Ashis K. Mondal

Endoplasmic Reticulum Stress Markers Are Associated with Obesity in Nondiabetic Subjects

OBJECTIVE Adipocyte and hepatocyte endoplasmic reticulum (ER) stress response is activated in dietary and genetic models of obesity in mice. We hypothesized that ER stress was also activated and associated with reduced insulin sensitivity (SI) in human obesity. RESEARCH DESIGN AND METHODS We recruited 78 healthy, nondiabetic individuals over a spectrum of body mass index (BMI) who underwent oral and iv glucose tolerance tests, and fasting sc adipose and muscle biopsies. We tested expression of 18 genes and levels of total and phosphorylated eukaryotic initiation factor 2alpha, c-jun, and c-Jun N-terminal kinase 1 in adipose tissue. We compared gene expression in stromal vascular and adipocyte fractions in paired samples from 22 individuals, and tested clustering on gene and protein markers. RESULTS Adipocyte expression of most markers of ER stress, including chaperones downstream of activating transcription factor 6, were significantly correlated with BMI and percent fat (r>0.5; P<0.00001). Phosphorylation of eukaryotic initiation factor 2alpha but not of c-Jun N-terminal kinase 1 or c-jun was increased with obesity. ER stress response (as elsewhere) was also increased with obesity in a second set of 86 individuals, and in the combined sample (n=161). The increase was only partially attributable to the stromal vascular fraction and macrophage infiltration. ER stress markers were only modestly correlated with S(I). Clustering algorithms supported ER stress activation with high BMI but not low SI. CONCLUSIONS Multiple markers of ER stress are activated in human adipose with obesity, particularly for protective chaperones downstream of activating transcription factor 6alpha.

Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class.

From the fruitfly, Drosophila melanogaster, ten members of the cluster of Delta-class glutathione S-transferases (GSTs; formerly denoted as Class I GSTs) and one member of the Epsilon-class cluster (formerly GST-3) have been cloned, expressed in Escherichia coli, and their catalytic properties have been determined. In addition, nine more members of the Epsilon cluster have been identified through bioinformatic analysis but not further characterized. Of the 11 expressed enzymes, seven accepted the lipid peroxidation product 4-hydroxynonenal as substrate, and nine were active in glutathione conjugation of 1-chloro-2,4-dinitrobenzene. Since the enzymically active proteins included the gene products of DmGSTD3 and DmGSTD7 which were previously deemed to be pseudogenes, we investigated them further and determined that both genes are transcribed in Drosophila. Thus our present results indicate that DmGSTD3 and DmGSTD7 are probably functional genes. The existence and multiplicity of insect GSTs capable of conjugating 4-hydroxynonenal, in some cases with catalytic efficiencies approaching those of mammalian GSTs highly specialized for this function, indicates that metabolism of products of lipid peroxidation is a highly conserved biochemical pathway with probable detoxification as well as regulatory functions.

Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipose and in palmitate-induced stress in human liver and adipose cell lines

Obesity and elevated cytokine secretion result in a chronic inflammatory state and may cause the insulin resistance observed in type 2 diabetes. Recent studies suggest a key role for endoplasmic reticulum stress in hepatocytes and adipocytes from obese mice, resulting in reduced insulin sensitivity. To address the hypothesis that thiazolidinediones, which improve peripheral insulin sensitivity, act in part by reducing the endoplasmic reticulum stress response, we tested subcutaneous adipose tissue from 20 obese volunteers treated with pioglitazone for 10 wk. We also experimentally induced endoplasmic reticulum stress using palmitate, tunicamycin, and thapsigargin in the human HepG2 liver cell line with or without pioglitazone pretreatment. We quantified endoplasmic reticulum stress response by measuring both gene expression and phosphorylation. Pioglitazone significantly improved insulin sensitivity in human volunteers (P = 0.002) but did not alter markers of endoplasmic reticulum stress. Differences in pre- and posttreatment endoplasmic reticulum stress levels were not correlated with changes in insulin sensitivity or body mass index. In vitro, palmitate, thapsigargin, and tunicamycin but not oleate induced endoplasmic reticulum stress in HepG2 cells, including increased transcripts CHOP, ERN1, GADD34, and PERK, and increased XBP1 splicing along with phosphorylation of eukaryotic initiation factor eIF2alpha, JNK1, and c-jun. Although patterns of endoplasmic reticulum stress response differed among palmitate, tunicamycin, and thapsigargin, pioglitazone pretreatment had no significant effect on any measure of endoplasmic reticulum stress, regardless of the inducer. Together, our data suggest that improved insulin sensitivity with pioglitazone is not mediated by a reduction in endoplasmic reticulum stress.

