Human preadipocytes display a depot-specific susceptibility to apoptosis

Adipose tissue mass is determined by both the number and volume of adipose cells. Adipose cell number reflects the balance of cell acquisition and cell loss, whereas adipose cell volume represents the balance of lipolysis and lipogenesis. It is well recognized that insulin resistance, NIDDM, and other metabolic disorders are associated more strongly with increased omental adiposity than with subcutaneous adiposity. Depot-related differences exist in adipocyte responses to lipolytic and lipogenic stimuli, in adipocyte apoptosis, and in the potential for preadipocyte replication and differentiation. In the present study, we address the question of whether there might also be a site-specific difference in the susceptibility of human preadipocytes to apoptosis. Paired samples of human omental and subcutaneous preadipocytes from 12 individuals were cultured, and apoptosis was induced by serum deprivation or treatment with tumor necrosis factor (TNF)-alpha for 4 h. Cells were then stained with acridine orange, and apoptotic indices were calculated as the fraction of cells showing nuclear condensation. Under both conditions, in 9 of 11 subjects, apoptotic indices were substantially greater in preadipocytes from the omental depot than in those from the subcutaneous depot, and mean apoptotic indices were more than twofold higher in omental cells (serum-free medium: P < 0.05; TNF-alpha: P < 0.02; paired t test). Omental preadipocytes are therefore more susceptible to two different apoptotic stimuli than subcutaneous preadipocytes, demonstrating another intrinsic site-specific difference between human adipose cells of the two depots. These results suggest that the regulation of adipose tissue distribution in humans could involve depot-specific differences in rates of preadipocyte apoptosis.     

Cholinergic amacrine cells are not required for the progression and atropine-mediated suppression of form-deprivation myopia

Expression of extracellular matrix (ECM) components during differentiation of pre-existing preadipocytes and preadipocytes recruited by dexamethasone (DEX) was examined with immunocytochemistry in primary cultures of adipose tissue stromal vascular (S-V) cells. Immunocytochemistry showed that a small proportion of preadipocytes (AD-3+) in 24-h cultures (d 0 to 1) contained lipid or expressed ECM. Two days of insulin treatment markedly increased preadipocyte ECM expression, and preadipocytes were "rounder" than those not treated with insulin. Dexamethasone with insulin increased preadipocyte recruitment two- to fivefold in completely serum-free cultures and in cultures serum-free after seeding and plating in serum for 1 to 3 d. Double staining demonstrated that ECM expression and lipid accretion were tightly coupled and lagged significantly behind preadipocyte recruitment (AD-3 expression). Double staining (lipid and AD-3) also demonstrated remarkable and unexpected cytological traits indicating a "reticuloendothelial" nature of newly recruited preadipocytes. Time-lapse phase contrast microscopy verified these observations and demonstrated that small adipocytes and preadipocytes migrated and formed cell-to-cell contacts while aggregating and clustering. Large clusters of lipid-free preadipocytes developed in DEX-treated cultures, but not in cultures treated with DEX + insulin. However, the influence of DEX on preadipocyte recruitment and ECM expression was independent of insulin. Preadipocytes on ECM substrata accumulated lipid but were "flat" and did not express ECM components, regardless of insulin or DEX treatment. These studies clearly indicate that preadipocytes express ECM components after recruitment, and the ECM may be critical for morphological development of adipocytes.     

Primary pancreatic non-Hodgkin''s lymphoma in a renal transplant recipient

Dormant preadipocytes isolated from adipose tissue are able to differentiate into adipocytes in vitro. A few adipogenic hormones (glucocorticoids or prostacyclin, IGF-I and insulin) are sufficient to trigger the differentiation program. In preadipose and adipose cells, glucocorticoids play a cardinal role by regulating the expression of numerous genes and by increasing the production of prostacyclin which acts as an intracrine/autocrine/paracrine effector. Fatty acids and metabolites (including prostacyclin) enhance adipocyte differentiation via the activation of nuclear peroxisome proliferator-activated receptors (PPARs). PPARs then modulate positively the expression of various lipid-related genes involved in triacylglycerol accumulation. These in vitro observations emphasize the importance of the hypothalamic-pituitary-adrenal axis and provide also a link which may take place in vivo between high-fat diets and the excess of adipose tissue development.     

