Publications by authors named "Anita M Hennige"

45 Publications

Challenges in tackling energy expenditure as obesity therapy - from preclinical models to clinical application.

Mol Metab 2021 Apr 17:101237. Epub 2021 Apr 17.

CardioMetabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany. Electronic address:

Background: A chronic imbalance of energy intake and energy expenditure results in excess fat storage. The obesity often caused by this overweight is detrimental to the health of millions of people. Understanding both sides of the energy balance equation and their counter-regulatory mechanisms is critical to the development of effective therapies to treat this epidemic.

Scope Of The Review: Behaviors surrounding ingestion have been reviewed extensively. This review focuses more specifically on energy expenditure regarding bodyweight control, with a particular emphasis on the organs and attractive metabolic processes known to reduce bodyweight. Additionally, previous and current attempts at anti-obesity strategies focusing on energy expenditure are highlighted. Precise measurements of energy expenditure, which consist of cellular, animal, and human models, as well as measurements of their translatability, are required to provide the most effective therapies.

Major Conclusions: A precise understanding of the components surrounding energy expenditure, including tailored approaches based on genetic, biomarker, or physical characteristics, must be integrated into future anti-obesity treatments. Further comprehensive investigations are required to define suitable treatments, especially since the complex nature of the human perspective remains poorly understood.
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http://dx.doi.org/10.1016/j.molmet.2021.101237DOI Listing
April 2021

The Gly385(388)Arg Polymorphism of the FGFR4 Receptor Regulates Hepatic Lipogenesis Under Healthy Diet.

J Clin Endocrinol Metab 2019 06;104(6):2041-2053

Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry, University of Tübingen, Tübingen, Germany.

Context: The effect of a lifestyle intervention to reduce liver fat content in nonalcoholic fatty liver disease in humans is influenced by genetics. We hypothesized that the amino acid exchange in human Gly388Arg (mouse homolog: Gly385Arg) in fibroblast growth factor receptor 4 (FGFR4), which regulates bile acid, lipid, and glucose metabolism, could determine hepatic lipid accumulation and insulin sensitivity. Mechanisms of this substitution were studied in mice under normal chow and high-fat diets.

Design: In humans, the Gly388Arg polymorphism was studied for its relationship with changes in liver fat content and insulin sensitivity during 9 months of a lifestyle intervention. We also studied a knock-in mouse strain with an Arg385 allele introduced into the murine FGFR4 gene under normal chow and high-fat diets.

Results: In humans, the FGFR4 Arg388 allele was not associated with liver fat content or insulin sensitivity in subjects who were overweight and obese before lifestyle intervention. However, it was associated with less decrease in liver fat content and less increase in insulin sensitivity during the intervention. In mice receiving normal chow, the FGFR4 Arg385 allele was associated with elevated hepatic triglyceride content, altered hepatic lipid composition, and increased hepatic expression of genes inducing de novo lipogenesis and glycolysis. Body fat mass and distribution, glucose tolerance, and insulin sensitivity were unaltered. The FGFR4 Arg385 allele had no effect on glucose or lipid metabolism under the high-fat diet.

Conclusion: Our data indicate that the FGFR4 Arg388(385) allele affects hepatic lipid and glucose metabolism specifically during healthy caloric intake.
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http://dx.doi.org/10.1210/jc.2018-01573DOI Listing
June 2019

Maternal whole blood cell miRNA-340 is elevated in gestational diabetes and inversely regulated by glucose and insulin.

Sci Rep 2018 01 22;8(1):1366. Epub 2018 Jan 22.

Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Zentrum München at the Eberhard Karls University Tübingen, Tübingen, Germany.

The number of pregnancies complicated by gestational diabetes (GDM) is increasing worldwide. To identify novel characteristics of GDM, we studied miRNA profiles of maternal and fetal whole blood cells (WBCs) from GDM and normal glucose tolerant (NGT) pregnant women matched for body mass index and maternal age. After adjustment for maternal weight gain and pregnancy week, we identified 29 mature micro-RNAs (miRNAs) up-regulated in GDM, one of which, i.e., miRNA-340, was validated by qPCR. mRNA and protein expression of PAIP1, a miRNA-340 target gene, was found down-regulated in GDM women, accordingly. In lymphocytes derived from the mothers' blood and treated in vitro, insulin increased and glucose reduced miRNA-340 expression. In fetal cord blood samples, no associations of miRNA-340 with maternal GDM were observed. Our results provide evidence for insulin-induced epigenetic, i.e., miRNA-dependent, programming of maternal WBCs in GDM.
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http://dx.doi.org/10.1038/s41598-018-19200-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778051PMC
January 2018

Sustained Treatment with Insulin Detemir in Mice Alters Brain Activity and Locomotion.

PLoS One 2016 2;11(9):e0162124. Epub 2016 Sep 2.

Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry, University of Tuebingen, Tuebingen, Germany.

Aims: Recent studies have identified unique brain effects of insulin detemir (Levemir®). Due to its pharmacologic properties, insulin detemir may reach higher concentrations in the brain than regular insulin. This might explain the observed increased brain stimulation after acute insulin detemir application but it remained unclear whether chronic insulin detemir treatment causes alterations in brain activity as a consequence of overstimulation.

Methods: In mice, we examined insulin detemir's prolonged brain exposure by continuous subcutaneous (s.c.) application using either micro-osmotic pumps or daily s.c. injections and performed continuous radiotelemetric electrocorticography and locomotion recordings.

Results: Acute intracerebroventricular injection of insulin detemir activated cortical and locomotor activity significantly more than regular insulin in equimolar doses (0.94 and 5.63 mU in total), suggesting an enhanced acute impact on brain networks. However, given continuously s.c., insulin detemir significantly reduced cortical activity (theta: 21.3±6.1% vs. 73.0±8.1%, P<0.001) and failed to maintain locomotion, while regular insulin resulted in an increase of both parameters.

