Assessment of diet-induced obese rats as an obesity model by comparative functional genomics

Objective: We applied a comparative functional genomics approach to evaluate whether diet‐induced obese (DIO) rats serve as an effective obesity model. Methods and Procedures: Gene‐expression profiles of epididymal fat from DIO and lean rats were generated using microarrays and compared with the published array data of obese and non‐obese human subcutaneous adipocytes. Results: Caloric intake and fuel efficiency were significantly higher in DIO rats, which resulted in increased body weight and adiposity. Circulating glucose, cholesterol, triglyceride, insulin, and leptin levels in DIO rats were significantly higher than those in the lean controls. DIO rats also exhibited impaired insulin sensitivity. A direct comparison of gene‐expression profiles from DIO and lean rats and those from obese and non‐obese humans revealed that global gene‐expression patterns in DIO rat fat resemble those of obese human adipocytes. Differentially expressed genes between obese and non‐obese subjects in both human and rat studies were identified and associated with biological pathways by mapping genes to Gene Ontology (GO) categories. Immune response–related genes and angiogenesis‐related genes exhibited significant upregulation in both obese humans and DIO rats when compared with non‐obese controls. However, genes in fatty acid metabolism and oxidation exhibited a broad downregulation only in obese human adipocytes but not in DIO rat epididymal fat. Discussion: Our study based on gene‐expression profiling suggested that DIO rats in general represent an appropriate obesity model. However, the discrepancies in gene‐expression alterations between DIO rats and obese humans, particularly in the metabolic pathways, may explain the limitations of using DIO rodent models in obesity research and drug discovery.     

Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes

Enlarged fat cells exhibit modified metabolic capacities, which could be involved in the metabolic complications of obesity at the whole body level. We show here that sterol regulatory element-binding protein 2 (SREBP-2) and its target genes are induced in the adipose tissue of several models of rodent obesity, suggesting cholesterol imbalance in enlarged adipocytes. Within a particular fat pad, larger adipocytes have reduced membrane cholesterol concentrations compared with smaller fat cells, demonstrating that altered cholesterol distribution is characteristic of adipocyte hypertrophy per se. We show that treatment with methyl-beta-cyclodextrin, which mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces insulin resistance. We also produced cholesterol depletion by mevastatin treatment, which activates SREBP-2 and its target genes. The analysis of 40 adipocyte genes showed that the response to cholesterol depletion implicated genes involved in cholesterol traffic (caveolin 2, scavenger receptor BI, and ATP binding cassette 1 genes) but also adipocyte-derived secretion products (tumor necrosis factor alpha, angiotensinogen, and interleukin-6) and proteins involved in energy metabolism (fatty acid synthase, GLUT 4, and UCP3). These data demonstrate that altering cholesterol balance profoundly modifies adipocyte metabolism in a way resembling that seen in hypertrophied fat cells from obese rodents or humans. This is the first evidence that intracellular cholesterol might serve as a link between fat cell size and adipocyte metabolic activity.     

Differences in lipogenesis and lipolysis in obese and non-obese adult human adipocytes

It has been proposed that differences in adipocyte function and/or metabolism between obese and lean individuals may manifest themselves in functional adipose tissue abnormalities that lead to metabolic disorders in obesity. We studied lipogenesis and lipolysis of omental adipocytes from obese (OB) and non-obese (NOB) humans. The specific activity of the lipogenic marker enzyme G3PDH was 50% lower in total adipocytes of OB compared to that of NOB subjects. Omental adipocytes from OB subjects also had lower basal lipolytic levels, and a lower lipolytic response to beta-adrenergic stimulus. Cholesterol depletion of adipocyte plasma membrane using methyl b-cyclodextrin caused a lipolytic effect on adipocytes of both groups together, but when obese and lean subjects were analyzed separately, the response was significant only in the obese. We present evidence of a different lipogenic and lipolytic profile in obese individuals' omental adipocytes, and propose a relevant role of plasma membrane cholesterol, where the impact of its removal in OB and NOB adipocyte lipolysis differs.     

Uptake of thyroxine by cultured human adipocyte precursors isolated from lean and obese subjects.

