Anita M. Hennige

Fetuin-A Induces Cytokine Expression and Suppresses Adiponectin Production

Background The secreted liver protein fetuin-A (AHSG) is up-regulated in hepatic steatosis and the metabolic syndrome. These states are strongly associated with low-grade inflammation and hypoadiponectinemia. We, therefore, hypothesized that fetuin-A may play a role in the regulation of cytokine expression, the modulation of adipose tissue expression and plasma concentration of the insulin-sensitizing and atheroprotective adipokine adiponectin. Methodology and Principal Findings Human monocytic THP1 cells and human in vitro differenttiated adipocytes as well as C57BL/6 mice were treated with fetuin-A. mRNA expression of the genes encoding inflammatory cytokines and the adipokine adiponectin (ADIPOQ) was assessed by real-time RT-PCR. In 122 subjects, plasma levels of fetuin-A, adiponectin and, in a subgroup, the multimeric forms of adiponectin were determined. Fetuin-A treatment induced TNF and IL1B mRNA expression in THP1 cells (p<0.05). Treatment of mice with fetuin-A, analogously, resulted in a marked increase in adipose tissue Tnf mRNA as well as Il6 expression (27- and 174-fold, respectively). These effects were accompanied by a decrease in adipose tissue Adipoq mRNA expression and lower circulating adiponectin levels (p<0.05, both). Furthermore, fetuin-A repressed ADIPOQ mRNA expression of human in vitro differentiated adipocytes (p<0.02) and induced inflammatory cytokine expression. In humans in plasma, fetuin-A correlated positively with high-sensitivity C-reactive protein, a marker of subclinical inflammation (r = 0.26, p = 0.01), and negatively with total- (r = −0.28, p = 0.02) and, particularly, high molecular weight adiponectin (r = −0.36, p = 0.01). Conclusions and Significance We provide novel evidence that the secreted liver protein fetuin-A induces low-grade inflammation and represses adiponectin production in animals and in humans. These data suggest an important role of fatty liver in the pathophysiology of insulin resistance and atherosclerosis.

The cerebrocortical response to hyperinsulinemia is reduced in overweight humans: A magnetoencephalographic study

Animal studies have shown that the brain is an insulin-responsive organ and that central nervous insulin resistance induces obesity and disturbances in glucose metabolism. In humans, insulin effects in the brain are poorly characterized. We used a magnetoencephalography approach during a two-step hyperinsulinemic euglycemic clamp to (i) assess cerebrocortical insulin effects in humans, (ii) compare these effects between 10 lean and 15 obese subjects, and (iii) test whether the insulin receptor substrate (IRS)-1 Gly972Arg polymorphism in the insulin-signaling cascade modifies these effects. Both spontaneous and stimulated (mismatch negativity) cortical activity were assessed. In lean humans, stimulated cortical activity (P = 0.046) and the beta and theta band of spontaneous cortical activity (P = 0.01 and 0.04) increased with insulin infusion relative to saline. In obese humans, these effects were suppressed. Moreover, the insulin effect on spontaneous cortical activity correlated negatively with body mass index and percent body fat (all r < −0.4; all P < 0.05) and positively with insulin sensitivity of glucose disposal (theta band, r = 0.48, P = 0.017). Furthermore, insulin increased spontaneous cortical activity (beta band) in carriers of wild-type IRS-1, whereas, in carriers of the 972Arg allele, this insulin effect was absent (P = 0.01). We conclude that, in lean humans, insulin modulates cerebrocortical activity, and that these effects are diminished in obese individuals. Moreover, cerebrocortical insulin resistance is found in individuals with the Gly972Arg polymorphism in IRS-1, which is considered a type 2 diabetes risk gene.

Direct Cross-talk of Interleukin-6 and Insulin Signal Transduction via Insulin Receptor Substrate-1 in Skeletal Muscle Cells

