IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Adipocyte telomere length associates negatively with adipocyte size, whereas adipose tissue telomere length associates negatively with the extent of fibrosis in severely obese women.

Telomere length can be considered as a biological marker for cell proliferation and aging. Obesity is associated with adipocyte hypertrophy and proliferation as well as with shorter telomeres in adipose tissue. As adipose tissue is a mixture of different cell types and the cellular composition of adipose tissue changes with obesity, it is unclear what determines telomere length of whole adipose tissue. We aimed to investigate telomere length in whole adipose tissue and isolated adipocytes in relation to adiposity, adipocyte hypertrophy and adipose tissue inflammation and fibrosis. Telomere length was measured by real-time PCR in visceral adipose tissue, and isolated adipocytes of 21 obese women with a waist ranging from 110 to 147 cm and age from 31 to 61 years. Telomere length in adipocytes was shorter than in whole adipose tissue. Telomere length of adipocytes but not whole adipose tissue correlated negatively with waist and adipocyte size, which was still significant after correction for age. Telomere length of whole adipose tissue associated negatively with fibrosis as determined by collagen content. Thus, in extremely obese individuals, adipocyte telomere length is a marker of adiposity, whereas whole adipose tissue telomere length reflects the extent of fibrosis and may indicate adipose tissue dysfunction.

Effects of pain controlling neuropeptides on human fat cell lipolysis.

OBJECTIVE: Neuropeptides NPFF and NPSF are involved in pain control, acting through the G-protein coupled receptors (GPR)74 (high affinity for NPFF) and GPR147 (equal affinity for NPFF and NPSF). GPR74 also inhibits catecholamine-induced adipocyte lipolysis and regulates fat mass in humans. The aim of this study was to compare the effects of NPFF and NPSF on noradrenaline-induced lipolysis and to determine the expression of their receptors in human fat cells. DESIGN: Adipose tissue was obtained during surgery. Adipocytes were prepared and kept in primary culture. Lipolysis, protein expression and gene expression were determined. RESULTS: NPFF counteracted noradrenaline-induced lipolysis, which was more marked after 48 h than after 4 h exposure and was solely attributed to inhibition of beta-adrenoceptor signalling. NPSF counteracted noradrenaline-induced lipolysis maximally after 4 h of exposure, which was attributed to a combination of inhibition of beta-adrenoceptor signalling and decreased activation of the protein kinase-A hormone sensitive lipase complex by cyclic AMP. Both neuropeptides were effective in nanomolar concentrations. NPFF and NPSF had no effects on the expression of genes involved in catecholamine signal transduction. Both GPR74 and GPR147 were expressed at the protein level in fat cells from various adipose regions. GPR74 mRNA levels were higher in adipose tissue from obese as compared with non-obese subjects. High gene expression of either receptor correlated with low noradrenaline-induced lipolysis (P<0.05). CONCLUSIONS: Pain controlling neuropeptides NPFF and NPSF may be important for the regulation of lipolysis in man probably acting through GPR74 and GPR147. At low concentrations they inhibit catecholamine-induced lipolysis through rapid and long-term post-transcriptional effects at several steps in adrenoceptor signalling in fat cells.

Primary differences in lipolysis between human omental and subcutaneous adipose tissue observed using in vitro differentiated adipocytes.

Catecholamine-induced lipolysis is elevated in omental as compared to subcutaneous adipocytes due to primary differences between the two cell types (i.e., they have different progenitor cells). Whether there is regional variation in atrial natriuretic peptide (ANP)-induced lipolysis is unknown. We studied whether beta-adrenoceptor signaling to lipolysis and ANP-induced lipolysis are involved in the primary differences in lipolysis. In vitro experiments on differentiated preadipocytes from human subcutaneous and omental adipose tissue were performed. The cells were kept in culture for a relative long duration, so any influence of local environment and circulation in the various adipose tissue depots could be excluded. Using beta1-, beta2-, and beta3-adenoceptor agonists, lipolysis was found to be significantly higher in omental as compared to subcutaneous differentiated preadipocytes. Forskolin and dibutyryl cAMP, which act at post-adrenoceptor levels, did not show any regional difference. There was no regional difference in ANP-induced lipolysis. Gene expression of beta1- and beta3-adrenoceptors was higher and beta2-adrenoceptor expression was lower in the omental cells. Omental fat cells have an increased beta-adrenoceptor-mediated lipolysis principally due to primary differences in the early event that couples beta-adrenoceptor subtypes to G-proteins. ANP-induced lipolysis is not subject to primary regional variation.