The mechanism of thyroxine uptake by human adipocyte precursors has been studied in primary culture. Also the rates of transport of this hormone into the isolated cells of adipose tissue were compared for lean and obese subjects. It was demonstrated that thyroxine transport into the human adipocyte precursor cells is an active, energy-dependent process characterized by very low rate (Km = 10 pmol/l, Vmax = 8 fmol FT4/10(6) cells/min.). By comparing the rates of thyroxine transport into the precursor cells of adipocytes isolated from adipose tissue of lean and obese subjects it was possible to demonstrate a clear tendency to the lowered rate of transport of thyroxine to the cells in obesity. The results of this study suggest that the lowered rate of thyroxine transport to preadipocytes and adipocytes observed in obesity may significantly influence the metabolic state of these cells.     

Sphingolipid Content of Human Adipose Tissue: Relationship to Adiponectin and Insulin Resistance

Ceramides (Cer) are implicated in obesity‐associated skeletal muscle and perhaps adipocyte insulin resistance. We examined whether the sphingolipid content of human subcutaneous adipose tissue and plasma varies by obesity and sex as well as the relationship between ceramide content and metabolic indices. Abdominal subcutaneous adipose biopsies were performed on 12 lean adults (males = 6), 12 obese adults (males = 6) for measurement of sphingolipid content and activity of the main ceramide metabolism enzymes. Blood was sampled for glucose, insulin (to calculate homeostasis model assessment‐estimated insulin resistance (HOMA_IR)) adiponectin, and interleukin‐6 (IL‐6) concentrations. Compared to lean controls, total ceramide content (pg/adipocyte) was increased by 31% (P < 0.05) and 34% (P < 0.05) in obese females and males, respectively. In adipocytes from obese adults sphingosine, sphinganine, sphingosine‐1‐phosphate, C14‐Cer, C16‐Cer, and C24‐Cer were all increased. C18:1‐Cer was increased in obese males and C24:1‐Cer in obese females. For women only, there was a negative correlation between C16‐Cer ceramide and plasma adiponectin (r = ?0.77, P = 0.003) and a positive correlation between total ceramide content and HOMA_IR (r = 0.74, P = 0.006). For men only there were significant (at least P < 0.05), positive correlations between adipocyte Cer‐containing saturated fatty acid and plasma IL‐6 concentration. We conclude that the sexual dimorphism in adipose tissue behavior in humans extends to adipose tissue sphingolipid content its association with adiponectin, IL‐6 and insulin resistance.     

A Modified Protocol to Maximize Differentiation of Human Preadipocytes and Improve Metabolic Phenotypes

Adipose stromal cells proliferate and differentiate into adipocytes, providing a valuable model system for studies of adipocyte biology. We compared differentiation protocols for human preadipocytes and report on their metabolic phenotypes. By simply prolonging the adipogenic induction period from the first 3 to 7 days, the proportion of cells acquiring adipocyte morphology increased from 30–70% to over 80% in human subcutaneous preadipocytes (passages 5–6). These morphological changes were accompanied by increases in the adipogenic marker expression and improved adipocyte metabolic phenotypes: enhanced responses to β‐adrenergically stimulated lipolysis and to insulin‐stimulated glucose metabolism into triglyceride (TG). Confirming previous studies, fetal bovine serum (FBS) dose‐dependently inhibited adipogenesis. However, in subcutaneous preadipocytes that differentiate well (donor‐dependant high capacity and subcultured fewer than two times), the use of 7d‐induction protocols in both 3% FBS and serum‐free conditions allowed >80% differentiation. Responsiveness to β‐adrenergically stimulated lipolysis was lower in 3% FBS. Rates of insulin‐stimulated glucose uptake were higher in adipocytes differentiated with 3% FBS, whereas the sensitivity to insulin was almost identical between the two groups. In summary, extending the length of the induction period in adipogenic cocktail improves the degree of differentiation and responses to key metabolic hormones. This protocol permits functional analysis of metabolic phenotypes in valuable primary human adipocyte cultures through multiple passages.     

Secretome Analysis of Hypoxia‐Induced 3T3‐L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte‐secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3‐L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin‐1 and ‐2, and matrix metalloproteinase‐11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3‐L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.     

