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Dissociation Between Fatty Liver and Insulin Resistance in Humans Carrying a Variant of the Patatin-Like Phospholipase 3 Gene Konstantinos Kantartzis 1 , Andreas Peter 1 , Fausto Machicao 1 , Jürgen Machann 2 , Silvia Wagner 3 , Ingmar Königsrainer 3 , Alfred Königsrainer 3 , Fritz Schick 2 , Andreas Fritsche 1 , Hans-Ulrich Häring 1 and Norbert Stefan 1 1 Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, University of Tübingen, Tübingen, Germany; 2 Section on Experimental Radiology, University of Tübingen, Tübingen, Germany; 3 Department of General, Visceral and Transplant Surgery, University of Tübingen, Tübingen, Germany. Corresponding author: N. Stefan, norbert.stefan{at}med.uni-tuebingen.de . Abstract OBJECTIVE In a genome-wide association scan, the rs738409 C>G single nucleotide polymorphism (SNP) in the patatin-like phospholipase 3 gene ( PNPLA3 ) was strongly associated with increased liver fat but not with insulin resistance estimated from fasting values. We investigated whether the SNP determines liver fat independently of visceral adiposity and whether it may even play a role in protecting from insulin resistance. RESEARCH DESIGN AND METHODS Liver fat was measured by 1 H magnetic resonance spectroscopy and total and visceral fat by magnetic resonance tomography in 330 subjects. Insulin sensitivity was estimated during an oral glucose tolerance test and the euglycemic-hyperinsulinemic clamp ( n = 222). PNPLA3 and tumor necrosis factor-α mRNA and triglyceride content were measured in liver biopsies from 16 subjects. RESULTS Liver fat correlated strongly with insulin sensitivity ( P < 0.0001) independently of age, sex, total fat, and visceral fat. G allele carriers of the SNP rs738409 had higher liver fat ( P < 0.0001) and an odds ratio of 2.38 (95% CI 1.37–4.20) for having fatty liver compared to C allele homozygotes. Interestingly, insulin sensitivity (oral glucose tolerance test: P = 0.99; clamp: P = 0.32), serum C-reactive protein levels, lipids, or liver enzymes (all P > 0.14) were not different among the genotypes. Additional adjustment for liver fat actually revealed increased insulin sensitivity in more obese carriers of the G allele ( P = 0.01). In liver biopsies triglyceride content correlated positively with expression of the proinflammatory gene tumor necrosis factor-α in C allele homozygotes ( n = 6, P = 0.027) but not in G allele carriers ( n = 10, P = 0.149). CONCLUSIONS PNPLA3 may be an important key to understand the mechanisms discriminating fatty liver with and without metabolic consequences. Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received February 24, 2009. Accepted July 17, 2009. © 2009 American Diabetes Association

α 2 -Heremans-Schmid Glycoprotein/ Fetuin-A Is Associated With Insulin Resistance and Fat Accumulation in the Liver in Humans Norbert Stefan , MD 1 , Anita M. Hennige , MD 1 , Harald Staiger , PHD 1 , Jürgen Machann , PHD 2 , Fritz Schick , PHD 2 , Stefan M. Kröber , MD 3 , Fausto Machicao , PHD 1 , Andreas Fritsche , MD 1 and Hans-Ulrich Häring , MD 1 1 Department of Internal Medicine, University of Tübingen, Tübingen, Germany 2 Section on Experimental Radiology, University of Tübingen, Tübingen, Germany 3 Department of Pathology, University of Tübingen, Tübingen, Germany Address correspondence and reprint requests to Norbert Stefan, MD, Department of Internal Medicine, Otfried-Müller-Str. 10, D-72076 Tübingen, Germany. E-mail: norbert.stefan{at}med.uni-tuebingen.de Abstract OBJECTIVE —The α 2 -Heremans-Schmid glycoprotein (AHSG; fetuin-A in animals) impairs insulin signaling in vitro and in rodents. Whether AHSG is associated with insulin resistance in humans is under investigation. In an animal model of diet-induced obesity that is commonly associated with hepatic steatosis, an increase in Ahsg mRNA expression was observed in the liver. Therefore, we hypothesized that the AHSG plasma protein, which is exclusively secreted by the liver in humans, may not only be associated with insulin resistance but also with fat accumulation in the liver. RESEARCH DESIGN AND METHODS —Data from 106 healthy Caucasians without type 2 diabetes were included in cross-sectional analyses. A subgroup of 47 individuals had data from a longitudinal study. Insulin sensitivity was measured by a euglycemic-hyperinsulinemic clamp, and liver fat was determined by 1 H magnetic resonance spectroscopy. RESULTS —AHSG plasma levels, adjusted for age, sex, and percentage of body fat, were higher in subjects with impaired glucose tolerance compared with subjects with normal glucose tolerance ( P = 0.006). AHSG plasma levels were negatively associated with insulin sensitivity ( r = −0.22, P = 0.03) in cross-sectional analyses. Moreover, they were positively associated with liver fat ( r = 0.27, P = 0.01). In longitudinal analyses, under weight loss, a decrease in liver fat was accompanied by a decrease in AHSG plasma concentrations. Furthermore, high AHSG levels at baseline predicted less increase in insulin sensitivity ( P = 0.02). CONCLUSIONS —We found that high AHSG plasma levels are associated with insulin resistance in humans. Moreover, AHSG plasma levels are elevated in subjects with fat accumulation in the liver. This is consistent with a potential role of AHSG as a link between fatty liver and insulin resistance. AHSG, α2-Heremans-Schmid glycoprotein Footnotes Additional information on this article can be found in an online appendix at http://care.diabetesjournals.org . A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted January 2, 2006. Received October 11, 2005. DIABETES CARE

