 29.0±0.3kg/m 2 ,C C [n=267] 29.5±0.5 kg/m 2 ,m eans±SEM, p<0.001) as a result of increased body weight (AA 79.8± 0.9 kg, AC 84.7±1.0 kg, CC 84.8±1.5 kg, p<0.001). This finding replicates the previously reported weight difference in our population [1–4]. However, the mechanism by which FTO polymorphisms affect body weight in humans is still unclear. In mice, FTO is expressed in multiple tissues, including the brain. It is located on a region on chromosome 8 that is deleted by the Fused toes mutation [6]. The fused toes mutation causes a complex phenotype that features partial syndactyly of forelimbs and defects in brain morpho