Romy Kursawe

Hepatic Acetyl CoA Links Adipose Tissue Inflammation to Hepatic Insulin Resistance and Type 2 Diabetes

Impaired insulin-mediated suppression of hepatic glucose production (HGP) plays a major role in the pathogenesis of type 2 diabetes (T2D), yet the molecular mechanism by which this occurs remains unknown. Using a novel in vivo metabolomics approach, we show that the major mechanism by which insulin suppresses HGP is through reductions in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT) leading to reductions in pyruvate carboxylase flux. This mechanism was confirmed in mice and rats with genetic ablation of insulin signaling and mice lacking adipose triglyceride lipase. Insulins ability to suppress hepatic acetyl CoA, PC activity, and lipolysis was lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion. Taken together, these data identify WAT-derived hepatic acetyl CoA as the main regulator of HGP by insulin and link it to inflammation-induced hepatic insulin resistance associated with obesity and T2D.

SirT1 regulates adipose tissue inflammation.

OBJECTIVE Macrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation. RESEARCH DESIGN AND METHODS We used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects. RESULTS We found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration. CONCLUSIONS Reduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.

A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents†‡

The genetic factors associated with susceptibility to nonalcoholic fatty liver disease (NAFLD) in pediatric obesity remain largely unknown. Recently, a nonsynonymous single‐nucleotide polymorphism (rs738409), in the patatin‐like phospholipase 3 gene (PNPLA3) has been associated with hepatic steatosis in adults. In a multiethnic group of 85 obese youths, we genotyped the PNLPA3 single‐nucleotide polymorphism, measured hepatic fat content by magnetic resonance imaging and insulin sensitivity by the insulin clamp. Because PNPLA3 might affect adipogenesis/lipogenesis, we explored the putative association with the distribution of adipose cell size and the expression of some adipogenic/lipogenic genes in a subset of subjects who underwent a subcutaneous fat biopsy. Steatosis was present in 41% of Caucasians, 23% of African Americans, and 66% of Hispanics. The frequency of PNPLA3(rs738409) G allele was 0.324 in Caucasians, 0.183 in African Americans, and 0.483 in Hispanics. The prevalence of the G allele was higher in subjects showing hepatic steatosis. Surprisingly, subjects carrying the G allele showed comparable hepatic glucose production rates, peripheral glucose disposal rate, and glycerol turnover as the CC homozygotes. Carriers of the G allele showed smaller adipocytes than those with CC genotype (P = 0.005). Although the expression of PNPLA3, PNPLA2, PPARγ2(peroxisome proliferator‐activated receptor gamma 2), SREBP1c(sterol regulatory element binding protein 1c), and ACACA(acetyl coenzyme A carboxylase) was not different between genotypes, carriers of the G allele showed lower leptin (LEP)(P = 0.03) and sirtuin 1 (SIRT1) expression (P = 0.04). Conclusion: A common variant of the PNPLA3 gene confers susceptibility to hepatic steatosis in obese youths without increasing the level of hepatic and peripheral insulin resistance. The rs738409 PNPLA3 G allele is associated with morphological changes in adipocyte cell size. (HEPATOLOGY 2010.)

Glucose dysregulation and hepatic steatosis in obese adolescents: is there a link?

