Paska A. Permana

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Adipogenic Human Adenovirus Ad-36 Induces Commitment, Differentiation, and Lipid Accumulation in Human Adipose-Derived Stem Cells

Human adenovirus Ad‐36 is causatively and correlatively linked with animal and human obesity, respectively. Ad‐36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad‐36‐induced adipogenesis in human obesity, the effect of the virus on commitment, differentiation, and lipid accumulation was investigated in vitro in primary human adipose‐derived stem/stromal cells (hASC). Ad‐36 infected hASC in a time‐ and dose‐dependent manner. Even in the presence of osteogenic media, Ad‐36‐infected hASC showed significantly greater lipid accumulation, suggestive of their commitment to the adipocyte lineage. Even in the absence of adipogenic inducers, Ad‐36 significantly increased hASC differentiation, as indicated by a time‐dependent expression of genes within the adipogenic cascade—CCAAT/Enhancer binding protein‐β, peroxisome proliferator‐activated receptor‐γ, and fatty acid‐binding protein—and consequentially increased lipid accumulation in a time‐ and viral dose‐dependent manner. Induction of hASC to the adipocyte state by Ad‐36 was further supported by increased expression of lipoprotein lipase and the accumulation of its extracellular fraction. hASC from subjects harboring Ad‐36 DNA in their adipose tissue due to natural infection had significantly greater ability to differentiate compared with Ad‐36 DNA‐negative counterparts, which offers a proof of concept. Thus, Ad‐36 has the potential to induce adipogenesis in hASC, which may contribute to adiposity induced by the virus.

Amyloid precursor protein expression is upregulated in adipocytes in obesity.

The aim of this study was to determine whether amyloid precursor protein (APP) is expressed in human adipose tissue, dysregulated in obesity, and related to insulin resistance and inflammation. APP expression was examined by microarray expression profiling of subcutaneous abdominal adipocytes (SAC) and cultured preadipocytes from obese and nonobese subjects. Quantitative real‐time PCR (QPCR) was performed to confirm differences in APP expression in SAC and to compare APP expression levels in adipose tissue, adipocytes, and stromal vascular cells (SVCs) from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) specimens. Adipose tissue samples were also examined by western blot and immunofluorescence confocal microscopy. Microarray studies demonstrated that APP mRNA expression levels were higher in SAC (∼2.5‐fold) and preadipocytes (∼1.4) from obese subjects. Real‐time PCR confirmed increased APP expression in SAC in a separate group of obese compared with nonobese subjects (P = 0.02). APP expression correlated to in vivo indices of insulin resistance independently of BMI and with the expression of proinflammatory genes, such as monocyte chemoattractant protein‐1 (MCP‐1) (R = 0.62, P = 0.004), macrophage inflammatory protein‐1α (MIP‐1α) (R = 0.60, P = 0.005), and interleukin‐6 (IL‐6) (R = 0.71, P = 0.0005). Full‐length APP protein was detected in adipocytes by western blotting and APP and its cleavage peptides, Aβ40 and Aβ42, were observed in SAT and VAT by immunofluorescence confocal microscopy. In summary, APP is highly expressed in adipose tissue, upregulated in obesity, and expression levels correlate with insulin resistance and adipocyte cytokine expression levels. These data suggest a possible role for APP and/or Aβ in the development of obesity‐related insulin resistance and adipose tissue inflammation.

Interaction Between an 11βHSD1 Gene Variant and Birth Era Modifies the Risk of Hypertension in Pima Indians

11&bgr;-Hydroxysteroid dehydrogenase type 1 (11&bgr;HSD1) is a candidate gene for hypertension, diabetes, and obesity through altered glucocorticoid production. This study explored the association of 11&bgr;HSD1 gene variants with diabetes, hypertension, and obesity in a longitudinal population study of American Indians (N=918; exams=5508). In multivariate mixed models assuming an additive effect of genotype, a 5′ upstream variant (rs846910) was associated with blood pressure (diastolic blood pressure &bgr;=1.58 mm Hg per copy of the A allele, P=0.0008; systolic blood pressure &bgr;=2.28 mm Hg per copy of the A allele, P=0.004; mean arterial blood pressure &bgr;=1.83 mm Hg per copy of the A allele, P=0.0006) and hypertension (odds ratio=1.27 per copy of the A allele, P=0.02). However, birth date modified these associations (test for interaction: diastolic blood pressure P=0.16; systolic blood pressure P=0.007; mean arterial blood pressure P=0.01), such that the magnitude and direction of association between genotype and blood pressure changed with time. Finally, in models controlling for potential confounding by population stratification, we observed evidence of within-family effects for blood pressure (diastolic blood pressure &bgr;=1.77 mm Hg per copy of the A allele, P=0.004; systolic blood pressure &bgr;=2.04 mm Hg per copy of the A allele, P=0.07; mean arterial blood pressure &bgr;=1.85 mm Hg per copy of the A allele, P=0.01) and for hypertension (odds ratio=1.26 per copy of the A allele; P=0.08). No association was observed for obesity. Associations with diabetes were similar in magnitude as reported previously but were not statistically significant. These data demonstrate association between genetic variability at 11&bgr;HSD1 with hypertension, but these effects are modified by environmental factors.