Expression and regulation of neuronal apoptosis inhibitory protein during adipocyte differentiation

We have used the 3T3-L1 and 3T3-F442A preadipocyte cell lines to examine the expression and regulation of neuronal apoptosis inhibitory protein (NAIP) during adipocyte differentiation. When 3T3-L1 preadipocytes differentiated into adipocytes, they developed resistance to apoptosis induced by growth factor deprivation, as assessed by terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end labeling. Protein expression of NAIP was markedly elevated in 3T3-L1 and 3T3-F442A adipocytes compared with that in their fibroblast-like precursors. NAIP was also present in rat white adipocytes. In 3T3-L1 cells, the increase in NAIP occurred by day 4 of the 8-day differentiation protocol, which includes exposure of confluent preadipocytes to insulin, dexamethasone, and isobutylmethylxanthine. Incubation of confluent 3T3-L1 preadipocytes with any of these components alone had no effect on NAIP expression. When 3T3-C2 cells, a control cell line that does not differentiate, were subjected to the differentiation protocol, the low NAIP levels remained unaltered. Addition of rapamycin, a p70 S6 kinase inhibitor that blocks adipocyte differentiation, to the 3T3-L1 differentiation medium prevented the rise in NAIP expression. These data demonstrate for the first time that NAIP is expressed in adipocyte cell lines and primary adipocytes. The differentiation-dependent augmentation of NAIP protein levels in 3T3-L1 adipocytes is closely correlated with the development of resistance to apoptosis induced by growth factor deprivation, suggesting a potential role for NAIP in these cells.     

The status of perioperative pain therapy in Germany. Results of a representative, anonymous survey of 1,000 surgical clinic. Pain Study Group

Conditions that trigger preadipocyte differentiation in vivo have yet to be elucidated. To investigate the role of endogenous arachidonic acid (AA) metabolites on adipose tissue growth, rat preadipocytes in primary culture were induced to differentiate using medium conditioned by isolated mature adipocytes (ACM). Differentiation was determined by assay of glycerol-3-phosphate dehydrogenase (GPDH). When collected in the presence of indomethacin (10 nmol/L) to inhibit prostaglandin (PG) synthesis by adipocytes, ACM induced greater differentiation (GPDH activity, 405 +/- 68 nmol NADH used/min/mg protein) than when indomethacin was added postcollection to inhibit preadipocyte PG synthesis (205 +/- 24, P < .05) or ACM alone (304 +/- 55). This suggested that PGs released by adipocytes inhibited differentiation, whereas those released by preadipocytes appeared to act in an autocrine manner to stimulate differentiation. However, 24-hour collections of ACM contained 125 pmol/L PGE2 and 900 pmol/L PGI2, concentrations too low to promote differentiation when added exogenously. Nordihydroguaiaretic acid (NDGA; 10 pmol/L), an inhibitor of lipoxygenase (LOX), stimulated the ACM-induced increase in GPDH activity (ACM, 99 +/- 13; ACM + NDGA, 369 +/- 130). In contrast, when differentiation was induced by a hormonal cocktail (MIX), including insulin and corticosterone, NDGA decreased GPDH activity (MIX, 329 +/- 66; MIX + NDGA, 142 +/- 40; P < .03). We concluded that preadipocyte differentiation within adipose tissue may be subject to both positive and negative regulators derived from AA metabolism resulting from both LOX and cyclooxygenase (COX) activity.     

Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth

Adipose tissue growth results from de novo adipocyte recruitment (hyperplasia) and increased size of preexisting adipocytes. Adipocyte hyperplasia accounts for the severalfold increase in adipose tissue mass that occurs throughout life, yet the mechanism of adipocyte hyperplasia is unknown. We studied the potential of macrophage colony-stimulating factor (MCSF) to mediate adipocyte hyperplasia because of the profound effects MCSF exerts on pluripotent cell recruitment and differentiation in other tissues. We found that MCSF mRNA and protein were expressed by human adipocytes and that adipocyte MCSF expression was upregulated in rapidly growing adipose tissue that encircled acutely inflamed bowel and in adipose tissue from humans gaining weight (4-7 kg) with overfeeding. Localized overexpression of adipocyte MCSF was then induced in rabbit subcutaneous adipose tissue in vivo using adenoviral-mediated gene transfer. Successful overexpression of MCSF was associated with 16-fold increases in adipose tissue growth compared with a control adenovirus expressing beta-galactosidase. This occurred in the absence of increased cell size and in the presence of increased nuclear staining for MIB-1, a marker of proliferation. We conclude that MCSF participates in adipocyte hyperplasia and the physiological regulation of adipose tissue growth.     

Neurological monitoring of neurotoxicity induced by paclitaxel/cisplatin chemotherapy

Thiazolidinediones are potent antidiabetic compounds, in both animal and human models, which act by enhancing peripheral sensitivity to insulin. Thiazolidinediones are high-affinity ligands for peroxisome proliferator-activated receptor-gamma, a key factor for adipocyte differentiation, and they are efficient promoters of adipocyte differentiation in vitro. Thus, it could be questioned whether a thiazolidinedione therapy aimed at improving insulin sensitivity would promote the recruitment of new adipocytes in vivo. To address this problem, we have studied the in vivo effect of pioglitazone on glucose metabolism and gene expression in the adipose tissue of an animal model of obesity with insulin resistance, the obese Zucker (fa/fa) rat. Pioglitazone markedly improves insulin action in the obese Zucker (fa/fa) rat, but doubles its weight gain after 4 weeks of treatment. The drug induces a large increase of glucose utilization in adipose tissue, where it stimulates the expression of genes involved in lipid metabolism such as the insulin-responsive GLUT, fatty acid synthase, and phosphoenolpyruvate carboxykinase genes, but decreases the expression of the ob gene. These changes are related to both an enhanced adipocyte differentiation, as shown by the large increase in the number of small adipocytes in the retroperitoneal fat pad, and a direct effect of pioglitazone on specific gene expression (phosphoenolpyruvate carboxykinase and ob genes) in mature adipocytes.     

Changes in N-acetylaspartate and myo-inositol detected in the cerebral cortex of hamsters with Creutzfeldt-Jakob disease

Thiazolidinediones are potent antidiabetic compounds, which act by enhancing peripheral insulin sensitivity. They are also activators of the peroxisome proliferator activated receptor gamma in adipose tissue. Pioglitazone induces in vivo adipocyte differentiation in the obese Zucker fa/fa rat and hence the capacity of adipose tissue to utilize glucose. Nevertheless, muscles are the major site for insulin-mediated glucose disposal. The increase of muscle glucose utilization under thiazolidinedione treatment could be secondary to local adipose tissue differentiation. This possibility is supported by the fact that a thiazolidinedione-induced myoblast conversion into adipocytes has been described in vitro. To address this problem, we have studied the in vivo effect of a pioglitazone treatment on insulin-induced glucose utilization and the expression of genes exclusively expressed in mature adipocytes in three muscles differing by their fibre composition in Zucker (fa/fa) rats. Whereas pioglitazone treatment increased insulin-stimulated glucose utilization to the same extent in all muscle types, an adipocyte differentiation was only present in the oxidative muscle, the soleus. Soleus muscle was also the only one in which the presence of genes specific for adipose tissue could be detected before the pioglitazone treatment. There was no detectable expression of adipocyte specific genes in the extensor digitorum longus or in the epitrochlearis muscles before or after the drug treatment. We conclude that pioglitazone effects on muscle glucose metabolism cannot be due to a local adipocyte differentiation, and that the conversion of myoblasts into adipocytes under thiazolidinedione stimulation observed in vitro is, if it exists, a marginal phenomenon in vivo.     