The exercise-induced interleukin (IL)-6 production and secretion within skeletal muscle fibers has raised the question of a putative tissue-specific function of IL-6 in the energy metabolism of the muscle during and after the exercise. In the present study, we followed the hypothesis that IL-6 signaling may directly interact with insulin receptor substrate (IRS)-1, a keystone in the insulin signaling cascade. We showed that IL-6 induces a rapid recruitment of IRS-1 to the IL-6 receptor complex in cultured skeletal muscle cells. Moreover, IL-6 induced a rapid and transient phosphorylation of Ser-318 of IRS-1 in muscle cells and in muscle tissue, but not in the liver of IL-6-treated mice, probably via the IL-6-induced co-recruitment of protein kinase C-δ. This Ser-318 phosphorylation improved insulin-stimulated Akt phosphorylation and glucose uptake in myotubes since transfection with an IRS-1/Glu-318 mutant simulating a permanent phospho-Ser-318 modification increased Akt phosphorylation and glucose uptake. Noteworthily, two inhibitory mechanisms of IL-6 on insulin action, phosphorylation of the inhibitory Ser-307 residue of IRS-1 and induction of SOCS-3 expression, were only found in liver but not in muscle of IL-6-treated mice. Thus, the data provided evidence for a possible molecular mechanism of the physiological metabolic effects of IL-6 in skeletal muscle, thereby exerting short term beneficial effects on insulin action.

Insulin Modulates Food-Related Activity in the Central Nervous System

CONTEXT Previous data suggest a key role of central nervous insulin action in regulating energy homeostasis. OBJECTIVE We therefore investigated whether insulin modulates brain responses to food and nonfood pictures in a functional magnetic resonance imaging study. DESIGN AND PATIENTS Nine healthy, normal-weight subjects underwent two functional magnetic resonance imaging measurements to compare the effects of insulin and placebo administration during a visual recognition task with food and nonfood pictures. Insulin was administered intranasally to raise insulin concentrations in the cerebrospinal fluid without altering systemic effects in the periphery. Metabolic parameters were continuously determined during the experiments. MAIN OUTCOME MEASURE We measured the changes in brain activity after intranasal insulin administration. RESULTS Food pictures were detected faster when compared to nonfood pictures in all conditions without any effect of placebo or insulin. After insulin application, functional magnetic resonance imaging measurements showed a significantly reduced activity in the presence of food pictures compared to placebo in the right and left fusiform gyrus, the right hippocampus, the right temporal superior cortex, and the right frontal middle cortex. The brain activation induced by nonfood pictures remained unaffected by insulin. CONCLUSION We demonstrate that intranasal insulin led to a reduction of activity in brain areas related to object processing and memory and may have an effect on brain activation with regard to the processing of food pictures. This effect might be part of a mechanism that terminates food intake in the postprandial state.

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.

Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1

Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine‐318 as a protein kinase C (PKC)‐dependent phosphorylation site in Irs1 (Ser‐318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity‐related insulin resistance, promotes phosphorylation of Ser‐318 in Irs1 by a janus kinase 2, Irs2, and PKC‐dependent pathway. Mutation of Ser‐318 to alanine abrogates the inhibitory effect of leptin on insulin‐induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser‐318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet‐induced obesity Ser‐318 phosphorylation levels were already up‐regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser‐318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser‐318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser‐318 in Irs1 mediates the inhibitory signal of leptin on the insulin‐signaling cascade in obese subjects.—Hennige A. M., Stefan N., Kapp K., Lehmann R., Weigert C., Beck A., Moeschel K., Mushack J., Schleicher E., and Häring H. U. Leptin down‐regulates insulin action through phosphorylation of serine‐318 in insulin receptor substrate 1. FASEB J. 20, E381–E389 (2006)

Acute regulation of metabolic genes and insulin receptor substrates in the liver of mice by one single bout of treadmill exercise

Acute exercise performance represents a major metabolic challenge for the skeletal muscle, but also for the liver as the most important source of energy. However the molecular adaptation of the liver to one single bout of exercise is largely unknown. C57BL/6 mice performed a 60 min treadmill run at high aerobic intensity. Liver, soleus and white gastrocnemius muscle were removed immediately after exercise. The single bout of exercise resulted in a very rapid and pronounced induction of hepatic metabolic enzymes and regulators of metabolism or transcription: glucose‐6‐phosphatase (G6Pase; 3‐fold), pyruvate dehydrogenase kinase‐4 (PDK4; 4.8‐fold), angiopoietin‐like 4 (2.1‐fold), insulin receptor substrate (IRS)‐2 (5.1‐fold), peroxisome proliferator activated receptor‐γ coactivator 1α (PGC‐1α; 3‐fold). In soleus and white gastrocnemius muscle the up‐regulation of IRS‐2 and PDK4 was less pronounced compared with the liver and no significant induction of PGC‐1α could be detected at this early time point. Activation of AMPK was found in both liver and white gastrocnemius muscle as phosphorylation of Thr‐172. The induction of endogenous insulin secretion by a glucose load directly after the exercise bout resulted in a significantly higher PKB/Akt phosphorylation in the liver of exercised mice. The markedly enhanced IRS‐2 protein amount, and presumably reduced serine/threonine phosphorylation of the IRS proteins induced by the acute exercise could be responsible for this enhanced action of insulin. In conclusion, acute exercise induced a rapid and pronounced transcriptional adaptation in the liver, and regulated hepatic IRS proteins leading to improved cellular insulin signal transduction.