Endothelin-1 stimulates human adipocyte lipolysis through the ET A receptor.

OBJECTIVE: Levels of the vascular peptide endothelin-1 (ET-1) are significantly elevated in obesity. Adipose tissue-derived ET-1 attenuates insulin-mediated antilipolysis in human visceral adipocytes through the activation of the ET receptor B (ET(B)R), thereby linking ET-1 to insulin resistance. Whether ET-1 has direct effects on lipolysis in human adipocytes is not known. RESEARCH DESIGN AND SUBJECTS: Endothelin-1 receptor (ETR) mRNA expression was determined by quantitative PCR in 130 non-obese and obese subjects. ET-1 mRNA in different adipose tissue regions was also assessed. ETR protein expression was analyzed by western blotting in 37 subjects. The effect of ET-1 on lipolysis was assessed in freshly isolated adipocytes and in vitro differentiated adipocytes from human donors. RESULTS: Freshly isolated human adipocytes incubated with different concentrations of ET-1 showed no acute effect on lipolysis. In contrast, a 24 h incubation in primary cultures of human adipocytes resulted in a significant 50% increase in lipolysis. This effect was concentration dependent and could be mimicked by an agonist of the ET(A) receptor but not with a selective ET(B)R agonist. Adipocyte differentiation was not affected by any of the agonists. In subcutaneous (s.c.) adipose tissue from 19 non-obese and 18 obese subjects, the protein expression of ET(A)R was significantly higher in obese subjects whereas there was no difference in ET(B)R expression. Interestingly, the differences in protein expression were not observed at the mRNA level as ET(A)R expression was similar between lean and obese subjects. CONCLUSION: Long-term but not acute incubation of human adipocytes with ET-1 results in a significant increase in lipolysis. This appears to be mediated through the activation of ET(A)R, demonstrating a yet another receptor-specific effect of ET-1. In addition, the protein expression of ET(A)R is increased in s.c. adipose tissue in obesity, possibly through post-transcriptional mechanisms. An increased effect of ET-1 could be a mechanism that contributes to increased basal lipolysis in human obesity.

Characterization of the human CIDEA promoter in fat cells.

BACKGROUND: Cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) is a protein that regulates lipolysis in human adipocytes through cross-talk involving tumor necrosis factor-alpha (TNF-alpha). TNF-alpha downregulates CIDEA mRNA although it is unclear whether this is mediated through transcriptional or post-transcriptional mechanisms. CIDEA has important metabolic effects in human fat cells and genetic variations in the human CIDEA gene have been correlated to the development of obesity. However, little is known about the factors regulating CIDEA expression in human adipocytes. We set out to describe the transcriptional control of human CIDEA. METHODS: A 1.1-kb genomic fragment upstream of the transcriptional start site (TSS) of human CIDEA was cloned and deletion fragments were generated. Transcriptional activity of the promoter was analyzed by luciferase reporter assays in in vitro-differentiated human adipocytes. The effect of TNF-alpha was assessed in human adipocytes and murine 3T3-L1 cells transfected with deletion fragments of the CIDEA promoter. Protein-DNA interactions were analyzed by electrophoretic mobility shift assays (EMSA). RESULTS: Basal transcriptional activity was found in a 97-bp region upstream of the TSS. We studied the effect of three common haplotypes in the promoter region but found no significant difference in transcriptional activity among them. Incubation of in vitro-differentiated human adipocytes as well as 3T3-L1 cells with TNF-alpha reduced the transcriptional activity of the human CIDEA promoter, demonstrating a direct effect on CIDEA transcription. EMSAs and mutational analysis indicated that this was mediated by a nuclear factor-kappaB (NF-kappaB) site at position -163/-151. CONCLUSION: We demonstrate that basal transcription of the human CIDEA gene is confined to the 97 first bases upstream of TSS and that TNF-alpha negatively regulates transcription of this gene, which at least in part involves NF-kappaB activation.

Strong association between mitochondrial DNA copy number and lipogenesis in human white adipose tissue.