Fatty liver is increasingly common in obese adolescents. We determined its association with glucose dysregulation in 118 (37M/81F) obese adolescents of similar age and percent total fat. Fast‐magnetic resonance imaging (MRI) and simple MRI were used to quantify hepatic fat content and abdominal fat distribution. All subjects had a standard oral glucose tolerance test. Insulin sensitivity was estimated by the Matsuda Index and homeostasis model assessment of insulin resistance. Baseline total and high molecular weight (HMW)‐adiponectin and interleukin (IL)‐6 levels were measured. The cohort was stratified according to tertiles of hepatic fat content. Whereas age and %fat were comparable across tertiles, ethnicity differed in that fewer Blacks and more Whites and Hispanics were in the moderate and high category of hepatic fat fraction (HFF). Visceral and the visceral‐to‐subcutaneous fat ratio increased and insulin sensitivity decreased across tertiles. Two‐hour plasma glucose rose with increasing hepatic steatosis (P < 0.008). 73.7% of the subjects in the high HFF had the metabolic syndrome compared to 19.5% and 30.6%, respectively, in the low and moderate categories. Both total and HMW‐adiponectin decreased, and IL‐6 increased with increasing hepatic steatosis. Conclusion: In obese adolescents, independent of total fat, increasing severity of fatty liver is associated with glucose dysregulation, metabolic syndrome, and with a proinflammatory milieu. (HEPATOLOGY 2009.)

Variant in the glucokinase regulatory protein (GCKR) gene is associated with fatty liver in obese children and adolescents

Recently, the single nucleotide polymorphism (SNP) identified as rs1260326, in the glucokinase regulatory protein (GCKR), was associated with hypertriglyceridemia in adults. Because accumulation of triglycerides in hepatocytes represents the hallmark of steatosis, we aimed to investigate whether this variant might be associated with fatty liver (hepatic fat content, HFF%). Moreover, because recently rs738409 in the PNPLA3 and rs2854116 in the APOC3 were associated with fatty liver, we explored how the GCKR SNP and these two variants jointly influence hepatosteatosis. We studied 455 obese children and adolescents (181 Caucasians, 139 African Americans, and 135 Hispanics). All underwent an oral glucose tolerance test and fasting lipoprotein subclasses measurement by proton nuclear magnetic resonance. A subset of 142 children underwent a fast gradient magnetic resonance imaging to measure the HFF%. The rs1260326 was associated with elevated triglycerides (Caucasians P = 0.00014; African Americans P = 0.00417), large very low‐density lipoprotein (VLDL) (Caucasians P = 0.001; African Americans, P = 0.03), and with fatty liver (Caucasians P = 0.034; African Americans P = 0.00002; and Hispanics P = 0.016). The PNPLA3, but not the APOC3 rs2854116 SNP, was associated with fatty liver but not with triglyceride levels. There was a joint effect between the PNPLA3 and GCKR SNPs, explaining 32% of HFF% variance in Caucasians (P = 0.00161), 39.0% in African Americans (P = 0.00000496), and 15% in Hispanics (P = 0.00342). Conclusion: The rs1260326 in GCKR is associated with hepatic fat accumulation along with large VLDL and triglyceride levels. GCKR and PNPLA3 act together to convey susceptibility to fatty liver in obese youths. (Hepatology 2012)

Deletion of the α-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity

OBJECTIVE Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis. Here, we tested the hypothesis that Txnip-null (knockout) mice are protected from insulin resistance induced by a high-fat diet. RESEARCH DESIGN AND METHODS Txnip gene-deleted (knockout) mice and age-matched wild-type littermate control mice were maintained on a standard chow diet or subjected to 4 weeks of high-fat feeding. Mice were assessed for body composition, fat development, energy balance, and insulin responsiveness. Adipogenesis was measured from ex vivo fat preparations, and in mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes after forced manipulation of Txnip expression. RESULTS Txnip knockout mice gained significantly more adipose mass than controls due to a primary increase in both calorie consumption and adipogenesis. Despite increased fat mass, Txnip knockout mice were markedly more insulin sensitive than controls, and augmented glucose transport was identified in both adipose and skeletal muscle. RNA interference gene-silenced preadipocytes and Txnip−/− MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation. As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator–activated receptor-γ (PPARγ) response, we investigated Txnips regulation of PPARγ function; manipulation of Txnip expression directly regulated PPARγ expression and activity. CONCLUSIONS Txnip deletion promotes adiposity in the face of high-fat caloric excess; however, loss of this α-arrestin protein simultaneously enhances insulin responsiveness in fat and skeletal muscle, revealing Txnip as a novel mediator of insulin resistance and a regulator of adipogenesis.