Insulin Promotes Glycogen Storage and Cell Proliferation in Primary Human Astrocytes

Introduction In the human brain, there are at least as many astrocytes as neurons. Astrocytes are known to modulate neuronal function in several ways. Thus, they may also contribute to cerebral insulin actions. Therefore, we examined whether primary human astrocytes are insulin-responsive and whether their metabolic functions are affected by the hormone. Methods Commercially available Normal Human Astrocytes were grown in the recommended medium. Major players in the insulin signaling pathway were detected by real-time RT-PCR and Western blotting. Phosphorylation events were detected by phospho-specific antibodies. Glucose uptake and glycogen synthesis were assessed using radio-labeled glucose. Glycogen content was assessed by histochemistry. Lactate levels were measured enzymatically. Cell proliferation was assessed by WST-1 assay. Results We detected expression of key proteins for insulin signaling, such as insulin receptor β-subunit, insulin receptor substrat-1, Akt/protein kinase B and glycogen synthase kinase 3, in human astrocytes. Akt was phosphorylated and PI-3 kinase activity increased following insulin stimulation in a dose-dependent manner. Neither increased glucose uptake nor lactate secretion after insulin stimulation could be evidenced in this cell type. However, we found increased insulin-dependent glucose incorporation into glycogen. Furthermore, cell numbers increased dose-dependently upon insulin treatment. Discussion This study demonstrated that human astrocytes are insulin-responsive at the molecular level. We identified glycogen synthesis and cell proliferation as biological responses of insulin signaling in these brain cells. Hence, this cell type may contribute to the effects of insulin in the human brain.

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms β2 and θ

Aims/hypothesis. Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor β-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity.¶Methods. Tyrosine kinase assay and transfection studies.¶Results. In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms β2 and θ. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C.¶Conclusion/interpretation. The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms β2 and θ on insulin receptor autophosphorylation. [Diabetologia (2000) 43: 443–449]

Overexpression of Kinase-Negative Protein Kinase Cδ in Pancreatic β-Cells Protects Mice From Diet-Induced Glucose Intolerance and β-Cell Dysfunction

OBJECTIVE In vitro models suggest that free fatty acid–induced apoptotic β-cell death is mediated through protein kinase C (PKC)δ. To examine the role of PKCδ signaling in vivo, transgenic mice overexpressing a kinase-negative PKCδ (PKCδKN) selectively in β-cells were generated and analyzed for glucose homeostasis and β-cell survival. RESEARCH DESIGN AND METHODS Mice were fed a standard or high-fat diet (HFD). Blood glucose and insulin levels were determined after glucose loads. Islet size, cleaved caspase-3, and PKCδ expression were estimated by immunohistochemistry. In isolated islet cells apoptosis was assessed with TUNEL/TO-PRO3 DNA staining and the mitochondrial potential by rhodamine-123 staining. Changes in phosphorylation and subcellular distribution of forkhead box class O1 (FOXO1) were analyzed by Western blotting and immunohistochemistry. RESULTS PKCδKN mice were protected from HFD-induced glucose intolerance. This was accompanied by increased insulin levels in vivo, by an increased islet size, and by a reduced staining of β-cells for cleaved caspase-3 compared with wild-type littermates. In accordance, long-term treatment with palmitate increased apoptotic cell death of isolated islet cells from wild-type but not from PKCδKN mice. PKCδKN overexpression protected islet cells from palmitate-induced mitochondrial dysfunction and inhibited nuclear accumulation of FOXO1 in mouse islet and INS-1E cells. The inhibition of nuclear accumulation of FOXO1 by PKCδKN was accompanied by an increased phosphorylation of FOXO1 at Ser256 and a significant reduction of FOXO1 protein. CONCLUSIONS Overexpression of PKCδKN in β-cells protects from HFD-induced β-cell failure in vivo by a mechanism that involves inhibition of fatty acid–mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of FOXO1 activation.