AIMS/HYPOTHESIS: Recent studies suggest a link between insulin resistance and mitochondrial function in white fat cells. The aim of this study was to evaluate adipocyte mitochondrial DNA (mtDNA) copy number in relation to adipocyte and clinical variables that are related to insulin sensitivity. METHODS: We studied a group of 148 healthy volunteers with a large inter-individual variation in BMI. Relative amounts of mtDNA and nuclear DNA were determined by quantitative RT-PCR. The mtDNA:nuclear DNA ratio reflects the tissue concentration of mtDNA per cell. RESULTS: The mtDNA copy number was enriched in adipocytes of adipose tissue and decreased slightly by ageing (p = 0.015) and increasing BMI (p = 0.004); however, it was not influenced by sex, energy-restricted diets or marked long-term weight reduction. Adipose mtDNA copy number was not independently related to resting energy expenditure, overall insulin sensitivity or adipocyte lipolysis. However, it showed a strong positive correlation with basal (p = 0.0012) and insulin-stimulated lipogenesis (p < 0.0001) in fat cells, independently of age and BMI, and a weak positive correlation with levels of mRNA from several genes involved in mitochondrial oxidative capacity (r = 0.2-0.3). CONCLUSIONS/INTERPRETATION: The mtDNA copy number in human white fat cells is fairly stable within healthy individuals. It is not influenced by sex or weight loss and is not important for overall insulin sensitivity or energy expenditure at rest. However, it is strongly related to adipocyte lipogenesis and weakly to mitochondrial oxidative capacity, suggesting that adipocyte mitochondria are, above all, local regulators.

The influence of preadipocyte differentiation capacity on lipolysis in human mature adipocytes.

The ability of catecholamines to maximally stimulate adipocyte lipolysis (lipolytic capacity) is decreased in obesity. It is not known whether the lipolytic capacity is determined by the ability of adipocytes to differentiate. The aim of the study was to investigate if lipolytic capacity is related to preadipocyte differentiation and if the latter can predict lipolysis in mature adipocytes. IN VITRO experiments were performed on differentiating preadipocytes and isolated mature adipocytes from human subcutaneous adipose tissue. In preadipocytes, noradrenaline-induced lipolysis increased significantly until terminal differentiation (day 12). However, changes in the expression of genes involved in lipolysis (hormone sensitive lipase, adipocyte triglyceride lipase, the alpha2-and beta1-adrenoceptors, perilipin, and fatty acid binding protein) reached a plateau much earlier during differentiation (day 8). A significant positive correlation between lipolysis in differentiated preadipocytes and mature adipocytes was observed for noradrenaline (r=0.5, p<0.01). The late differentiation capacity of preadipocytes measured as glycerol-3-phosphate dehydrogenase activity was positively correlated with noradrenaline-induced lipolysis in preadipocytes (r=0.51, p<0.005) and mature fat cells (r=0.35, p<0.05). In conclusion, intrinsic properties related to terminal differentiation determine the ability of catecholamines to maximally stimulate lipolysis in fat cells. The inability to undergo full differentiation might in part explain the low lipolytic capacity of fat cells among the obese.

Differential function of the alpha2A-adrenoceptor and Phosphodiesterase-3B in human adipocytes of different origin.

OBJECTIVE: Human adipocytes can be obtained in vitro by differentiation of human preadipocytes or mesenchymal stem cells (hMSC). Although functionally similar to freshly isolated cells, no detailed comparison of the different cell types has been performed. The antilipolytic alpha2A-adrenoceptor (AR) and the cAMP-degrading enzyme Phosphodiesterase-3B (PDE3B) have been implicated in the fine-tuning of lipolysis but little is known regarding their role in human adipocytes nor whether their expression and/or function differs in fat cells from different precursors. METHODS: The effects of alpha2A-AR and PDE3B inhibition in mature adipocytes was determined and compared to that in differentiated preadipocytes and hMSC-derived fat cells. Gene expression was determined by real-time PCR and protein expression by Western blot. RESULTS: Noradrenaline (NA) stimulated lipolysis in preadipocytes and mature adipocytes but markedly reduced lipolysis in differentiated hMSC derived-adipocytes. This was due to a potent stimulation of alpha2A-AR since co-incubation with NA and the alpha2-AR-inhibitor yohimbine restored NA-induced lipolysis. The order of Yohimbine response was hMSC>preadipocytes>mature adipocytes. Although alpha2-AR mRNA expression was highest in mature adipocytes there was no difference in alpha2A-AR protein levels between the cell types. In contrast, Galphai2 mRNA and protein expression was significantly higher in MSC-derived adipocytes, suggesting that differences in the response to alpha2A-AR inhibition reside at the postreceptor level. Incubation with the cAMP-analog 8-bromo(8b) cAMP increased lipolysis in hMSC-derived fat cells while co-incubation with the PDE3-specific inhibitor OPC3911 did not alter the lipolytic effect. In contrast, OPC3911 increased 8bcAMP-induced lipolysis significantly in preadipocytes and mature adipocytes. The response to PDE3B inhibition was; mature adipocytes>preadipocytes>hMSC a finding that correlated significantly with both PDE3B mRNA expression and enzymatic activity. CONCLUSION: Although differentiated adipocytes of different origins display similar functional characteristics there are important differences in the regulation of lipolysis with a marked alpha2A-AR and less pronounced PDE3B effect in fat cells from MSCs.