Conclusions: The data suggest that permanently-increased insulin detemir levels in the brain convert its hyperstimulatory effects and finally mediate impairments in brain activity and locomotion. This observation might be considered when human studies with insulin detemir are designed to target the brain in order to optimize treatment regimens.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162124PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010192PMC
August 2017

Variation in the Phosphoinositide 3-Kinase Gamma Gene Affects Plasma HDL-Cholesterol without Modification of Metabolic or Inflammatory Markers.

PLoS One 2015 10;10(12):e0144494. Epub 2015 Dec 10.

Department of Internal Medicine, Division of Endocrinology, Diabetology, Angiology, Nephrology and Clinical Chemistry, University Hospital Tübingen, Tübingen, Germany.

Objective: Phosphoinositide 3-kinase γ (PI3Kγ) is a G-protein-coupled receptor-activated lipid kinase mainly expressed in leukocytes and cells of the cardiovascular system. PI3Kγ plays an important signaling role in inflammatory processes. Since subclinical inflammation is a hallmark of atherosclerosis, obesity-related insulin resistance, and pancreatic β-cell failure, we asked whether common genetic variation in the PI3Kγ gene (PIK3CG) contributes to body fat content/distribution, serum adipokine/cytokine concentrations, alterations in plasma lipid profiles, insulin sensitivity, insulin release, and glucose homeostasis.

Study Design: Using a tagging single nucleotide polymorphism (SNP) approach, we analyzed genotype-phenotype associations in 2,068 German subjects genotyped for 10 PIK3CG SNPs and characterized by oral glucose tolerance tests. In subgroups, data from hyperinsulinaemic-euglycaemic clamps, magnetic resonance spectroscopy of the liver, whole-body magnetic resonance imaging, and intravenous glucose tolerance tests were available, and peripheral blood mononuclear cells (PBMCs) were used for gene expression analysis.

Results: After appropriate adjustment, none of the PIK3CG tagging SNPs was significantly associated with body fat content/distribution, adipokine/cytokine concentrations, insulin sensitivity, insulin secretion, or blood glucose concentrations (p>0.0127, all; Bonferroni-corrected α-level: 0.0051). However, six non-linked SNPs displayed at least nominal associations with plasma HDL-cholesterol concentrations, two of them (rs4288294 and rs116697954) reaching the level of study-wide significance (p = 0.0003 and p = 0.0004, respectively). More precisely, rs4288294 and rs116697954 influenced HDL2-, but not HDL3-, cholesterol. With respect to the SNPs' in vivo functionality, rs4288294 was significantly associated with PIK3CG mRNA expression in PBMCs.

Conclusions: We could demonstrate that common genetic variation in the PIK3CG locus, possibly via altered PIK3CG gene expression, determines plasma HDL-cholesterol concentrations. Since HDL2-, but not HDL3-, cholesterol is influenced by PIK3CG variants, PI3Kγ may play a role in HDL clearance rather than in HDL biogenesis. Even though the molecular pathways connecting PI3Kγ and HDL metabolism remain to be further elucidated, this finding could add a novel aspect to the pathophysiological role of PI3Kγ in atherogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675530PMC
June 2016

Protein kinase Cδ regulates nuclear export of FOXO1 through phosphorylation of the chaperone 14-3-3ζ.

Diabetologia 2015 Dec 12;58(12):2819-31. Epub 2015 Sep 12.

Division of Endocrinology, Diabetology, Angiology, Nephrology and Clinical Chemistry, Department of Internal Medicine IV, University Hospital Tübingen, D-72076, Tübingen, Germany.

Aims/hypothesis: Forkhead box protein O1 (FOXO1) is a transcription factor essential for beta cell fate. Protein kinase B-dependent phosphorylation of FOXO1 at S256 (P-FOXO1) enables its binding to 14-3-3 dimers and nuclear export. Dephosphorylated FOXO1 enters nuclei and activates pro-apoptotic genes. Since our previous observations suggest that protein kinase C delta (PKCδ) induces nuclear accumulation of FOXO1, the underlying mechanism was examined.

Methods: In human islets, genetically modified mice and INS-1E cells apoptosis was assessed by TUNEL staining. Subcellular translocation of proteins was examined by confocal microscopy and signalling pathways were analysed by western blotting and overlay assay.

Results: In PKCδ-overexpressing (PKCδ-tg) mouse islet cells and INS-1E cells FOXO1 accumulated in nuclei, surprisingly, as P-FOXO1. PKCδ-tg decelerated IGF-1-dependent stimulation of nuclear export, indicating that changes in export caused nuclear retention of P-FOXO1. Nuclear accumulation of P-FOXO1 was accompanied by increased phosphorylation of 14-3-3ζ at S58 and reduced dimerisation of 14-3-3ζ. Palmitic acid further augmented phosphorylation of 14-3-3ζ and triggered nuclear accumulation of FOXO1 in both INS-1E and human islet cells. Furthermore, the overexpression of a phosphomimicking mutant of 14-3-3ζ (S58D) enhanced nuclear FOXO1. In accordance with the nuclear accumulation of P-FOXO1, PKCδ overexpression alone did not increase apoptotic cell death. Additionally, insulin secretion and glucose homeostasis in PKCδ-overexpressing mice remained unaffected.

Conclusions/interpretation: These results suggest that PKCδ-mediated phosphorylation of 14-3-3ζ contributes to the nuclear retention of FOXO1, even when FOXO1 is phosphorylated as under non-stress conditions. P-FOXO1 does not induce pro-apoptotic genes, but may rather exert beneficial effects on beta cells.
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http://dx.doi.org/10.1007/s00125-015-3744-zDOI Listing
December 2015

The brain response to peripheral insulin declines with age: a contribution of the blood-brain barrier?

PLoS One 2015 12;10(5):e0126804. Epub 2015 May 12.

German Center for Diabetes Research (DZD), Tuebingen, Germany; Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tuebingen (IDM), Tuebingen, Germany.

Objectives: It is a matter of debate whether impaired insulin action originates from a defect at the neural level or impaired transport of the hormone into the brain. In this study, we aimed to investigate the effect of aging on insulin concentrations in the periphery and the central nervous system as well as its impact on insulin-dependent brain activity.