Ethnic Differences in the Responsiveness of Adipocyte Lipolytic Activity to Insulin

Objective: The goal of this study was to quantify differences in lipid metabolism and insulin sensitivity in black and white subjects to explain ethnic clinicopathological differences in type 2 diabetes. Research Methods and Procedures: The in vitro lipolytic activity of adipocytes isolated from obese black and white women was measured in the presence of insulin and isoproterenol. Insulin resistance was assessed in vivo using the euglycemic hyperinsulinemic clamp technique. Results: Fasting plasma levels of insulin and nonesterified fatty acid (NEFA) in black and white women were 67 ± 5 pM vs. 152 ± 20 pM (p < 0.01) and 863 ± 93 μM vs. 412 ± 34 μM (p < 0.01), respectively. Euglycemic hyperinsulinemic clamp studies showed that obese black subjects were more insulin‐resistant than their white counterparts (glucose infusion rates: 1.3 ± 0.2 vs. 2.2 ± 0.3 mg/kg per min; p < 0.05). Isolated adipocytes from white women were more responsive to insulin than those from black women with 0.7 nM insulin causing a 55 ± 4% inhibition of isoproterenol‐stimulated lipolysis compared with 27 ± 10% in black women (p < 0.05). Discussion: The low responsiveness of adipocyte lipolytic activity to insulin in black women in the presence of a relative insulinopenia may account for the high plasma NEFA levels seen in these women, which may, in turn, account for their higher in vivo insulin resistance. High NEFA levels may also contribute to the low insulin secretory activity observed in the obese black females. These data suggest that the pathogenesis of insulin resistance and type 2 diabetes within the black obese community is strongly influenced by their adipocyte metabolism.     

Fat Depot‐specific Impact of Visceral Obesity on Adipocyte Adiponectin Release in Women

Our objective was to examine omental and subcutaneous adipocyte adiponectin release in women. We tested the hypothesis that adiponectin release would be reduced to a greater extent in omental than in subcutaneous adipocytes of women with visceral obesity. Omental and subcutaneous adipose tissue samples were obtained from 52 women undergoing abdominal hysterectomies (age: 47.1 ± 4.8 years; BMI: 26.7 ± 4.7 kg/m²). Adipocytes were isolated and their adiponectin release in the medium was measured over 2 h. Measures of body fat accumulation and distribution were obtained using dual‐energy X‐ray absorptiometry and computed tomography, respectively. Adiponectin release by omental and subcutaneous adipocytes was similar in lean individuals; however, in subsamples of obese or visceral obese women, adiponectin release by omental adipocytes was significantly reduced while that of subcutaneous adipocytes was not affected. Omental adipocyte adiponectin release was significantly and negatively correlated with total body fat mass (r = ?0.47, P < 0.01), visceral adipose tissue area (r = ?0.50, P < 0.01), omental adipocyte diameter (r = ?0.43, P < 0.01), triglyceride levels (r = ?0.32, P ≤ 0.05), cholesterol/high‐density lipoprotein (HDL)‐cholesterol (r = ?0.31, P ≤ 0.05), fasting glucose (r = ?0.39, P ≤ 0.01), fasting insulin (r = ?0.36, P ≤ 0.05), homeostasis model assessment index (r = ?0.39, P ≤ 0.01), and positively associated with HDL‐cholesterol concentrations (r = 0.33, P ≤ 0.05). Adiponectin release from subcutaneous cells was not associated with any measure of adiposity, lipid profile, or glucose homeostasis. In conclusion, compared to subcutaneous adipocyte adiponectin release, omental adipocyte adiponectin release is reduced to a greater extent in visceral obese women and better predicts obesity‐associated metabolic abnormalities.     

Polybrominated diphenyl ethers as endocrine disruptors of adipocyte metabolism

Objective: Obesity is thought to result from poor diet and insufficient exercise. An additional factor may be endocrine‐disrupting environmental chemicals that contaminate the air, water, and food supply. We tested the hypothesis that a class of lipid‐soluble flame retardant chemicals known to accumulate in adipose tissue, polybrominated diphenyl ethers (PBDEs), disrupts insulin and isoproterenol sensitivity of isolated rat adipocytes. Research Methods and Procedures: Six‐week‐old Sprague‐Dawley rats were gavaged daily with 14 mg/kg body weight (BW) pentabrominated diphenyl ether (penta‐BDE) in corn oil (n = 24) or corn oil alone (n = 24). At 2 and 4 weeks of treatment, epididymal fat pad adipocytes were isolated, and isoproterenol‐stimulated lipolysis, insulin‐stimulated glucose oxidation, and adipocyte size were measured. Results: There was no alteration in adipocyte metabolism after 2 weeks of in vivo penta‐BDE treatment, but after 4 weeks of treatment, adipocytes averaged a 30% increase in isoproterenol‐stimulated lipolysis and a 59% decrease in insulin‐stimulated glucose oxidation, compared with control. There were no differences in average rat BW and adipocyte size between treated and control rats, but plasma total thyroxine level in 2‐ and 4‐week treated rats was 30% of control. Discussion: Daily exposure of rats to 14 mg/kg BW penta‐BDE for 4 weeks has no effect on animal or adipocyte size but significantly alters insulin and isoproterenol‐stimulated metabolism of isolated adipocytes. These alterations, hallmark features of metabolic obesity, suggest the need for further research on the contribution of lipid‐soluble, endocrine‐disrupting environmental chemicals to the obesity epidemic.     

Identification of endocannabinoids and related compounds in human fat cells

Objective: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2‐arachidonoylglycerol and anandamide (N‐arachidonoylethanolamine), are up‐regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids. Research Methods and Procedures: The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator‐activated receptor (PPAR)‐γ activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high‐performance liquid chromatography (HPLC)‐atmospheric pressure chemical ionization (APCI)‐mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme‐linked immunosorbent assay (ELISA), and PPAR‐γ protein expression examined by Western blotting. Results: We show that 2‐arachidonoylglycerol, anandamide, and both anandamide analogs, N‐palmitoylethanolamine and N‐oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.042 ± 0.004 to 0.531 ± 0.048 pM/mg lipid extract. N‐palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down‐regulated by leptin but not affected by adiponectin and PPAR‐γ agonist ciglitazone. N‐palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR‐γ protein expression in adipocytes. Discussion: This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.     

Fenoxycarb promotes adipogenesis in 3T3‐L1 preadipocytes

Lipophilic insect hormones and their analogs affect mammalian physiology by regulating the expression of metabolic genes. Therefore, we determined the effect of fenoxycarb, a juvenile hormone analog, on lipid metabolism in adipocytes. Here, we demonstrated that fenoxycarb dose‐dependently promoted lipid accumulation in 3T3‐L1 adipocytes during adipocyte differentiation and that its lipogenic effect was comparable to that of rosiglitazone, a well‐known ligand for peroxisome proliferator‐activated receptor gamma (PPARγ). Furthermore, fenoxycarb stimulated PPARγ activity without affecting other nuclear receptors, such as liver X receptor (LXR), farnesoid X‐activated receptor (FXR) and Nur77. In addition, fenoxycarb treatment increased the expression of PPARγ and fatty acid transporter protein 1 (FATP1) in 3T3‐L1 adipocytes, suggesting that fenoxycarb may facilitate adipocyte differentiation by enhancing PPARγ signaling, the master regulator of adipogenesis. Together, our results suggest that fenoxycarb promoted lipid accumulation in adipocytes, in part, by stimulating PPARγ.     

An in vitro model for hypertrophic adipocytes: Time‐dependent adipocyte proteome and secretome changes under high glucose and high insulin conditions

Obesity is the consequence of a positive energy balance and characterized by enlargement of the adipose tissue, which in part is due to hyperplasia and hypertrophy of the adipocytes. Not much is known about the transition of normal mature adipocytes to the hypertrophic state, which in vivo is very hard to study. Here, we have maintained mature human SGBS cells as a surrogate for adipocytes, changes of morphological and molecular metabolism of the adipocytes were monitored over the first 4 days and the last 4 days. In total, 393 cellular proteins and 246 secreted proteins were identified for further analysis. During the first 4 days of high glucose and insulin, the adipocytes seemed to prefer pyruvate as energy source, whereas beta‐oxidation was down‐regulated supporting lipid loading. Over time, lipid droplet fusion instead of lipid uptake became relatively important for growth of lipid droplets during the last 4 days. Moreover, ECM production shifted towards ECM turnover by the up‐regulation of proteases over eight days. The present in vitro system provides insight into the metabolic changes of adipocytes under conditions of high glucose and insulin, which may help to understand the process of in vivo adipocyte hypertrophy during the development of obesity.     

Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.