An Integrative Genomics Approach Identifies Activation of Thioredoxin/Thioredoxin Reductase-1-Mediated Oxidative Stress Defense Pathway and Inhibition of Angiogenesis in Obese Nondiabetic Human Subjects

CONTEXT Obesity is a complex disease that involves both genetic and environmental perturbations to gene networks in adipose tissue and is proposed as a trigger for metabolic sequelae. OBJECTIVE We hypothesized that expression of adipose tissue transcripts in gene networks for adaptive response would correlate with the percent fat mass (PFAT) in healthy nondiabetic subjects to maintain metabolic equilibrium and would overlap with genes modulated in response to elevated fatty acid. DESIGN, SETTINGS, AND PATIENTS Genome-wide transcript profiles were determined in sc adipose tissue of 136 nondiabetics and in palmitate-induced cells. Genotype information and gene expression data in nondiabetic subjects were integrated to characterize the function of 41 obesity-associated polymorphisms. RESULTS Genes involved in inflammation-immune response, endoplasmic reticulum stress, and cell-extracellular matrix interactions were significantly correlated with PFAT. The NRF2 (nuclear factor erythroid 2-related factor-2)-mediated oxidative stress response pathway was strongly enriched among genes correlated with PFAT in adipose and also emerged as the most enriched pathway among genes differentially expressed by palmitate in vitro. Thioredoxin reductase-1 (TXNRD1) was the most strongly correlated gene (ρ = 0.65). Genes coregulated with TXNRD1 expression indicated a significant interaction network of genes involved in thioredoxin-mediated oxidative stress defense mechanisms and angiogenesis. Pro- and antiangiogenic factors were negatively and positively correlated, respectively, with obesity. Eight obesity genome-wide association study single-nucleotide polymorphisms (SNP) were associated with expression of 10 local transcripts. SNP rs6861681 was the strongest cis-eQTL (expression quantitative trait loci) for CPEB4 (P = 3.02 × 10⁻⁹). CONCLUSIONS Our study suggests a novel interaction of up-regulated TXN-TXNRD1 system-mediated oxidative stress defense mechanisms and down-regulated angiogenesis pathways as an adaptive response in obese nondiabetic subjects. A subset of obesity-associated SNP regulated expression of transcripts as cis-eQTL.

Effect of Endoplasmic Reticulum Stress on Inflammation and Adiponectin Regulation in Human Adipocytes

The endoplasmic reticulum (ER) of adipocytes plays a major role in the assembly and secretion of adipokines. The levels of serum adiponectin, secreted by adipocytes, are decreased in insulin resistance, diabetes, and obesity. The role of ER stress in downregulating adiponectin levels has been demonstrated in mouse models of obesity. Studies examining human adipose tissue have indicated that there is an increase in the ER stress transcript HSPA5 with increased body mass index (BMI). However, it is not established whether ER stress results in changes in adiponectin levels or multimerization in human adipocytes. We examined whether the induction of ER stress using tunicamycin, thapsigargin, or palmitate alters the messenger RNA (mRNA) and protein expression of adiponectin and the mRNA expression of chaperones ERP44 and ERO1 in adult-derived human adipocyte stem (ADHAS) cells. ER stress was measured using key indicators of ER stress-HSPA5, ERN1, CHOP, and GADD34, as well as changes in eIF2α phosphorylation. Because ER stress is suggested to be the proximal cause of inflammation in adipocytes, we further examined the change in inflammatory status by quantitating the change in Iκβ-α protein following the induction of ER stress. Our studies indicate that: (1) ER stress markers were increased to a higher degree using tunicamycin or thapsigargin compared to palmitate; (2) ER stress significantly decreased adiponectin mRNA in response to tunicamycin and thapsigargin, but palmitate did not decrease adiponectin mRNA levels. In all three instances, the induction of ER stress was accompanied by a decrease in adiponectin protein as well as adiponectin multimerization. All three inducers of ER stress increased tumor necrosis factor-α (TNF-α) mRNA and decreased Iκβ-α protein in adipocytes. The data suggest that ER stress modifies adiponectin secretion and induces inflammation in ADHAS cells.

Distinct gene expression profiles characterize cellular responses to palmitate and oleate.

Obese individuals are both insulin resistant and have high levels of circulating free fatty acids (FFAs). In cell culture, saturated but not unsaturated fatty acids induce endoplasmic reticulum (ER) stress. We hypothesized that chronic exposure to low dose fatty acids would significantly attenuate the acute stress response to a saturated fatty acid challenge and that unsaturated fatty acids (oleate) would be more protective than saturated fatty acids (palmitate). The ER stress response to palmitate was reduced after low dose fatty acid exposure in human hepatoma cells. Palmitate and oleate gave distinctive transcript responses, both acutely and after chronic low dose exposure. Differentially regulated pathways included lipid, cholesterol, fatty acid, and triglyceride metabolism, and IκB kinase and nuclear factor κB kinase inflammatory cascades. Oleate reduced palmitate-induced changes significantly more than low dose palmitate and completely blocked palmitate-induced phosphoinositide 3 kinase inhibitor (PIK3IP1) as well as induction of GADD45A and B. These changes are predicted to alter the PI3 kinase pathway and the pro-apoptotic p38 MAPK pathway. We recapitulated the oleate response by small interfering RNA-mediated block of PIK3IP1 stimulation with palmitate and significantly protected cells from palmitate-mediated ER stress. We show that transcriptional responses to oleate and palmitate are distinct, broad, and often discordant. We identified several potential candidates that may direct the transcriptional networks and demonstrate that PIK3IP1 partially accounts for the protective effects of oleate.