Identification of novel putative membrane proteins selectively expressed during adipose conversion of 3T3-L1 cells

Fat tissue plays a critical role in the regulation of energy metabolism. Here we report the proteomic identification of a novel _fa_t tissue-specific _l_ow molecular weight _p_rotein (Falp) which responds to insulin. Falp is preferentially expressed in adipocytes but not in preadipocytes, as shown by two-dimensional gel electrophoresis. Northern blot analysis shows that the Falp gene is predominantly expressed in brown and white fat tissues, but not in any other tissues examined. Human homologs of mouse Falp are found to exist as two alternatively spliced isoforms, which share the same N-terminus but have different C-termini. Both human and mouse Falp contain a conserved putative transmembrane domain. Immunofluorescent analyses of 3T3-L1 adipocytes show that Falp protein strictly localizes at a compact perinuclear membrane compartment. Treatment of cells with insulin induces the redistribution of Falp into numerous discrete spotty structures spreading throughout the cytoplasm. Whereas the function of Falp is currently unclear, its tissue specific expression and the responsiveness to insulin suggest that Falp might be involved in a process specifically restricted to adipose tissue function, such as vesicular transport and protein secretion.

mRNA concentrations of MIF in subcutaneous abdominal adipose cells are associated with adipocyte size and insulin action

Objective:To determine whether the mRNA concentrations of inflammation response genes in isolated adipocytes and in cultured preadipocytes are related to adipocyte size and in vivo insulin action in obese individuals.Design:Cross-sectional inpatient study.Subjects:Obese Pima Indians with normal glucose tolerance.Measurements:Adipocyte diameter (by microscope technique; n=29), expression of candidate genes (by quantitative real-time PCR) in freshly isolated adipocytes (monocyte chemoattractant protein (MCP) 1 and MCP2, macrophage inflammatory protein (MIP) 1α, MIP1β and MIP2, macrophage migration inhibitory factor (MIF), tumor necrosis factor α, interleukin (IL) 6 and IL8; n=22) and cultured preadipocytes (MCP1, MIP1α, MIF, IL6 and matrix metalloproteinase 2; n=33) from subcutaneous abdominal adipose tissue (by aspiration biopsy, n=34), body fat by dual-energy X-ray absorptiometry, glucose tolerance by 75 g oral glucose tolerance test and insulin action by euglycemic-hyperinsulinemic clamp (insulin infusion rate 40 mU m−2 min−1) (all n=34).Results:MIF was the only gene whose expression in both freshly isolated adipocytes and cultured preadipocytes was positively associated with adipocytes diameter and negatively associated with peripheral and hepatic insulin action (all P<0.05). In multivariate analysis, the association between adipocyte MIF mRNA concentrations and adipocytes diameter was independent of the percentage of body fat (P=0.03), whereas adipocyte MIF mRNA concentrations, but not adipocyte diameter, independently predicted peripheral insulin action. The mRNA expression concentrations of the MIF gene in adipocytes were not associated with plasma concentrations of MIF, but were negatively associated with plasma adiponectin concentrations (P=0.004). In multivariate analysis, adipocyte MIF RNA concentrations (P=0.03) but not plasma adiponectin concentrations (P=0.4) remained a significant predictor of insulin action.Conclusions:Increased expression of MIF gene in adipose cells may be an important link between obesity characterized by enlarged adipocytes and insulin resistance in normal glucose tolerant people.