Cellularity and Adipogenic Profile of the Abdominal Subcutaneous Adipose Tissue From Obese Adolescents: Association With Insulin Resistance and Hepatic Steatosis

OBJECTIVE We explored whether the distribution of adipose cell size, the estimated total number of adipose cells, and the expression of adipogenic genes in subcutaneous adipose tissue are linked to the phenotype of high visceral and low subcutaneous fat depots in obese adolescents. RESEARCH DESIGN AND METHODS A total of 38 adolescents with similar degrees of obesity agreed to have a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test and hyperinsulinemic euglycemic clamp) and imaging studies (MRI, DEXA, 1H-NMR). Subcutaneous periumbilical adipose cell-size distribution and the estimated total number of subcutaneous adipose cells were obtained from tissue biopsy samples fixed in osmium tetroxide and analyzed by Beckman Coulter Multisizer. The adipogenic capacity was measured by Affymetrix GeneChip and quantitative RT-PCR. RESULTS Subjects were divided into two groups: high versus low ratio of visceral to visceral + subcutaneous fat (VAT/[VAT+SAT]). The cell-size distribution curves were significantly different between the high and low VAT/(VAT+SAT) groups, even after adjusting for age, sex, and ethnicity (MANOVA P = 0.035). Surprisingly, the fraction of large adipocytes was significantly lower (P < 0.01) in the group with high VAT/(VAT+SAT), along with the estimated total number of large adipose cells (P < 0.05), while the mean diameter was increased (P < 0.01). From the microarray analyses emerged a lower expression of lipogenesis/adipogenesis markers (sterol regulatory element binding protein-1, acetyl-CoA carboxylase, fatty acid synthase) in the group with high VAT/(VAT+SAT), which was confirmed by RT-PCR. CONCLUSIONS A reduced lipo-/adipogenic capacity, fraction, and estimated number of large subcutaneous adipocytes may contribute to the abnormal distribution of abdominal fat and hepatic steatosis, as well as to insulin resistance in obese adolescents.

Reversal of Early Abnormalities in Glucose Metabolism in Obese Youth: Results of an Intensive Lifestyle Randomized Controlled Trial

OBJECTIVE The childhood obesity epidemic has been accompanied by an increasing prevalence of type 2 diabetes (T2D), particularly in minority children. Twenty to thirty percent of obese youth have “prediabetes,” a precursor to diabetes marked by insulin resistance, β-cell dysfunction, and impaired glucose tolerance. The Diabetes Prevention Program demonstrated that T2D could be prevented/delayed by intensive lifestyle modification in adults with prediabetes, but efficacy of similar interventions in youth has not been established. Therefore, we evaluated the effects of the Bright Bodies (BB) Healthy Lifestyle Program on 2-h oral glucose tolerance test (OGTT) glucose in comparison with adolescents receiving standard of care. RESEARCH DESIGN AND METHODS A parallel-group randomized controlled trial comparing BB with standard clinical care (CC) in obese adolescents (10–16 years old, Tanner stage >2) with elevated OGTT 2-h blood glucose (130–199 mg/dL) from a racially/ethnically diverse population. OGTTs, including cardiovascular and anthropometric assessments, were conducted at baseline and 6 months. Children attended BB twice per week for exercise and nutrition/behavior modification, and the CC group received CC from their pediatrician. Primary outcome was change in 2-h OGTT glucose and percentage conversion from elevated 2-h blood glucose to nonelevated (<130 mg/dL) 2-h blood glucose. Changes in outcomes were compared between groups using an ANCOVA, with adjustment for baseline outcome and multiple imputation for missing data. RESULTS Reductions in 2-h glucose were more favorable in BB compared with CC (−27.2 vs. −10.1 mg/dL; difference = −17.1, 95% CI; P = 0.005). Moreover, greater conversion to <130 mg/dL 2-h glucose occurred in BB than CC (P = 0.003), and other insulin sensitivity indices were significantly improved. CONCLUSIONS Compared with standard of care, the Yale BB Program is a more effective means of reducing the risk of T2D in obese adolescents with elevated 2-h glucose levels.