HMG-CoA reductase inhibitor cerivastatin inhibits interleukin-6 expression and secretion in human adipocytes.

Human adipose tissue is a main contributor to plasma levels of pro-inflammatory cytokine IL-6. How IL-6 expression is regulated in adipocytes remains unclear. In the current study, we investigated the effect of the HMG-CoA reductase inhibitor, cerivastatin, on the production of IL-6 from cultured human adipocytes. Cerivastatin reduced both IL-6 mRNA and secretion in a dose- and time-dependent manner. The inhibitory effect on IL-6 mRNA was prevented by the intermediates of the cholesterol synthesis pathway, mevalonate and geranyl-geranyl-phyrophosphate (GGPP) but not by farnesyl-pyrophosphate. This suggests the involvement of geranylgeranyl-modified intermediates in the effect of cerivastatin on IL-6. Moreover, cerivastatin induced an inactivation of the phosphorylation of the p65 subunit of NFkappaB which was prevented by GGPP. Our data suggest that cerivastatin exerts an anti-inflammatory effect by down-regulating IL-6 levels in adipocytes, which seems to be mediated by reduced production of GGPP and interference with the NFkappaB pathway.

Effect of BMI and age on adipose tissue cellularity and differentiation capacity in women.

OBJECTIVE: To study the relation between body mass index (BMI) and age on the one hand and total number of human (pre-) adipocytes and preadipocyte differentiation capacity on the other hand. SUBJECTS: In total, 189 women undergoing surgical mammary reduction, age range 16-73 y, BMI range 19.7-39.7 kg/m(2). MEASUREMENTS: Differentiation of preadipocytes in primary culture was assessed by morphological criteria, and determination of glycerol-3-phosphate dehydrogenase after stimulation of the cells by standardized adipogenic conditions containing isobutyl-methylxanthine, troglitazone or both compounds. The total number of stromal cells (ie preadipocytes) and fat cells per gram of adipose tissue and per body as well as mature fat cell volume were calculated from isolated stromal cells and adipocytes, respectively, and anthropometric measures. RESULTS: BMI correlated positively to age, mature fat cell size and total number of adipocytes and stromal cells per body (r varying from 0.22 to 0.54, each P<0.05). In contrast, BMI correlated negatively to the number of adipocytes and stromal cells per gram of adipose tissue and the capacity of preadipocytes to differentiate (r varying from -0.20 to -0.37, each P<0.05). No significant correlation was observed between BMI and the ratio of stromal cells to adipocytes. The sample was also divided into three groups: BMI <25 kg/m(2) (lean), BMI 25-29.9 kg/m(2) (overweight) and BMI >/=30 kg/m(2) (obese). The overweight group showed a larger fat cell size but no increase in total fat cell or stromal cell number when compared to the lean subjects. The obese subjects showed larger stromal and fat cell numbers when compared to the lean subjects. Age did not independently correlate to the number of stromal cells or adipocytes per gram of adipose tissue or total body, nor with the capacity of preadipocytes to undergo differentiation and the ratio of stromal cells to adipocytes. CONCLUSION: There seems to be a constant ratio between the number of adipose tissue stromal cells and adipocytes independently of BMI and age in humans. During adipose tissue expansion, there seems to be both a continuous increase in fat cell size, and in stromal cell and adipocyte number, but the increase in fat cell size apparently precedes the increase in fat cell number. The differentiation capacity of the stromal cells appears to decrease with increasing BMI.

Secretion of free and protein-bound leptin from subcutaneous adipose tissue of lean and obese women.