Methods: Insulin, glucose and albumin concentrations were determined in 160 paired human serum and cerebrospinal fluid (CSF) samples. Additionally, insulin was applied in young and aged mice by subcutaneous injection or intracerebroventricularly to circumvent the blood-brain barrier. Insulin action and cortical activity were assessed by Western blotting and electrocorticography radiotelemetric measurements.

Results: In humans, CSF glucose and insulin concentrations were tightly correlated with the respective serum/plasma concentrations. The CSF/serum ratio for insulin was reduced in older subjects while the CSF/serum ratio for albumin increased with age like for most other proteins. Western blot analysis in murine whole brain lysates revealed impaired phosphorylation of AKT (P-AKT) in aged mice following peripheral insulin stimulation whereas P-AKT was comparable to levels in young mice after intracerebroventricular insulin application. As readout for insulin action in the brain, insulin-mediated cortical brain activity instantly increased in young mice subcutaneously injected with insulin but was significantly reduced and delayed in aged mice during the treatment period. When insulin was applied intracerebroventricularly into aged animals, brain activity was readily improved.

Conclusions: This study discloses age-dependent changes in insulin CSF/serum ratios in humans. In the elderly, cerebral insulin resistance might be partially attributed to an impaired transport of insulin into the central nervous system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126804PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429020PMC
February 2016

Cinnamon extract improves insulin sensitivity in the brain and lowers liver fat in mouse models of obesity.

PLoS One 2014 18;9(3):e92358. Epub 2014 Mar 18.

German Center for Diabetes Research (DZD), Tuebingen, Germany; Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tuebingen (IDM), Tuebingen, Germany.

Objectives: Treatment of diabetic subjects with cinnamon demonstrated an improvement in blood glucose concentrations and insulin sensitivity but the underlying mechanisms remained unclear. This work intends to elucidate the impact of cinnamon effects on the brain by using isolated astrocytes, and an obese and diabetic mouse model.

Methods: Cinnamon components (eugenol, cinnamaldehyde) were added to astrocytes and liver cells to measure insulin signaling and glycogen synthesis. Ob/ob mice were supplemented with extract from cinnamomum zeylanicum for 6 weeks and cortical brain activity, locomotion and energy expenditure were evaluated. Insulin action was determined in brain and liver tissues.

Results: Treatment of primary astrocytes with eugenol promoted glycogen synthesis, whereas the effect of cinnamaldehyde was attenuated. In terms of brain function in vivo, cinnamon extract improved insulin sensitivity and brain activity in ob/ob mice, and the insulin-stimulated locomotor activity was improved. In addition, fasting blood glucose levels and glucose tolerance were greatly improved in ob/ob mice due to cinnamon extracts, while insulin secretion was unaltered. This corresponded with lower triglyceride and increased liver glycogen content and improved insulin action in liver tissues. In vitro, Fao cells exposed to cinnamon exhibited no change in insulin action.

Conclusions: Together, cinnamon extract improved insulin action in the brain as well as brain activity and locomotion. This specific effect may represent an important central feature of cinnamon in improving insulin action in the brain, and mediates metabolic alterations in the periphery to decrease liver fat and improve glucose homeostasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092358PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958529PMC
November 2014

Monitoring the stress-level of rats with different types of anesthesia: a tail-artery cannulation protocol.

J Pharmacol Toxicol Methods 2014 Jul-Aug;70(1):35-9. Epub 2014 Mar 14.

High-Field MR Center, Max-Planck-Institute for Biological Cybernetics, Spemannstr. 41, 72076 Tübingen, Germany.

Introduction: Functional MRI in rats under anesthesia can largely minimize motion artifacts and attenuate the stress of the animal. However, two issues remain to be clarified and improved. First, fMRI results obtained with different types of anesthesia during surgical preparation and imaging show a large variability, which could be caused by the variable stress level of the rodents. Second, the most common surgical procedure used for anesthesia, blood gas analysis and mean arterial blood-pressure (MABP) monitoring is the femoral vein and artery catheterization that makes longitudinal studies difficult.

Methods: In order to examine the variability of the stress level with three different anesthesia protocols using isoflurane (Iso), medetomidine-ketamine (MK) or propofol-remifentanil (PR), we measured the plasma corticosterone (CORT) concentration with (125)I-radioimmunoassay in blood samples collected prior to, immediately after and 60min after surgery. Tail-artery and vein catheterization was adapted for long-term monitoring of MABP with periodic blood sampling and is proposed as a less invasive and technically simple alternative to femoral vessel catheterization in fMRI preparation protocols.

Results: We show that the CORT concentration depends on the anesthesia protocol with both alternatives providing more efficient stress reduction than the protocol using Iso. However, only the protocol using PR achieved a significant hormone reduction during surgery. Stress was not reliably manifested in changes in heart-rate and breathing-rate. Anesthesia and strain related changes in these two physiological parameters may be assigned to the pharmacological effects of the premedication and anesthetic agents. The results indicate also that MABP can be monitored over a long period of time (e.g. functional imaging session) through an arterial access point in the rat tail after cannulation with the proposed procedure.

Discussion And Conclusion: Animals can experience stress during fMRI preparation protocols without obvious signs in commonly monitored physiological parameters. Our results challenge the efficiency of surgical protocols using Iso as mono-anesthetic agent, even when extended with topical analgesia. It was demonstrated that the CORT-based stress-level measurement through tail-artery cannulation can be used for developing anesthesia protocols (i.e. the presented PR protocol) when setting up future fMRI studies. The proposed surgical method for the tail is expected to facilitate longitudinal fMRI studies with permanent arterial access.
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http://dx.doi.org/10.1016/j.vascn.2014.03.003DOI Listing
March 2015

Evidence for altered transport of insulin across the blood-brain barrier in insulin-resistant humans.

Acta Diabetol 2014 Aug 27;51(4):679-81. Epub 2013 Dec 27.