Regional differences in triacylglycerol synthesis in adipose tissue and in cultured preadipocytes

The initial suspicion that obesity increases coronary risk has been much sharpened with the demonstration that risk is more tightly linked to abdominal than to peripheral obesity, and tighter yet again when the mass of omental adipose tissue is taken into account. These data suggest that important metabolic differences might exist between adipocytes from different regions, and indeed, it has long been appreciated that triacylglycerol hydrolysis can be stimulated to a greater extent in omental than in subcutaneous adipocytes. The present study focuses on triacylglycerol synthesis in human subcutaneous and omental adipocytes, a process which, by contrast, has received relatively little attention. Experiments were done on adipose tissue removed at laparotomy and on cultured preadipocytes. With the former, triacylglycerol synthesis was measured in the presence and absence of oleate added to the medium using radiolabeled glucose and oleate as tracers. The results demonstrate that under all conditions examined triacylglycerol synthesis in subcutaneous adipose tissue exceeded that in deep omental adipose tissue. To study the cells in more detail, preadipocytes were cultured and triacylglycerol synthesis was examined again under basal conditions and with stimulation with insulin and acylation stimulating protein (ASP). Under basal conditions, particularly when oleate was added to the medium, clear differences were present such that triacylglycerol synthesis was substantially greater in subcutaneous preadipocytes than in omentally derived preadipocytes. These differences were more pronounced when the cells were stimulated with either insulin or acylation stimulating protein. Overall, triacylglycerol synthetic capacity in subcutaneous tissue exceeded that in omental tissue. As a consequence, omental tissue as compared to subcutaneous adipose tissue would have a limited capacity to prevent fatty acids from reaching the liver and stimulating hepatic lipoprotein synthesis.     

New physiological importance of two classic residual products: carbon monoxide and bilirubin

The relationship between obese (ob) gene expression and preadipocyte differentiation was examined in primary cultures of porcine stromal-vascular (S-V) cells by Northern-blot analysis using a pig ob cDNA probe. Isolated adipocytes expressed high levels of ob gene, but S-V cells did not express the ob gene. Cultures were seeded with fetal bovine serum (FBS) plus dexamethasone (Dex) for 3 days followed by ITS (insulin 5 microg/ml, transferrin 5 microg/ml, and selenium 5 ng/ml) treatment for 6 days. Detectable levels of ob mRNA first appeared at day 1 with very low activity of glycerol phosphate dehydrogenase (GPDH). Levels of ob mRNA increased in parallel with preadipocyte number or GPDH activity at the later times in cultures. The depletion of preadipocytes by complement-mediated cytotoxicity at day 3 of culture resulted in markedly decreased ob mRNA expression. Immunocytochemical analysis showed that ob protein was localized in the cytosol of preadipocytes and adipocytes. These data indicated that the ob gene is expressed by preadipocytes and ob gene expression may be correlated with preadipocyte recruitment as well as fat cell size.     

Mutant alpha-subunit of the G protein G12 activates proliferation and inhibits differentiation of 3T3-F442A preadipocytes