Decreased Transcription of ChREBP-α/β Isoforms in Abdominal Subcutaneous Adipose Tissue of Obese Adolescents With Prediabetes or Early Type 2 Diabetes Associations With Insulin Resistance and Hyperglycemia

Insulin resistance associated with altered fat partitioning in liver and adipose tissues is a prediabetic condition in obese adolescents. We investigated interactions between glucose tolerance, insulin sensitivity, and the expression of lipogenic genes in abdominal subcutaneous adipose and liver tissue in 53 obese adolescents. Based on their 2-h glucose tests they were stratified in the following groups: group 1, 2-h glucose level <120 mg/dL; group 2, 2-h glucose level between 120 and 140 mg/dL; and group 3, 2-h glucose level >140 mg/dL. Liver and adipose tissue insulin sensitivity were greater in group 1 than in group 2 and group 3, and muscle insulin sensitivity progressively decreased from group 1 to group 3. The expression of the carbohydrate-responsive element-binding protein (ChREBP) was decreased in adipose tissue but increased in the liver (eight subjects) in adolescents with impaired glucose tolerance or type 2 diabetes. The expression of adipose ChREBPα and ChREBPβ was inversely related to 2-h glucose level and positively correlated to insulin sensitivity. Improvement of glucose tolerance in four subjects was associated with an increase of ChREBP/GLUT4 expression in the adipose tissue. In conclusion, early in the development of prediabetes/type 2 diabetes in youth, ChREBPβ expression in adipose tissue predicts insulin resistance and, therefore, might play a role in the regulation of glucose tolerance.

Circulating Levels of FGF-21 in Obese Youth: Associations With Liver Fat Content and Markers of Liver Damage

OBJECTIVE Fibroblast growth factor (FGF)-21 is highly expressed in the liver and regulates glucose and lipid metabolism in rodents. The effects of obesity and fatty liver on circulating FGF-21 levels have been described mainly in adults. Herein, we measured plasma FGF-21 levels in lean and obese adolescents with low and high hepatic fat content (HFF% <5.5% and HFF% ≥ 5.5%, respectively) and explored their relationship with hepatic fat content, measures of hepatic apoptosis, and insulin sensitivity. METHODS A total of 217 lean and obese adolescents with both low and high HFF% (lean = 31; obese low HFF% = 107; and obese high HFF% = 79) underwent an oral glucose tolerance test, a fast gradient magnetic resonance imaging to measure the %HFF and abdominal fat distribution. Cytokeratin 18 levels were measured as a biomarker of liver apoptosis. A subset of adolescents underwent a 2-step hyperinsulinemic-euglycemic clamp, and a liver biopsy (N = 14), to assess insulin sensitivity and steatohepatitis, respectively. RESULTS Compared to controls, FGF-21 levels were higher in obese youth, especially in those with high HFF (P < .001). FGF-21 significantly correlated with adiposity indexes (P < .001), visceral fat (r² = 0.240, P < .001), hepatic fat content (r² = 0.278, P < .001), cytokeratin 18 (r² = 0.217, P < .001), and alanine aminotransferase (r² = .164, P < .001). In subjects with steatoheaptitis, FGF-21 levels significantly correlated with the nonalcoholic fatty liver disease activity score (r² = 0.27, P = .04). Stepwise regression analysis indicated that these relationships are independent of body mass index, visceral fat, and insulin sensitivity. An inverse correlation was documented with insulin, hepatic resistance indexes, and adipose resistance indexes, which disappeared after adjusting for hepatic fat content. CONCLUSIONS Plasma FGF-21 levels are increased in obese adolescents, particularly in those with fatty liver. FGF-21 concentrations significantly and independently correlate with hepatic fat content and markers of hepatic apoptosis in obese youths.