Type 2 diabetes (T2D) associated polymorphisms regulate expression of adjacent transcripts in transformed lymphocytes, adipose, and muscle from Caucasian and African-American subjects.

CONTEXT Genome-wide association scans (GWAS) have identified novel single nucleotide polymorphisms (SNPs) that increase T2D susceptibility and indicated the role of nearby genes in T2D pathogenesis. OBJECTIVE We hypothesized that T2D-associated SNPs act as cis-regulators of nearby genes in human tissues and that expression of these transcripts may correlate with metabolic traits, including insulin sensitivity (S(I)). DESIGN, SETTINGS, AND PATIENTS Association of SNPs with the expression of their nearest transcripts was tested in adipose and muscle from 168 healthy individuals who spanned a broad range of S(I) and body mass index (BMI) and in transformed lymphocytes (TLs). We tested correlations between the expression of these transcripts in adipose and muscle with metabolic traits. Utilizing allelic expression imbalance (AEI) analysis we examined the presence of other cis-regulators for those transcripts in TLs. RESULTS SNP rs9472138 was significantly (P = 0.037) associated with the expression of VEGFA in TLs while rs6698181 was detected as a cis-regulator for the PKN2 in muscle (P = 0.00027) and adipose (P = 0.018). Significant association was also observed for rs17036101 (P = 0.001) with expression of SYN2 in adipose of Caucasians. Among 19 GWAS-implicated transcripts, expression of VEGFA in adipose was correlated with BMI (r = -0.305) and S(I) (r = 0.230). Although only a minority of the T2D-associated SNPs were validated as cis-eQTLs for nearby transcripts, AEI analysis indicated presence of other cis-regulatory polymorphisms in 54% of these transcripts. CONCLUSIONS Our study suggests that a small subset of GWAS-identified SNPs may increase T2D susceptibility by modulating expression of nearby transcripts in adipose or muscle.

The Effect of ACACB cis-Variants on Gene Expression and Metabolic Traits

Background Acetyl Coenzyme A carboxylase β (ACACB) is the rate-limiting enzyme in fatty acid oxidation, and continuous fatty acid oxidation in Acacb knock-out mice increases insulin sensitivity. Systematic human studies have not been performed to evaluate whether ACACB variants regulate gene expression and insulin sensitivity in skeletal muscle and adipose tissues. We sought to determine whether ACACB transcribed variants were associated with ACACB gene expression and insulin sensitivity in non-diabetic African American (AA) and European American (EA) adults. Methods ACACB transcribed single nucleotide polymorphisms (SNPs) were genotyped in 105 EAs and 46 AAs whose body mass index (BMI), lipid profiles and ACACB gene expression in subcutaneous adipose and skeletal muscle had been measured. Allelic expression imbalance (AEI) was assessed in lymphoblast cell lines from heterozygous subjects in an additional EA sample (n = 95). Selected SNPs were further examined for association with insulin sensitivity in a cohort of 417 EAs and 153 AAs. Results ACACB transcribed SNP rs2075260 (A/G) was associated with adipose ACACB messenger RNA expression in EAs and AAs (p = 3.8×10−5, dominant model in meta-analysis, Stouffer method), with the (A) allele representing lower gene expression in adipose and higher insulin sensitivity in EAs (p = 0.04). In EAs, adipose ACACB expression was negatively associated with age and sex-adjusted BMI (r = −0.35, p = 0.0002). Conclusions Common variants within the ACACB locus appear to regulate adipose gene expression in humans. Body fat (represented by BMI) may further regulate adipose ACACB gene expression in the EA population.

Phospholipid Biosynthesis Genes and Susceptibility to Obesity: Analysis of Expression and Polymorphisms

Recent studies have identified links between phospholipid composition and altered cellular functions in animal models of obesity, but the involvement of phospholipid biosynthesis genes in human obesity are not well understood. We analyzed the transcript of four phospholipid biosynthesis genes in adipose and muscle from 170 subjects. We examined publicly available genome-wide association data from the GIANT and MAGIC cohorts to investigate the association of SNPs in these genes with obesity and glucose homeostasis traits, respectively. Trait-associated SNPs were genotyped to evaluate their roles in regulating expression in adipose. In adipose tissue, expression of PEMT, PCYT1A, and PTDSS2 were positively correlated and PCYT2 was negatively correlated with percent fat mass and body mass index (BMI). Among the polymorphisms in these genes, SNP rs4646404 in PEMT showed the strongest association (p = 3.07E-06) with waist-to-hip ratio (WHR) adjusted for BMI. The WHR-associated intronic SNP rs4646343 in the PEMT gene showed the strongest association with its expression in adipose. Allele “C” of this SNP was associated with higher WHR (p = 2.47E-05) and with higher expression (p = 4.10E-04). Our study shows that the expression of PEMT gene is high in obese insulin-resistant subjects. Intronic cis-regulatory polymorphisms may increase the genetic risk of obesity by modulating PEMT expression.

An ACACB Variant Implicated in Diabetic Nephropathy Associates with Body Mass Index and Gene Expression in Obese Subjects

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m2 (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m2 (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m2; p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m2 for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN.