Melanie Weisser

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

OBJECTIVE— Long-chain fatty acids (LCFAs) contribute to metabolic homeostasis in part via gene regulation. This studys 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.

The tumour necrosis factor alpha –238 G → A and –308 G → A promoter polymorphisms are not associated with insulin sensitivity and insulin secretion in young healthy relatives of Type II Diabetic patients

Aims/hypothesis. Tumour necrosis factor-α (TNF-α) is believed to influence skeletal muscle insulin resistance. Two G → A transitions in the promoter region of TNF-α at position –238 and –308 have been identified that could play a part in transcriptional regulation of the gene. Insulin resistance is an independent familial trait that predicts the development of Type II (non-insulin-dependent) diabetes mellitus. We investigated the influence on insulin sensitivity and insulin secretion of both polymorphisms in a cohort of young healthy relatives of patients with Type II diabetes.¶Methods. We examined 109 first-degree relatives of Caucasian patients with a history of Type II diabetes, who underwent extensive metabolical and anthropometrical phenotyping, and determined the TNF-α–238 and –308 G→ A promoter polymorphisms.¶Results. For the –238 polymorphism, 83 probands (76.1 %) were homozygous for the G-allele, 25 probands (22.9 %) were heterozygous and 1 proband (0.9 %) was homozygous for the A-allele. For the –308 polymorphism, 83 probands (76.1 %) were homozygous for the G-allele, 24 probands (22.0 %) were heterozygous and 2 probands (1.18 %) were homozygous for the A-allele. Probands with and without the polymorphism did not differ in insulin sensitivity (p = 0.78), insulin-concentrations and C-peptide concentrations in oral glucose tolerance tests (p > 0.05).¶Conclusions/interpretation. We could not detect an association between insulin sensitivity or insulin secretion and TNF-α promoter polymorphisms in our cohort. The polymorphisms occur at the same frequencies in probands with either low or high insulin sensitivity. [Diabetologia (2000) 43: 181–184]

Induction of adiponectin gene expression in human myotubes by an adiponectin-containing HEK293 cell culture supernatant

Aims/hypothesisAdiponectin, an adipocytokine known to be down-regulated in obesity-linked disorders, is considered to be a potential key mediator of insulin sensitivity. In this study, we asked whether adiponectin is able to regulate ten selected genes possibly associated with insulin sensitivity in human skeletal muscle cells.MethodsTo this end, we treated in vitro differentiated human myotubes with the culture supernatant of HEK293 cells stably transfected with human recombinant adiponectin and assessed gene expression by RT-PCR. Intracellular adiponectin protein was quantified by radioimmunoassay and visualized by Western blotting.ResultsIn contrast to the control supernatant, the adiponectin-containing supernatant consistently induced expression of adiponectin mRNA in human myotubes from eight different donors (mean increase: 90-fold over control; n=8, p<0.001). This increase in mRNA was paralleled by a rise in intracellular adiponectin protein (mean increase: 8.3-fold over control; n=4, p<0.05). Expression of the other nine candidate genes was not altered. In human skin fibroblasts and HepG2 cells, the adiponectin-enriched supernatant did not induce relevant amounts of adiponectin mRNA.Conclusions/interpretationIn conclusion, we show here that adiponectin gene expression is specifically inducible in skeletal muscle cells.

Genetic variation within the ANGPTL4 gene is not associated with metabolic traits in white subjects at an increased risk for type 2 diabetes mellitus

Angiopoietin-like protein 4 (ANGPTL4) represents an adipokine with metabolic effects within adipose tissue, such as inhibition of lipoprotein lipase activity and stimulation of lipolysis. These effects were convincingly demonstrated in mice. Therefore, we asked whether genetic variation within the ANGPTL4 gene contributes to prediabetic phenotypes, such as dyslipidemia, insulin resistance, or beta-cell dysfunction, in white subjects at an increased risk for type 2 diabetes mellitus. We genotyped 629 subjects with and without a family history of diabetes for the 4 single nucleotide polymorphisms (SNPs) rs4076317, rs2278236, rs1044250, and rs11672433 and performed correlational analyses with metabolic traits. For metabolic characterization, all subjects underwent an oral glucose tolerance test; a subset was additionally characterized by hyperinsulinemic-euglycemic clamp. The 4 SNPs rs4076317, rs2278236, rs1044250, and rs11672433 cover 100% of common genetic variation (minor allele frequency>or=0.05) within the ANGPTL4 gene (r2>or=0.8). None of these SNPs revealed significant correlation with anthropometric data (sex, age, body mass index, body fat, and waist-hip ratio) or with family history of diabetes. Furthermore, no reliable correlations were found with fasting triglycerides, fasting nonesterified fatty acids, and area under the curve of nonesterified fatty acids during oral glucose tolerance test or with parameters of insulin sensitivity and insulin secretion. Finally, haplotype analysis revealed the existence of 8 common diplotypes. None of these, however, was significantly correlated with insulin sensitivity, insulin secretion, or plasma lipid measures. We conclude that common genetic variation within the ANGPTL4 gene may not play a major role in the development of prediabetic phenotypes in our white population.