Division of Endocrinology, Diabetology, Angiology, Nephrology and Clinical Chemistry, Department of Internal Medicine, Eberhard Karls University, Tübingen, Germany.

Eating behavior, body weight regulation, peripheral glucose metabolism, and cognitive function depend on adequate insulin action in the brain, and recent studies in humans suggested that impaired insulin action in the brain emerges upon fat intake, obesity, and genetic variants. As insulin enters into the brain in a receptor-mediated fashion, we hypothesized that whole-body insulin sensitivity might affect the transport of insulin into the brain and contribute to the aversive effect of insulin resistance in the central nervous system. In this study, we aimed to determine the ratio of insulin in the cerebrospinal fluid and serum to whole-body insulin sensitivity. Healthy human subjects participated in an oral glucose tolerance test to determine whole-body insulin sensitivity and underwent lumbar puncture. Blood and CSF concentrations of insulin were significantly correlated. The CSF/serum ratio for insulin was significantly associated with whole body insulin sensitivity with reduced insulin transported into the CSF in insulin-resistant subjects. Together, our data suggest that transport of insulin into the CSF relates to peripheral insulin sensitivity and impairs insulin action in the brain. This underlines the need for sensitizing measures in insulin-resistant subjects.
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http://dx.doi.org/10.1007/s00592-013-0546-yDOI Listing
August 2014

Genetic variation in NR1H4 encoding the bile acid receptor FXR determines fasting glucose and free fatty acid levels in humans.

J Clin Endocrinol Metab 2013 Jul 2;98(7):E1224-9. Epub 2013 May 2.

Department of Internal Medicine, Division of Endocrinology, University Hospital, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.

Context: Bile acid signaling via farnesoid X receptor (FXR) regulates glucose and lipid levels, fat mass, and hepatic steatosis in animal models.

Objective: To understand the role of FXR in human metabolism, we investigated associations of common single-nucleotide polymorphisms (SNPs) in the FXR-encoding gene NR1H4 with glucose and lipid metabolism, body fat mass, and liver fat content.

Design: We genotyped 2166 healthy German subjects for 7 tagging SNPs within NR1H4 (rs35735, rs1030454, rs11110415, rs11610264, rs17030285, rs4764980, and rs11110390) covering 100% of common genetic variation (minor allele frequency > 10%).

Outcome Measures: Subjects were metabolically characterized by an oral glucose tolerance test. In subgroups, hyperinsulinemic-euglycemic clamp and liver fat quantification by (1)H-magnetic resonance spectroscopy were performed.

Results: SNP rs4764980 was significantly associated with fasting glycemia (P = .0043) and nominally associated with fasting and postglucose load free fatty acid (FFA) levels (P = .01). Upon interrogation of publicly available Meta-Analyses of Glucose and Insulin-related traits Consortium data, the association of rs4764980 with fasting glycemia was replicated (Meta-Analyses of Glucose and Insulin-related traits Consortium, P = .005). Additionally, SNP rs11110390 showed significant associations with fasting (P = .0054) and postload (P = .0051) FFA levels. For none of the investigated SNPs, associations with insulin secretion or sensitivity, body fat mass, or liver fat content were detected.

Conclusions: We conclude that FXR contributes to fasting glucose and FFA levels in humans independent of unhealthy body fat accumulation. The receptor represents an interesting target to influence lipid and glucose metabolism.
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http://dx.doi.org/10.1210/jc.2013-1177DOI Listing
July 2013

Leptin affects insulin action in astrocytes and impairs insulin-mediated physical activity.

Cell Physiol Biochem 2012 19;30(1):238-46. Epub 2012 Jun 19.

University of Tuebingen, Department of Internal Medicine 4, Tuebingen, Germany.

Background/aims: Impaired insulin action is an early event in the pathogenesis of obesity and type 2-diabetes, and among the metabolic confounders in obese, hyperleptinaemia is constantly present; however its impact on insulin action in the brain and locomotor activity is unknown.

Methods: We examined insulin action by Western Blot analysis and glycogen synthesis in primary astrocytes and brain tissue and detected locomotion in C57BL/6 mice. The insulin-mediated desire to move was evaluated in healthy volunteers and correlated to leptin levels.

Results: Leptin treatment led to a significant decrease in insulin-mediated phosphorylation of the insulin receptor and Akt473 which was accompanied by a decline in glycogen synthesis in primary astrocytes and significantly decreased insulin-induced phosphorylation of the insulin receptor and insulin receptor substrate-2 in brain tissues of mice. Intracerebroventricular insulin failed to promote locomotion in the presence of elevated leptin levels. Lean human subjects reported an increase in the desire to move following insulin which failed in obese and there was an inverse correlation between the insulin-mediated desire to move and leptin levels.

Conclusions: Our data suggest a crosstalk of leptin and insulin in the brain which leads to a decline in locomotor activity. This might represent a molecular mechanism in obese to inhibit physical activity.
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http://dx.doi.org/10.1159/000339060DOI Listing
October 2012

Monounsaturated fatty acids prevent the aversive effects of obesity on locomotion, brain activity, and sleep behavior.

Diabetes 2012 Jul 9;61(7):1669-79. Epub 2012 Apr 9.

Department of Internal Medicine, Division of Endocrinology, Diabetology, and Vascular Disease, University of Tuebingen, Tuebingen, Germany.

Fat and physical inactivity are the most evident factors in the pathogenesis of obesity, and fat quality seems to play a crucial role for measures of glucose homeostasis. However, the impact of dietary fat quality on brain function, behavior, and sleep is basically unknown. In this study, mice were fed a diet supplemented with either monounsaturated fatty acids (MUFAs) or saturated fatty acids (SFAs) and their impact on glucose homeostasis, locomotion, brain activity, and sleep behavior was evaluated. MUFAs and SFAs led to a significant increase in fat mass but only feeding of SFAs was accompanied by glucose intolerance in mice. Radiotelemetry revealed a significant decrease in cortical activity in SFA-mice whereas MUFAs even improved activity. SFAs decreased wakefulness and increased non-rapid eye movement sleep. An intracerebroventricular application of insulin promoted locomotor activity in MUFA-fed mice, whereas SFA-mice were resistant. In humans, SFA-enriched diet led to a decrease in hippocampal and cortical activity determined by functional magnetic resonance imaging techniques. Together, dietary intake of MUFAs promoted insulin action in the brain with its beneficial effects for cortical activity, locomotion, and sleep, whereas a comparable intake of SFAs acted as a negative modulator of brain activity in mice and humans.
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http://dx.doi.org/10.2337/db11-1521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379681PMC
July 2012

Toll-like receptors 2 and 4 impair insulin-mediated brain activity by interleukin-6 and osteopontin and alter sleep architecture.

FASEB J 2012 May 25;26(5):1799-809. Epub 2012 Jan 25.

Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry, University of Tuebingen, Otfried-Mueller-Straße 10, D-72076 Tuebingen, Germany.

Impaired insulin action in the brain represents an early step in the progression toward type 2 diabetes, and elevated levels of saturated free fatty acids are known to impair insulin action in prediabetic subjects. One potential mediator that links fatty acids to inflammation and insulin resistance is the Toll-like receptor (TLR) family. Therefore, C3H/HeJ/TLR2-KO (TLR2/4-deficient) mice were fed a high-fat diet (HFD), and insulin action in the brain as well as cortical and locomotor activity was analyzed by using telemetric implants. TLR2/4-deficient mice were protected from HFD-induced glucose intolerance and insulin resistance in the brain and displayed an improvement in cortical and locomotor activity that was not observed in C3H/HeJ mice. Sleep recordings revealed a 42% increase in rapid eye movement sleep in the deficient mice during daytime, and these mice spent 41% more time awake during the night period. Treatment of control mice with a neutralizing IL-6 antibody improved insulin action in the brain as well as cortical activity and diminished osteopontin protein to levels of the TLR2/4-deficient mice. Together, our data suggest that the lack of functional TLR2/4 protects mice from a fat-mediated impairment in insulin action, brain activity, locomotion, and sleep architecture by an IL-6/osteopontin-dependent mechanism.
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http://dx.doi.org/10.1096/fj.11-191023DOI Listing
May 2012

Protein kinase C delta (PKCδ) affects proliferation of insulin-secreting cells by promoting nuclear extrusion of the cell cycle inhibitor p21Cip1/WAF1.

PLoS One 2011 27;6(12):e28828. Epub 2011 Dec 27.

Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, Department of Internal Medicine, University of Tübingen, Tübingen, Germany.

Background: High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions.

Methodology And Principal Findings: Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1). This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1) at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1) was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1) with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic.

Conclusions And Significance: These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1), which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246440PMC
May 2012

An Obesity Risk SNP (rs17782313) near the MC4R Gene Is Associated with Cerebrocortical Insulin Resistance in Humans.

J Obes 2011 3;2011:283153. Epub 2011 Jun 3.

Department of Internal Medicine IV, University of Tübingen, Otfried-Müller-Strasse 10, 72076 Tübingen, Germany.

Activation of melanocortin-4 receptor (MC4R) by insulin sensitive neurons is a central mechanism in body weight regulation, and genetic variants in the MC4R gene (e.g., rs17782313) are associated with obesity. By using magnetoencephalography, we addressed whether rs17782313 affects the cerebrocortical insulin response. We measured the cerebrocortical insulin response by using magnetoencephalography in a hyperinsulinemic euglycemic clamp (versus placebo) in 51 nondiabetic humans (26 f/25 m, age 35 ± 3 years, BMI 28 ± 1 kg/m(2)). The C-allele of rs17782313 was minor allele (frequency 23%), and the genotype distribution (TT 30, TC 19, CC 2) was in Hardy-Weinberg-Equilibrium. Insulin-stimulated cerebrocortical theta activity was decreased in the presence of the C-allele (TT 33 ± 16 fT; TC/CC -27 ± 20 fT; P = .023), and this effect remained significant after adjusting for BMI and peripheral insulin sensitivity (P = .047). Cerebrocortical theta activity was impaired in carriers of the obesity risk allele. Therefore, cerebral insulin resistance may contribute to the obesity effect of rs17782313.
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http://dx.doi.org/10.1155/2011/283153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136179PMC
November 2011

Insulin promotes glycogen storage and cell proliferation in primary human astrocytes.

PLoS One 2011 27;6(6):e21594. Epub 2011 Jun 27.

Division of Endocrinology, Diabetology, Angiology, Nephrology and Clinical Chemistry, Department of Internal Medicine, Eberhard Karls University Tübingen, Tübingen, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021594PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124526PMC
December 2011

The insulin-mediated modulation of visually evoked magnetic fields is reduced in obese subjects.

PLoS One 2011 May 11;6(5):e19482. Epub 2011 May 11.

Internal Medicine IV, Department of Endocrinology and Diabetes, Angiology, Nephrology and Clinical Chemistry, University Hospital, University of Tübingen, Tübingen, Germany.

Background: Insulin is an anorexigenic hormone that contributes to the termination of food intake in the postprandial state. An alteration in insulin action in the brain, named "cerebral insulin resistance", is responsible for overeating and the development of obesity.

Methodology/principal Findings: To analyze the direct effect of insulin on food-related neuronal activity we tested 10 lean and 10 obese subjects. We conducted a magnetencephalography study during a visual working memory task in both the basal state and after applying insulin or placebo spray intranasally to bypass the blood brain barrier. Food and non-food pictures were presented and subjects had to determine whether or not two consecutive pictures belonged to the same category. Intranasal insulin displayed no effect on blood glucose, insulin or C-peptide concentrations in the periphery; however, it led to an increase in the components of evoked fields related to identification and categorization of pictures (at around 170 ms post stimuli in the visual ventral stream) in lean subjects when food pictures were presented. In contrast, insulin did not modulate food-related brain activity in obese subjects.