Leptin circulates as a free (FL) and a protein-bound (BL) form, with the soluble leptin receptor (LR) as an important binding compound. Here we measured these components of leptin in serum and in the incubation medium of sc adipose tissue in healthy lean (n = 10) and obese (n = 13) female subjects using recently developed specific RIA systems. In addition, immunostaining for FL, BL, and LR in adipose tissue was performed. Serum FL levels were increased in the obese subjects (P < 0.0001), whereas BL and LR concentrations in serum of lean and obese subjects were similar. Both FL and BL were secreted from human preadipocytes and increased in parallel to the differentiation of the cells. In sc fat cell explants LR antibodies predominantly stained the fat cell membrane, whereas FL and BL antibodies revealed intracytoplasmatic adipocyte staining. The release of FL, BL, and LR from adipose tissue was increased in obese compared with lean subjects (P < 0.005 for FL; P < 0.02 for BL, and P < 0.01 for LR). In summary, fat cells are capable of releasing not only FL, but also BL and LR.

Relationship between IL-6, leptin and adiponectin and variables of fibrinolysis in overweight and obese hypertensive patients.

Impaired fibrinolysis is a common finding in obese humans. This condition is now considered as an established risk factor for thromboembolic complications. Furthermore, obesity is characterized by a specific pattern of circulating concentrations of fat-cell products interleukin-6 (IL-6), leptin, and adiponectin. The aim of our study was to investigate the relationship between these proteins and selected variables of the fibrinolytic system in 74 mildly hypertensive, overweight subjects. Circulating IL-6 and leptin levels showed a positive association with BMI (r = 0.24, p = 0.04 and r = 0.70, p < 0.0001), whereas adiponectin was not correlated to BMI. Interestingly, IL-6 was also positively associated with t-PA/PAI-1 complexes after adjustment for BMI and other anthropometric variables. Leptin was positively correlated with PAI-1 activity and antigen (r = 0.32, p = 0.006 and r = 0.37, p < 0.001, respectively) and negatively with t-PA activity (r = -0.27, p = 0.03). However, these associations lost significance after correction for BMI or HOMA, an insulin sensitivity index. In contrast, adiponectin levels were independently and negatively correlated with PAI-1 antigen (r = -0.26, p = 0.04, after correction for BMI). In conclusion, our study provides further evidence that IL-6, leptin, and adiponectin are associated with impaired fibrinolysis in overweight hypertensive humans.

Increased expression of eNOS protein in omental versus subcutaneous adipose tissue in obese human subjects.

OBJECTIVE: To investigate the expression of eNOS and iNOS mRNA and protein in adipose tissue from subcutaneous (s.c.) and omental adipose tissue of obese subjects. DESIGN: Subcutaneous and omental adipose tissue was obtained from subjects undergoing weight reduction surgery. Messenger RNA and protein levels were measured in tissue extracts and related to basal lipolysis, which was measured in isolated adipocytes from the same subjects. SUBJECTS: Eight overweight but otherwise healthy male subjects (age 43.4+/-10.3 y, BMI 39+/-3.5 kg/m(2), mean+/-s.e.m.). MEASUREMENTS: For mRNA detection a competitive reverse transcription polymerase chain reaction method was used while protein was detected by Western blot. Glycerol release was determined in isolated adipocytes using a standard luminometric assay. RESULTS: Tissue mRNA levels for eNOS in s.c. tissue were 6098+/-1969 amol/mg RNA and in omental tissue 6987+/-2914 amol/mg RNA (mean+/-s.e.m., P=0.75). iNOS mRNA levels were substantially lower; in s.c. tissue 227+/-127 amol/mg RNA and in omental tissue 245+/-162 amol/mg RNA (P=0.8). In Western blot, eNOS protein levels in s.c. and omental tissue were 1.88+/-2.0 and 7.47+/-4.11 (OD/mm(2) 100 microg total protein, P=0.0063), respectively. iNOS protein was expressed at significantly lower levels and barely detectable in both s.c. and omental tissue. Basal rate of lipolysis was two times higher in s.c. compared to omental fat cells (P=0.028). CONCLUSIONS: eNOS protein is markedly increased in omental compared to s.c. adipose tissue in human obese subjects, probably due to post-transcriptional mechanisms. Since basal lipolysis is much lower in omental vs s.c. adipose tissue it is possible that regionally increased NO production, primarily by eNOS, may be involved in the site difference of basal lipolysis in obese subjects.