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. 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). 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.

Recently, the novel myokine irisin was described to drive adipose tissue 'browning', to increase energy expenditure, and to improve obesity and insulin resistance in high fat-fed mice. Here, we assessed whether common single nucleotide polymorphisms (SNPs) in the FNDC5 locus, encoding the irisin precursor, contribute to human prediabetic phenotypes (overweight, glucose intolerance, insulin resistance, impaired insulin release). A population of 1,976 individuals was characterized by oral glucose tolerance tests and genotyped for FNDC5 tagging SNPs. Subgroups underwent hyperinsulinaemic-euglycaemic clamps, magnetic resonance imaging/spectroscopy, and intravenous glucose tolerance tests. From 37 young and 14 elderly participants recruited in two different centres, muscle biopsies were obtained for the preparation of human myotube cultures. After appropriate adjustment and Bonferroni correction for the number of tested variants, SNPs rs16835198 and rs726344 were associated with in vivo measures of insulin sensitivity. Via interrogation of publicly available data from the Meta-Analyses of Glucose and Insulin-related traits Consortium, rs726344's effect on insulin sensitivity was replicated. Moreover, novel data from human myotubes revealed a negative association between FNDC5 expression and appropriately adjusted in vivo measures of insulin sensitivity in young donors. This finding was replicated in myotubes from elderly men. This study provides evidence that the FNDC5 gene, encoding the novel myokine irisin, determines insulin sensitivity in humans. Our gene expression data point to an unexpected insulin-desensitizing effect of irisin.

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. 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. 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. 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.

Muscle-Derived Angiopoietin-Like Protein 4 Is Induced by Fatty Acids via Peroxisome Proliferator–Activated Receptor (PPAR)-δ and Is of Metabolic Relevance in Humans Harald Staiger 1 , Carina Haas 1 , Jürgen Machann 2 , Roman Werner 1 , Melanie Weisser 1 , Fritz Schick 2 , Fausto Machicao 1 , Norbert Stefan 1 , Andreas Fritsche 1 and Hans-Ulrich Häring 1 1 Department of Internal Medicine, Division of Endocrinology, Diabetology, Angiology, Nephrology, and Clinical Chemistry, Eberhard-Karls-University Tübingen, Tübingen, Germany 2 Department of Experimental Radiology, Eberhard-Karls-University Tübingen, Tübingen, Germany Corresponding author: Harald Staiger, harald.staiger{at}med.uni-tuebingen.de Abstract OBJECTIVE— Long-chain fatty acids (LCFAs) contribute to metabolic homeostasis in part via gene regulation. This study's objective was to identify novel LCFA target genes in human skeletal muscle cells (myotubes). RESEARCH DESIGN AND METHODS— In vitro methods included culture and treatment of human myotubes and C2C12 cells, gene array analysis, real-time RT-PCR, Western blotting, ELISA, chromatin immunoprecipitation, and RNA interference. Human subjects (two cohorts) were characterized by oral glucose tolerance test, hyperinsulinemic-euglycemic clamp, magnetic resonance imaging and spectroscopy, and standard blood analyses (glucose, insulin, C-peptide, and plasma lipids). RESULTS— We show here that ANGPTL4 (encoding angiopoietin-like protein 4) represents a prominent LCFA-responsive gene in human myotubes. LCFA activated peroxisome proliferator-activated receptor (PPAR)-δ, but not PPAR-α or -γ, and pharmacological activation of PPAR-δ markedly induced ANGPTL4 production and secretion. In C2C12 myocytes, knockdown of PPARD , but not of PPARG , blocked LCFA-mediated ANGPTL4 induction, and LCFA treatment resulted in PPAR-δ recruitment to the ANGPTL4 gene. In addition, pharmacological PPAR-δ activation induced LIPE (encoding hormone-sensitive lipase), and this response crucially depended on ANGPTL4, as revealed by ANGPTL4 knockdown. In a human cohort of 108 thoroughly phenotyped subjects, plasma ANGPTL4 positively correlated with fasting nonesterified fatty acids ( P = 0.0036) and adipose tissue lipolysis ( P = 0.0012). Moreover, in 38 myotube donors, plasma ANGPTL4 levels and adipose tissue lipolysis in vivo were reflected by basal myotube ANGPTL4 expression in vitro ( P = 0.02, both). CONCLUSIONS— ANGPTL4 is produced by human myotubes in response to LCFA via PPAR-δ, and muscle-derived ANGPTL4 seems to be of systemic relevance in humans. Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 15 December 2008. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted December 6, 2008. Received October 8, 2007. DIABETES

Individual Stearoyl-CoA Desaturase 1 Expression Modulates Endoplasmic Reticulum Stress and Inflammation in Human Myotubes and Is Associated With Skeletal Muscle Lipid Storage and Insulin Sensitivity In Vivo Andreas Peter 1 , Cora Weigert 1 , Harald Staiger 1 , Fausto Machicao 1 , Fritz Schick 2 , Jürgen Machann 2 , Norbert Stefan 1 , Claus Thamer 1 , Hans-Ulrich Häring 1 and Erwin Schleicher 1 1 Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology, and Clinical Chemistry, University of Tübingen, Tübingen, Germany; 2 Section of Experimental Radiology, University of Tübingen, Tübingen, Germany. Corresponding author: Andreas Peter, andreas.peter{at}med.uni-tuebingen.de . Abstract OBJECTIVE Increased plasma levels of free fatty acids occur in obesity and type 2 diabetes and contribute to the development of insulin resistance. Saturated fatty acids (SFAs) such as palmitate especially have lipotoxic effects leading to endoplasmatic reticulum (ER) stress, inflammation, and insulin resistance. Stearoyl-CoA desaturase 1 (SCD1) plays a key role in preventing lipotoxic effects, as it converts SFAs to less harmful monounsaturated fatty acids. Here, we tested the hypothesis that individual differences in the regulation of SCD1 expression by palmitate exist and influence insulin sensitivity and the cellular response to palmitate. RESEARCH DESIGN AND METHODS Palmitate-induced gene expression was studied in primary human myotubes of 39 metabolically characterized individuals, as well as in an SCD1-overexpressing cell culture model. RESULTS SCD1 mRNA expression and inducibility by palmitate in cultured myotubes showed a broad interindividual variation, presumably due to inheritable characteristics of the donors. Overexpression of SCD1 prevented the inflammatory and ER stress response to palmitate exposure. In primary human myotubes, high SCD1 inducibility was associated with a low inflammatory (interleukin [IL]-6, IL-8, and chemokine [CXC motif] ligand 3 [CXCL3]) and ER stress (CCAAT/enhancer binding protein [C/EBP] homologous protein, activating transcription factor 3 [ATF3], and X-box binding protein 1 [XBP1]) response to palmitate exposure. Finally, palmitate-stimulated SCD1 mRNA expression, positively correlated with intramyocellular lipid (IMCL) content of the donors, was measured by 1 H-magnetic resonance spectroscopy. After adjustment for IMCL, SCD1 expression and inducibility were positively correlated with insulin sensitivity. CONCLUSIONS We hypothesize that myocellular SCD1 inducibility by palmitate is an individual characteristic that modulates lipid storage, palmitate-induced inflammation, ER stress, and insulin resistance. This may describe individuals with increased capability of innoxious free fatty acid handling and benign triglyceride storage. Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received February 9, 2009. Accepted May 19, 2009. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. © 2009 by the American Diabetes Association.