We studied the G protein alpha-subunit Galpha12 in various tissues and cell lines. Significant amounts of Galpha12 were detected by immunoblots in liver, chromaffin cells, RINm5F cells, 3T3-F442A cells, and preadipocytes, but not in adipocytes, sperm, kidney, NB2A cells, or brain. To study the role of Galpha12 in adipose tissue differentiation, the preadipocyte cell line 3T3-F442A was transfected with wild-type Galpha12 or a constitutively activated mutant of Galpha12. Stable expression of the activated mutant of Galpha12 stimulated cell growth and inhibited preadipocyte differentiation. In contrast, wild-type Galpha12 overexpression inhibited preadipocyte differentiation, without any effect on cell proliferation. The role of Galpah12 on the Raf/MEK/mitogen-activating protein kinase (MAPK) cascade was studied. In confluent preadipocytes, expression of the activated mutant of Galpha12 induced an increase in B-Raf expression, but no change in MAPK activity. Differentiation was associated with a decrease in MAPK activity in control 3T3-F442A cells. Wild-type Galpha12 overexpression prevented the decrease in MAPK activity and induced MEK1, but not B-Raf, expression. Moreover, the activated mutant of Galpha12 induced an increase in MAPK activity and in the expression of both MEK1 and B-Raf. These data indicate that the activated mutant of Galpha12 stimulates the proliferation of 3T3-F442A preadipocytes, possibly through an increase in B-Raf expression, independently of the MEK/MAPK pathway, but prevents differentiation, probably through an increase in MEK1 expression and MAPK activity.     

Rapid auditory processing in normal and disordered language development

A cell type, preadipocytes, isolated from the stroma of adult human adipose tissue appears capable of differentiating, in culture, into a cell with morphological features similar to that observed in terminally differentiated human adipocytes cultured under similar conditions. During this process of differentiation, preadipocytes develop extensive rough endoplasmic reticulum with prominent cisternae, the chromatin of most nuclei becomes decondensed and lipid bodies accumulate to levels observed in cultured adipocytes. Fibroblasts derived from non-adipose tissue do not undergo the same morphological changes when cultured under similar conditions.     

Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids

The peroxisome proliferator activated receptor (PPAR gamma) plays a key role in adipogenesis and adipocyte gene expression and is the receptor for the thiazolidinedione class of insulin-sensitizing drugs. The tissue expression and potential for regulation of human PPAR gamma gene expression in vivo are unknown. We have cloned a partial human PPAR gamma cDNA, and established an RNase protection assay that permits simultaneous measurements of both PPAR gamma1 and PPAR gamma2 splice variants. Both gamma1 and gamma2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma1 was detected at lower levels in liver and heart, whereas both gamma1 and gamma2 mRNAs were expressed at low levels in skeletal muscle. To examine the hypothesis that obesity is associated with abnormal adipose tissue expression of PPAR gamma, we quantitated PPARgamma mRNA splice variants in subcutaneous adipose tissue of 14 lean and 24 obese subjects. Adipose expression of PPARgamma 2 mRNA was increased in human obesity (14.25 attomol PPAR gamma2/18S in obese females vs 9.9 in lean, P = 0.003). This increase was observed in both male and females. In contrast, no differences were observed in PPAR gamma1/18S mRNA expression. There was a strong positive correlation (r = 0.70, P < 0.001) between the ratio of PPAR gamma2/gamma1 and the body mass index of these patients. We also observed sexually dimorphic expression with increased expression of both PPAR gamma1 and PPAR gamma2 mRNAs in the subcutaneous adipose tissue of women compared with men. To determine the effect of weight loss on PPAR gamma mRNA expression, seven additional obese subjects were fed a low calorie diet (800 Kcal) until 10% weight loss was achieved. Mean expression of adipose PPAR gamma2 mRNA fell 25% (P = 0.0250 after a 10% reduction in body weight), but then increased to pretreatment levels after 4 wk of weight maintenance. Nutritional regulation of PPAR gamma1 was not seen. In vitro experiments revealed a synergistic effect of insulin and corticosteroids to induce PPAR gamma expression in isolated human adipocytes in culture. We conclude that: (a) human PPAR gamma mRNA expression is most abundant in adipose tissue, but lower level expression of both splice variants is seen in skeletal muscle; to an extent that is unlikely to be due to adipose contamination. (b) RNA derived from adipose tissue of obese humans has increased expression of PPAR gamma 2 mRNA, as well as an increased ratio of PPAR gamma2/gamma1 splice variants that is proportional to the BMI; (c) a low calorie diet specifically down-regulates the expression of PPAR gamma2 mRNA in adipose tissue of obese humans; (d) insulin and corticosteroids synergistically induce PPAR gamma mRNA after in vitro exposure to isolated human adipocytes; and (e) the in vivo modulation of PPAR gamma2 mRNA levels is an additional level of regulation for the control of adipocyte development and function, and could provide a molecular mechanism for alterations in adipocyte number and function in obesity.     

Effects of antibodies to adipocytes on body weight, food intake, and adipose tissue cellularity in obese rats

Female Wistar rats were fed on a high fat diet for 18 weeks, during which their energy intake increased by 25% and body weight by 50% due to a doubling of adipose tissue tissue stores. Animals were then treated with increasing doses of a sheep polyclonal antiserum to rat adipocytes on days 1-4 and 7 after which they remained untreated for 14 weeks. Antibody treatment reduced body weight by 10% and the weight of parametrial and subcutaneous adipose tissue by 30-40%. This decrease was explicable entirely in terms of a decrease in the number of adipocytes presumably due to adipocyte lysis. These favourable changes in body fat mass were accompanied by improvement in at least one metabolic factor associated with obesity - serum leptin concentrations were significantly reduced in treated animals compared with high fat controls. Genetically obese Zucker rats also showed decreases in the number of adipocytes after treatment with antibodies but in contrast to diet-induced obese rats, they showed a compensatory increase in adipocyte volume which attenuated the effects on body fat mass. These results demonstrate for the first time, the potential to treat diet-induced obesity with antibodies to adipocytes by producing long-term reductions in the number of adipocytes, with minimal side-effects.     

Differential expression of adenosine A1 and A2 receptors in preadipocytes and adipocytes

Multiple physiological functions have been described to be affected by adenosine in numerous cell types. A comparative study of the expression of adenosine receptors has been performed in preadipocytes and adipocytes from rat epididymal fat pad. The results show that, in agreement with its well known antilipolytic effect, adenosine induces a negative modulation of adenylate cyclase via the A1 receptor present in adipocytes. By contrast, the A2 receptor subtype, which is positively coupled to adenylate cyclase, is herein demonstrated to be only expressed in adipose precursor cells. This expression allows, in chemically defined medium, the adenosine analogue NECA, by means of its ability to elevate cAMP concentration, to potentiate differentiation. These findings emphasize the role that adenosine might play as a bimodal regulatory extracellular signal in adipose tissue development.     

Increased adrenal renin in transgenic hypertensive rats, TGR(mREN2)27, and its regulation by cAMP, angiotensin II, and calcium

We investigated the effect of the tumor necrosis factor-alpha (TNF alpha) on the differentiation of human adipocyte precursor cells in primary culture. Adipocyte precursors convert into fat cells within 12-16 days in a chemically defined, hormone-supplemented medium. Exposure of cultured preadipocytes to TNF alpha resulted in a dose- and time-dependent decrease in the number of developing fat cells and the activity of glycerol-3-phosphate dehydrogenase (GPDH), an established marker of adipocyte differentiation. A 24-h incubation with TNF alpha at a concentration of 5 nM suppressed GPDH activity by 55% compared to that in control cultures. Continuous exposure of the cells to TNF alpha completely blocked expression of the adipocyte phenotype and GPDH activity. The inhibitory action of TNF alpha was not associated with a change in cell number, as assessed by cell counting. The addition of 5 nM TNF alpha for 24 h to newly developed fat cells caused a rapid reduction of GPDH activity by approximately 50%. A 14-day exposure of differentiated cells to TNF alpha was followed by complete suppression of GPDH and a marked delipidation of the cells, including morphological changes, leading to the development of long spindle-shaped cytoplasmatic extensions. These results clearly demonstrate that TNF alpha inhibits the differentiation of human adipocyte precursor cells and, in addition, promotes the delipidation of mature fat cells. It is suggested that TNF alpha may be involved in the physiological control of human adipose tissue cellularity and function.