Conclusions/significance: We demonstrated that intranasal insulin increases the cerebral processing of food pictures in lean whereas this was absent in obese subjects. This study further substantiates the presence of a "cerebral insulin resistance" in obese subjects and might be relevant in the pathogenesis of obesity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019482PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092755PMC
May 2011

Genetic ablation of cGMP-dependent protein kinase type I causes liver inflammation and fasting hyperglycemia.

Diabetes 2011 May 4;60(5):1566-76. Epub 2011 Apr 4.

Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry, University of Tübingen, Tübingen, Germany.

Objective: The nitric oxide/cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway regulates cell functions that play a pivotal role in the pathogenesis of type 2 diabetes. However, the impact of a dysfunction of this pathway for glucose metabolism in vivo is unknown.

Research Design And Methods: The expression of cGKI in tissues relevant to insulin action was analyzed by immunohistochemistry. The metabolic consequences of a genetic deletion of cGKI were studied in mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice).

Results: In wild-type mice, cGKI protein was detected in hepatic stellate cells, but not in hepatocytes, skeletal muscle, fat cells, or pancreatic β-cells. Compared with control animals, cGKI-SM mice had higher energy expenditure in the light phase associated with lower body weight and fat mass and increased insulin sensitivity. Mutant mice also showed higher fasting glucose levels, whereas insulin levels and intraperitoneal glucose tolerance test results were similar to those in control animals. Interleukin (IL)-6 signaling was strongly activated in the liver of cGKI-SM mice as demonstrated by increased levels of IL-6, phospho-signal transducer and activator of transcription 3 (Tyr 705), suppressor of cytokine signaling-3, and serum amyloid A2. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in the liver was impaired in cGKI-SM mice. The fraction of Mac-2-positive macrophages in the liver was significantly higher in cGKI-SM mice than in control mice. In contrast with cGKI-SM mice, conditional knockout mice lacking cGKI only in the nervous system were normal with respect to body weight, energy expenditure, fasting glucose, IL-6, and insulin action in the liver.

Conclusions: Genetic deletion of cGKI in non-neuronal cells results in a complex metabolic phenotype, including liver inflammation and fasting hyperglycemia. Loss of cGKI in hepatic stellate cells may affect liver metabolism via a paracrine mechanism that involves enhanced macrophage infiltration and IL-6 signaling.
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http://dx.doi.org/10.2337/db10-0760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292332PMC
May 2011

Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser675 and Ser907.

Am J Physiol Endocrinol Metab 2011 May 23;300(5):E824-36. Epub 2010 Nov 23.

Dept. of Internal Medicine, Div. of Pathobiochemistry and Clinical Chemistry, Univ. of Tuebingen, Otfried-Mueller-Straße 10, 72076 Tuebingen, Germany.

The identity of specific serine phosphorylation residues of insulin receptor substrate (IRS)-2 and their impact on insulin signal transduction are largely unknown. Ser(675) and Ser(907) of mouse IRS-2 are adjacent to PI 3-kinase or Grb2 binding domains, respectively. Using monoclonal phosphosite-specific antibodies, we demonstrated the phosphorylation of both serines after stimulation of Fao hepatoma cells with insulin, anisomycin, or phorbol esters. Phosphorylation of both sites was a late and prolonged event during insulin treatment and was also detected in liver tissue of insulin-treated as well as refed mice. Inhibition and siRNA-mediated knockdown of ERK1/2 indicated that the insulin-induced phosphorylation of Ser(907) was ERK dependent. Phosphorylation of Ser(907) did not prevent the insulin-induced association of IRS-2 with Grb2, but phosphorylation of the adjacent Tyr(911) was proved to be crucial in HEK 293 cells expressing IRS-2 Ala mutants. The insulin-induced phosphorylation of Ser(675) was prevented by inhibition and siRNA-mediated knockdown of mTOR but not of p70(S6K1). Mutation of Ser(675) to Ala did not affect downstream insulin signaling but increased the half-life of the protein, suggesting an involvement of phospho-Ser(675) in an accelerated degradation of IRS-2. Moreover, the insulin-induced degradation of IRS-2 was blocked by inhibition of mTOR. We conclude that the two novel insulin-dependent serine phosphorylation sites of IRS-2 were not involved in the regulation of the adjacent PI 3-kinase and Grb2 binding domains but might be implicated in the ERK- and mTOR-mediated negative feedback control.
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http://dx.doi.org/10.1152/ajpendo.00409.2010DOI Listing
May 2011

Enforced expression of protein kinase C in skeletal muscle causes physical inactivity, fatty liver and insulin resistance in the brain.

J Cell Mol Med 2010 Apr;14(4):903-13

University of Tuebingen, Department of Internal Medicine, Tübingen, Germany.

Among the multitude of dysregulated signalling mechanisms that comprise insulin resistance in divergent organs, the primary events in the development of type 2 diabetes are not well established. As protein kinase C (PKC) activation is consistently present in skeletal muscle of obese and insulin resistant subjects, we generated a transgenic mouse model that overexpresses constitutively active PKC-beta(2) in skeletal muscle to test whether activation of PKC is sufficient to cause an aversive whole-body phenotype. Upon this genetic modification, increased serine phosphorylation in Irs1 was observed and followed by impaired (3)H-deoxy-glucose uptake and muscle glycogen content, and transgenic mice exhibited insulin and glucose intolerance as they age. Muscle histochemistry revealed an increase in lipid deposition (intramyocellular lipids), and transgenic mice displayed impaired expression of transcriptional regulators of genes involved in fatty acid oxidation (peroxisome proliferator-activated receptor-gamma, PGC-1beta, acyl-CoA oxidase) and lipolysis (hormone-sensitive lipase). In this regard, muscle of transgenic mice exhibited a reduced capacity to oxidize palmitate and contained less mitochondria as determined by citrate synthase activity. Moreover, the phenotype included a profound decrease in the daily running distance, intra-abdominal and hepatic fat accumulation and impaired insulin action in the brain. Together, our data suggest that activation of a classical PKC in skeletal muscle as present in the pre-diabetic state is sufficient to cause disturbances in whole-body glucose and lipid metabolism followed by profound alterations in oxidative capacity, ectopic fat deposition and physical activity.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00629.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823122PMC
April 2010

Insulin modulates food-related activity in the central nervous system.

J Clin Endocrinol Metab 2010 Feb 8;95(2):748-55. Epub 2009 Dec 8.

MEG-Zentrum, Otfried-Müller-Strasse 47, 72076 Tübingen, Germany.

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.
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http://dx.doi.org/10.1210/jc.2009-1677DOI Listing
February 2010

Overexpression of kinase-negative protein kinase Cdelta in pancreatic beta-cells protects mice from diet-induced glucose intolerance and beta-cell dysfunction.

Diabetes 2010 Jan 13;59(1):119-27. Epub 2009 Oct 13.

Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology, and Clinical Chemistry, University of Tũbingen, Tũbingen, Germany.

Objective: In vitro models suggest that free fatty acid-induced apoptotic beta-cell death is mediated through protein kinase C (PKC)delta. To examine the role of PKCdelta signaling in vivo, transgenic mice overexpressing a kinase-negative PKCdelta (PKCdeltaKN) selectively in beta-cells were generated and analyzed for glucose homeostasis and beta-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 PKCdelta 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: PKCdeltaKN 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 beta-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 PKCdeltaKN mice. PKCdeltaKN 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 PKCdeltaKN was accompanied by an increased phosphorylation of FOXO1 at Ser256 and a significant reduction of FOXO1 protein.

Conclusions: Overexpression of PKCdeltaKN in beta-cells protects from HFD-induced beta-cell failure in vivo by a mechanism that involves inhibition of fatty acid-mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of FOXO1 activation.
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http://dx.doi.org/10.2337/db09-0512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797912PMC
January 2010

The insulin effect on cerebrocortical theta activity is associated with serum concentrations of saturated nonesterified Fatty acids.

J Clin Endocrinol Metab 2009 Nov 9;94(11):4600-7. Epub 2009 Oct 9.

Department of Internal Medicine IV, University Hospital, University of Tübingen, 72076 Tübingen, Germany.

Context: Insulin action in the brain contributes to adequate regulation of body weight, neuronal survival, and suppression of endogenous glucose production. We previously demonstrated by magnetoencephalography in lean humans that insulin stimulates activity in beta and theta frequency bands, whereas this effect was abolished in obese individuals.

Objective: The present study aims to define metabolic signals associated with the suppression of the cerebrocortical response in obese humans.

Design And Setting: We determined insulin-mediated modulation of spontaneous cerebrocortical activity by magnetoencephalography during a hyperinsulinemic euglycemic clamp and related it to measures of ectopic fat deposition and mediators of peripheral insulin resistance. Visceral fat mass and intrahepatic lipid content were quantified by magnetic resonance imaging and spectroscopy. Multiple regression analysis was used to analyze associations of cerebrocortical insulin sensitivity and metabolic markers related to obesity.

Participants: Forty-nine healthy, nondiabetic humans participated in the study.

Results: In a multiple regression, insulin-mediated stimulation of theta activity was negatively correlated to body mass index, visceral fat mass, and intrahepatic lipid content. Although fasting saturated nonesterified fatty acids mediated the correlations of theta activity with abdominal and intrahepatic lipid stores, adipocytokines displayed no independent correlation with insulin-mediated cortical activity in the theta frequency band.

Conclusions: Thus, insulin action at the level of cerebrocortical activity in the brain is diminished in the presence of elevated levels of saturated nonesterified fatty acids.
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http://dx.doi.org/10.1210/jc.2009-0469DOI Listing
November 2009

Enhanced insulin sensitivity of gene-targeted mice lacking functional KCNQ1.

Am J Physiol Regul Integr Comp Physiol 2009 Jun 15;296(6):R1695-701. Epub 2009 Apr 15.

Division of Diabetology, Endocrinology, Angiology, Nephrology, and Clinical Chemistry, University of Tuebingen, Tuebingen D-72076, Germany.

The pore-forming K+-channel alpha-subunit KCNQ1 is expressed in a wide variety of tissues including heart, skeletal muscle, liver, and epithelia. Most recent evidence revealed an association of the KCNQ1 gene with the susceptibility to type 2 diabetes. KCNQ1 participates in the regulation of cell volume, which is, in turn, critically important for the regulation of metabolism by insulin. The present study explored the influence of KCNQ1 on insulin-induced cellular K+ uptake and glucose metabolism. Insulin (100 nM)-induced K+ uptake was determined in isolated perfused livers from KCNQ1-deficient mice (kcnq1(-/-)) and their wild-type littermates (kcnq1(+/+)). Moreover, plasma glucose and insulin levels, intraperitoneal glucose (3 g/kg) tolerance, insulin (0.15 U/kg)-induced hypoglycemia, and peripheral uptake of radiolabeled 3H-deoxy-glucose were determined in both genotypes. Insulin-stimulated hepatocellular K+ uptake was significantly more sustained in isolated perfused livers from kcnq1(-/-) mice than from kcnq1(+/+)mice. The decline of plasma glucose concentration following an intraperitoneal injection of insulin was again significantly more sustained in kcnq1(-/-) than in kcnq1(+/+) mice. Both fasted and nonfasted plasma glucose and insulin concentrations were significantly lower in kcnq1(-/-) than in kcnq1(+/+)mice. Following an intraperitoneal glucose injection, the peak plasma glucose concentration was significantly lower in kcnq1(-/-) than in kcnq1(+/+)mice. Uptake of 3H-deoxy-glucose into skeletal muscle, liver, kidney and lung tissue was significantly higher in kcnq1(-/-) than in kcnq1(+/+)mice. In conclusion, KCNQ1 counteracts the stimulation of cellular K+ uptake by insulin and thereby influences K+-dependent insulin signaling on glucose metabolism. The observations indicate that KCNQ1 is a novel molecule affecting insulin sensitivity of glucose metabolism.
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http://dx.doi.org/10.1152/ajpregu.90839.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692790PMC
June 2009

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

J Physiol 2009 Jan 10;587(1):241-52. Epub 2008 Nov 10.