Beta-cell apoptosis and failure to induce beta-cell regeneration are hallmarks of type 2-like diabetes in mouse models. Here we show that islets from obese, diabetes-susceptible New Zealand Obese (NZO) mice, in contrast to diabetes-resistant C57BL/6J (B6)-ob/ob mice, do not proliferate in response to an in-vivo glucose challenge but lose their beta-cells. Genome-wide RNAseq based transcriptomics indicated an induction of 22 cell cycle-associated genes in B6-ob/ob islets that did not respond in NZO islets. Of all genes differentially expressed in islets of the two strains, seven mapped to the diabesity QTL Nob3, and were hypomorphic in either NZO (Lefty1, Apoa2, Pcp4l1, Mndal, Slamf7, Pydc3) or B6 (Ifi202b). Adenoviral overexpression of Lefty1, Apoa2, and Pcp4l1 in primary islet cells increased proliferation, whereas overexpression of Ifi202b suppressed it. We conclude that the identified genes in synergy with obesity and insulin resistance participate in adaptive islet hyperplasia and prevention from severe diabetes in B6-ob/ob mice.

Dipeptidyl-peptidase 4 (DPP-4) cleaves and inactivates the insulinotropic hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide, collectively termed incretins. DPP-4 inhibitors entered clinical practice as approved therapeutics for type-2 diabetes in 2006. However, inter-individual variance in the responsiveness to DPP-4 inhibitors was reported. Thus, we asked whether genetic variation in the DPP4 gene affects incretin levels, insulin secretion, and glucose tolerance in participants of the TÜbingen Family study for type-2 diabetes (TÜF). Fourteen common (minor allele frequencies ≥0.05) DPP4 tagging single nucleotide polymorphisms (SNPs) were genotyped in 1,976 non-diabetic TÜF participants characterized by oral glucose tolerance tests and bioimpedance measurements. In a subgroup of 168 subjects, plasma incretin levels were determined. We identified a variant, i.e., SNP rs6741949, in intron 2 of the DPP4 gene that, after correction for multiple comparisons and appropriate adjustment, revealed a significant genotype-body fat interaction effect on glucose-stimulated plasma GLP-1 levels (p = 0.0021). Notably, no genotype-BMI interaction effects were detected (p = 0.8). After stratification for body fat content, the SNP negatively affected glucose-stimulated GLP-1 levels (p = 0.0229), insulin secretion (p = 0.0061), and glucose tolerance (p = 0.0208) in subjects with high body fat content only. A common variant, i.e., SNP rs6741949, in the DPP4 gene interacts with body adiposity and negatively affects glucose-stimulated GLP-1 levels, insulin secretion, and glucose tolerance. Whether this SNP underlies the reported inter-individual variance in responsiveness to DPP-4 inhibitors, at least in subjects with high body fat content, remains to be shown.

The role of free fatty acid receptor 1 (FFAR1/GPR40) in glucose homeostasis is still incompletely understood. Small receptor agonists stimulating insulin secretion are undergoing investigation for the treatment of type 2 diabetes. Surprisingly, genome-wide association studies did not discover diabetes risk variants in FFAR1. We reevaluated the role of FFAR1 in insulin secretion using a specific agonist, FFAR1-knockout mice and human islets. Nondiabetic individuals were metabolically phenotyped and genotyped. In vitro experiments indicated that palmitate and a specific FFAR1 agonist, TUG-469, stimulate glucose-induced insulin secretion through FFAR1. The proapoptotic effect of chronic exposure of β-cells to palmitate was independent of FFAR1. TUG-469 was protective, whereas inhibition of FFAR1 promoted apoptosis. In accordance with the proapoptotic effect of palmitate, in vivo cross-sectional observations demonstrated a negative association between fasting free fatty acids (NEFAs) and insulin secretion. Because NEFAs stimulate secretion through FFAR1, we examined the interaction of genetic variation in FFAR1 with NEFA and insulin secretion. The inverse association of NEFA and secretion was modulated by rs1573611 and became steeper for carriers of the minor allele. In conclusion, FFAR1 agonists support β-cell function, but variation in FFAR1 influences NEFA effects on insulin secretion and therefore could affect therapeutic efficacy of FFAR1 agonists.