Differential Expression of Matrix Metalloproteinase 3 (MMP3) in Preadipocytes/Stromal Vascular Cells From Nonobese Nondiabetic Versus Obese Nondiabetic Pima Indians

Prior microarray studies comparing global gene expression patterns in preadipocytes/stromal vascular cells isolated from nonobese nondiabetic versus obese nondiabetic Pima Indians showed that matrix metalloproteinase 9 (MMP9) is upregulated in obese subjects. The current study targeted analysis of nine additional MMP genes that cluster to a region on chromosome 11q22 that is linked to BMI and percent body fat. Differential-display PCR showed that MMP3 is downregulated in preadipocytes/stromal vascular cells from obese subjects, and real-time PCR showed that MMP3 expression levels are negatively correlated with percent body fat. To determine whether variants within MMP3 are responsible for its altered expression, MMP3 was sequenced, and seven representative variants were genotyped in 1,037 Pima subjects for association analyses. Two variants were associated with both BMI and type 2 diabetes, and two additional variants were associated with type 2 diabetes alone; however, none of these variants were associated with MMP3 expression levels. We propose that the MMP3 pathway is altered in human obesity, but this alteration may be the result of a combination of genetic variation within the MMP3 locus itself, as well as variation in additional factors, either primary or secondary to obesity, that regulate expression of the MMP3 gene.

Pioglitazone reduces inflammatory responses of human adipocytes to factors secreted by monocytes/macrophages

Infiltration of monocyte-derived macrophages into adipose tissue may contribute to tissue and systemic inflammation and insulin resistance. We hypothesized that pioglitazone (Pio) could specifically reduce the inflammatory response of adipocytes to factors released by monocytes/macrophages. We show that macrophage factors (Mphi-factors) greatly increase expression levels of proinflammatory adipokines, chemokines, and adhesion molecules in human subcutaneous and visceral adipose tissue (SAT and VAT) as well as in adipocytes (up to several hundredfold of control). Compared with SAT, VAT showed enhanced basal and Mphi-factor-induced inflammatory responses. Mphi-factors also induced greater lipolysis in adipocytes, as assessed by concentrations of glycerol released from the cells (196 +/- 13 vs. 56 +/- 7 microM in control, P < 0.05). Pretreatment of adipose tissue or adipocytes with Pio reduced these responses to Mphi-factors (by 13-86%, P < 0.05) and prevented Mphi-factor suppression of adiponectin expression. Furthermore, Pio pretreatment of adipocytes and macrophages tended to further reduce inflammatory responses of adipocytes to Mphi-factors and monocyte adhesion to Mphi-factor-activated adipocytes. In support of these in vitro data, media conditioned by monocytes isolated from impaired glucose-tolerant subjects treated with Pio (compared with placebo) induced release of lower concentrations of proinflammatory adipokines and glycerol (100 +/- 7 vs. 150 +/- 15 microM, P < 0.05) from adipocytes. In summary, Pio decreases inflammatory responses in adipose tissue/cells induced by monocytes/macrophages by acting on either or both cell types. These beneficial effects of Pio may attenuate proinflammatory responses resulting from monocyte/macrophage infiltration into adipose tissue and suppress tissue inflammation resulting from the interaction between both cell types.

Pioglitazone Inhibits the Expression of Inflammatory Cytokines From Both Monocytes and Lymphocytes in Patients With Impaired Glucose Tolerance

Objective—The current study determines whether pioglitazone (PIO) therapy reduces both monocyte and lymphocyte inflammatory activity and their ability to induce inflammation in other tissues. Methods and Results—Monocyte and lymphocyte cytokine gene and protein expression of interleukin (IL)-6 were first shown to be greater in subjects with impaired glucose tolerance (IGT) than in subjects with normal glucose tolerance. Sixty-six IGT subjects were then randomized to 4,5 months of placebo or PIO therapy. After receiving PIO, subjects had lower triglycerides and higher HDL cholesterol (P<0.05) than did subjects receiving placebo. Monocyte gene and protein expression of IL-1&bgr;, IL-6, and IL-8 (and IL-2, IL-6 and IL-8 from lymphocytes) was significantly lower after PIO therapy in the resting state, as well as after lipopolysaccharide (LPS) stimulation (P<0.05 for all). Moreover, IL-6, IL-8, and MCP-1 gene expression were decreased by nearly 50% in human adipocytes exposed to conditioned media from monocytes or lymphocytes from PIO treated subjects. Conclusion—These results demonstrate that PIO therapy in IGT can reduce proinflammatory gene and protein expression from both monocytes and lymphocytes. This intervention also reduces the inflammatory cross-talk between these immune cells and adipose tissue, which could in turn contribute to the metabolic improvements resulting from PIO therapy.

11β-Hydroxysteroid Dehydrogenase Type 1 in Adipose Tissue and Prospective Changes in Body Weight and Insulin Resistance

Objective: Increased mRNA and activity levels of 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in human adipose tissue (AT) are associated with obesity and insulin resistance. The aim of our study was to investigate whether 11βHSD1 expression or activity in abdominal subcutaneous AT of non‐diabetic subjects are associated with subsequent changes in body weight and insulin resistance [homeostasis model assessment of insulin resistance (HOMA‐IR)].