Increased adipose angiotensinogen gene expression in human obesity.

OBJECTIVE: Adipose angiotensinogen has been suggested as a stimulator of adipose tissue growth and development. Therefore, the association of subcutaneous adipose angiotensinogen gene expression with human obesity was studied. RESEARCH METHODS AND PROCEDURES: The study group consisted of 17 men, undergoing either gastric banding for obesity or elective laparoscopic cholecystectomy (7 obese, 10 non-obese men; body mass index 22 to 51 kg/m2; age 26 to 68 years). Subcutaneous adipose angiotensinogen mRNA and 18S ribosomal RNA (reference gene) levels were measured using competitive quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Adipose angiotensinogen mRNA expression was about two times increased in obesity. The levels of 18S rRNA did not differ between the two groups. Body weight correlated independently and positively with adipose angiotensinogen mRNA expression after adjusting for differences in age and height. DISCUSSION: Adipose angiotensinogen gene expression is elevated in obesity in men.

Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans.

Studies have shown evidence of production of nitric oxide (NO) in adipose tissue, as well as inhibition of lipolysis by NO. We have analyzed nitric oxide synthase (NOS) expression in subcutaneous adipose tissue from 13 nonobese and 18 obese male subjects. Using a competitive reverse transcription polymerase chain reaction method, endothelial (eNOS) and inducible (iNOS), but not neuronal (nNOS), nitric oxide synthase mRNA expression was detected in isolated fat cells and pieces of adipose tissue. Tissue mRNA levels for eNOS were 3,814 +/- 825 and 5,956 +/- 476 amol/mg RNA (P = 0.043), and for iNOS 306 +/- 38 and 332 +/- 48 amol/mg RNA, for nonobese and obese individuals, respectively. Western blotting revealed similar eNOS protein levels in isolated fat cells and adipose tissue pieces. Protein levels for eNOS in nonobese and obese individuals, respectively, were (in optical density [OD] units per mm(2) per 100 microgram of total protein) 0.11 +/- 0.08 and 2.80 +/- 1.30 (P = 0.043). iNOS protein was detectable, but not measurable, at low levels in a subset of obese patients (3 of 10). iNOS protein levels could not be detected in nonobese individuals. Hormone-sensitive lipase (HSL), the key regulating enzyme in lipolysis, is reduced in obesity. The expression of HSL protein in subcutaneous adipose tissue was studied in the same subset of patients; in agreement with previous results, HSL levels were reduced in obese subjects: 4.64 +/- 1.10 and 1.27 +/- 0.35 (P = 0.012) in nonobese and obese subjects, respectively. In conclusion, this study shows that eNOS and iNOS, but not nNOS, are present in human subcutaneous adipose tissue. Gene expression and protein levels of eNOS are increased, whereas HSL protein levels are decreased in obesity. It is speculated that increased NO production, preferably by eNOS, and decreased HSL levels may cause decreased subcutaneous adipose tissue lipolysis in obesity. synthases in subcutaneous adipose tissue of nonobese and obese humans.

Secretion of tumor necrosis factor-alpha shows a strong relationship to insulin-stimulated glucose transport in human adipose tissue.

Some animal models suggest that tumor necrosis factor (TNF)-alpha is a key component in obesity-linked insulin resistance because it inhibits insulin receptor signaling and glucose transport in insulin-sensitive tissues. However, in vivo data in humans have given conflicting results regarding the relationship between circulating TNF-alpha levels and insulin sensitivity. In the present study, the potential local role of TNF-alpha on insulin action in human subcutaneous adipose tissue was studied in 42 obese women (BMI 39+/-10 kg/m2). We found a strong inverse correlation between adipose TNF-alpha secretion and maximum insulin-stimulated glucose transport in adipocytes that was independent of fat cell volume, age, and BMI (P < 0.001, r = 0.58). As much as one-third of the variation in insulin-stimulated glucose transport could be accounted for by variations in TNF-alpha secretion. There was no significant correlation (r = 0.11) between secretion of adipose plasminogen activator inhibitor 1 and glucose transport. Furthermore, subcutaneous adipose tissue of 4 obese women (BMI 40+/-4) incubated with TNF-A for 24 h showed a one-third concentration-dependent inhibition of insulin-stimulated glucose transport (P < 0.01). In conclusion, adipose TNF-alpha may be an important specific and local factor in adipose tissue that influences the ability of insulin to stimulate glucose transport in human fat cells, at least in obese women.

The association of human adipose angiotensinogen gene expression with abdominal fat distribution in obesity.

OBJECTIVE: To investigate in obese subjects the relationship between angiotensinogen gene expression in the abdominal omental and subcutaneous adipose tissue on the one hand and body fat distribution as measured by waist-to-hip ratio (WHR) on the other hand and to compare angiotensinogen gene expression between the two adipose tissue regions. SUBJECTS: Twenty obese subjects undergoing weight reduction surgery with adjustable gastric banding (12 men, eight women; WHR 0.89-1.09; body mass index (BMI) 29-51 kg/m2, age 26-54 y). MEASUREMENTS: Omental and subcutaneous adipose angiotensinogen mRNA and 18S ribosomal RNA (reference gene) levels were measured by competitive quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Angiotensinogen mRNA levels were one-third higher in the omental than in the subcutaneous adipose tissue region (P=0.02). The 18S rRNA levels did not differ significantly between the two adipose tissue regions. WHR correlated positively and significantly with angiotensinogen mRNA in both the subcutaneous and the omental adipose tissue (r=0.5). This relationship was independent of age and BMI. However, WHR did not correlate with 18S rRNA in any of the adipose tissue regions. CONCLUSION: The angiotensinogen gene in adipose tissue might be involved in the development of upper-body obesity.

Regional variation in plasminogen activator inhibitor-1 expression in adipose tissue from obese individuals.

High plasma plasminogen activator inhibitor-1 (PAI-1) activity is a frequent finding in obesity and adipose tissue has recently been suggested to be a source of circulating PAI-1 in humans. In the present study, differences in adipose tissue gene expression and protein secretion rate of PAI-1 between subcutaneous and visceral adipose tissue was analysed in specimens obtained from 22 obese individuals. The secretion rate of PAI-1 was two-fold higher in subcutaneous adipose tissue than in visceral adipose tissue (292 +/- 50 vs 138 +/- 24 ng PAI-1/10(7) cells, P <0.05). In accordance with the secretion data, subcutaneous adipose tissue contained about three-fold higher levels of PAI-1 mRNA than visceral adipose tissue (2.43 +/- 0.37 vs 0.81 +/- 0.12 attomole PAI-1 mRNA/microg total RNA, P <0.00 ). PAI-1 secretion from subcutaneous but not from visceral adipose tissue correlated significantly with cell size (r = 0.43, P<0.05). In summary, subcutaneous adipose tissue secreted greater amounts of PAI-1 and had a higher PAI-1 gene expression than visceral adipose tissue from the same obese individuals. Bearing in mind that subcutaneous adipose tissue is the largest fat depot these finding may be important for the coagulation abnormalities associated with obesity.

Role of gender and genetic variance in plasminogen activator inhibitor-1 secretion from human adipose tissue.

Gender and the 4G/5G polymorphism in the plasminogen activator inhibitor 1 (PAI-1) gene are believed to play a role in the regulation of plasma PAI-1 activity. Adipose tissue has been found to be an important source of PAI-1. The possible influence of gender and the 4G/5G polymorphism in the PAI-1 gene on PAI-1 secretion from abdominal subcutaneous adipose tissue was investigated in 59 women and 32 men. The subjects were apparently healthy, although they differed markedly inter-individually in body mass index (21-53 kg/m2). The 4G/5G polymorphism did not influence the adipose secretion rate of PAI-1 or plasma PAI-1 activity. There was no gender difference in the adipose secretion of PAI-1. In multiple regression, including body mass index (BMI), waist-to-hip ratio (WHR), plasma insulin and plasma triglycerides as the independent and adipose PAI-1 secretion as the dependent variable, only BMI and plasma triglycerides correlated independently with adipose PAI-1 secretion (r = 0.54, p <0.05; r = 0.51, p <0.05, respectively). Men had a two times higher plasma PAI-1 activity than women (p <0.05). This gender difference was mainly due to gender differences in WHR. In multiple regression analysis, BMI and WHR were identified to be independently correlated with plasma PAI-1 activity (r = 0.60, p <0.05; r = 0.52, p = 0.01, respectively). In conclusion, neither gender nor the 4G/5G polymorphism in the PAI-1 gene are associated with secretion of PAI-1 from abdominal subcutaneous adipose tissue.