Division of Pathobiochemistry and Clinical Chemistry, University of Tuebingen, Otfried-Mueller-Strasse 10, D-72076 Tuebingen, Germany.

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-gamma coactivator 1alpha (PGC-1alpha; 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-1alpha 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.
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http://dx.doi.org/10.1113/jphysiol.2008.160275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670037PMC
January 2009

Neuronal functions, feeding behavior, and energy balance in Slc2a3+/- mice.

Am J Physiol Endocrinol Metab 2008 Nov 9;295(5):E1084-94. Epub 2008 Sep 9.

Department of Pharmacology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany.

Homozygous deletion of the gene of the neuronal glucose transporter GLUT3 (Slc2a3) in mice results in embryonic lethality, whereas heterozygotes (Slc2a3+/-) are viable. Here, we describe the characterization of heterozygous mice with regard to neuronal function, glucose homeostasis, and, since GLUT3 might be a component of the neuronal glucose-sensing mechanism, food intake and energy balance. Levels of GLUT3 mRNA and protein in brain were reduced by 50% in Slc2a3+/- mice. Electrographic features examined by electroencephalographic recordings give evidence for slightly but significantly enhanced cerebrocortical activity in Slc2a3+/- mice. In addition, Slc2a3+/- mice were slightly more sensitive to an acoustic startle stimulus (elevated startle amplitude and reduced prepulse inhibition). However, systemic behavioral testing revealed no other functional abnormalities, e.g., in coordination, reflexes, motor abilities, anxiety, learning, and memory. Furthermore, no differences in body weight, blood glucose, and insulin levels were detected between wild-type and Slc2a3+/- littermates. Food intake as monitored randomly or after intracerebroventricular administration of 2-deoxyglucose or d-glucose, or food choice for carbohydrates/fat was not affected in Slc2a3+/- mice. Taken together, our data indicate that, in contrast to Slc2a1, a single allele of Slc2a3 is sufficient for maintenance of neuronal energy supply, motor abilities, learning and memory, and feeding behavior.
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http://dx.doi.org/10.1152/ajpendo.90491.2008DOI Listing
November 2008

Effect of insulin detemir, compared to human insulin, on 3T3-L1 adipogenesis.

Regul Pept 2008 Nov 24;151(1-3):160-3. Epub 2008 May 24.

Department of Internal Medicine, Division of Endocrinology, Diabetology, Angiology, Nephrology, and Clinical Chemistry, University Hospital Tübingen, D-72076 Tübingen, Germany.

Insulin detemir (DET) represents a myristic acid (MA)-coupled insulin derivative with protracted action due to reversible albumin binding. As compared to human insulin (HI), DET provokes no or only minor body weight gain in vivo. Therefore, we compared DET's and HI's adipogenic effects. 3T3-L1 preadipocytes were differentiated with 5 nmol/l HI, 5 nmol/l DET (=DET(equimolar)), or 20 nmol/l DET (=DET(equipotent); equipotent in terms of the reported metabolic potency in vitro). Due to differentiation-suppressive effects, albumin was excluded from the studies. During the induction period, only HI allowed clonal expansion. Moreover, HI induced a 200-fold increase in specific glycerol-3-phosphate dehydrogenase activity, whereas DET(equimolar) and DET(equipotent) were markedly less adipogenic (P
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http://dx.doi.org/10.1016/j.regpep.2008.05.005DOI Listing
November 2008

IGF-1 protects against dexamethasone-induced cell death in insulin secreting INS-1 cells independent of AKT/PKB phosphorylation.

Cell Physiol Biochem 2008 24;21(5-6):455-62. Epub 2008 Apr 24.

Institute of Physiology, University of Tübingen, Germany.

Appropriate insulin secretion depends on beta-cell mass that is determined by the balance between cell proliferation and death. IGF-1 stimulates proliferation and protects against apoptosis. In contrast, glucocorticoids promote cell death. In this study we examined molecular interactions of the glucocorticoid dexamethasone (dexa) with IGF-1 signalling pathways in insulin secreting INS-1 cells. IGF-1 (50 ng/ml) increased the growth rate and stimulated BrdU incorporation, while dexa (100 nmol/l) inhibited cell growth, BrdU incorporation and induced apoptosis. Dexa-induced cell death was partially antagonized by IGF-1. This protection was further increased by LY294002 (10 micromol/l), an inhibitor of PI3 kinase. In contrast, MAP kinase inhibitor PD98059 (10 micromol/l) significantly reduced the protective effect of IGF-1. The analysis of signalling pathways by Western blotting revealed that dexa increased IRS-2 protein abundance while the expression of PI3K, PKB and ERK remained unchanged. Despite increased IRS-2 protein,IRS-2 tyrosine phosphorylation stimulated by IGF-1 was inhibited by dexa. Dexa treatment reduced basal PKB phosphorylation. However, IGF-1-mediated stimulation of PKB phosphorylation was not affected by dexa, but ERK phosphorylation was reduced. LY294002 restored IGF-1-induced ERK phosphorylation. These data suggest that dexa induces apoptosis in INS-1 cells by inhibiting phosphorylation of IRS-2, PKB and ERK. IGF-1 counteracts dexa-mediated apoptosis in the presence of reduced PKB but increased ERK phosphorylation.
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http://dx.doi.org/10.1159/000129638DOI Listing
August 2008

Fetuin-A induces cytokine expression and suppresses adiponectin production.

PLoS One 2008 Mar 12;3(3):e1765. Epub 2008 Mar 12.

Department of Internal Medicine, Division of Endocrinology, Nephrology, Vascular Disease and Clinical Chemistry, University of Tübingen, Tübingen, Germany.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001765PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258416PMC
March 2008