Induction of preadipocyte differentiation by mature fat cells in the rat.

In this study we investigated the influence of mature adipocytes, derived from rat adipose tissue, on the replication and differentiation of preadipocytes in primary culture. Mature fat did not inhibit preadipocyte replication within the 6-d period studied. Preadipocyte differentiation, as assessed by both cytoplasmic lipid accretion and an increase in glycerophosphate dehydrogenase (GPDH) activity, was significantly stimulated by the presence of mature fat tissue or isolated adipocytes. The proportion of cells containing visible lipid droplets by oil red O staining was 47 +/- 10 to 58 +/- 10% (depending on the site of origin of the preadipocytes) when cocultured with mature fat compared with less than 1 to 2 +/- 1% when cultured in medium alone, while GPDH activity was 344 +/- 9 compared with 43 +/- 3 nM NADH/min per mg protein, respectively. This effect was not due to release of triacylglycerols from damaged adipocytes. Fatty acids added to the medium promoted lipid accumulation but did not stimulate a rise in GPDH activity. We concluded that mature adipocytes may release factor(s) that promote preadipocyte differentiation (and maturation).

Development of insulin resistance in the JCR:LA-cp rat: role of triacylglycerols and effects of MEDICA 16.

The JCR:LA-cp rat develops an extreme obese/insulin-resistant syndrome such that by 12 weeks of age, there is no longer any insulin-mediated glucose turnover. At 4 weeks of age, obese and lean rats have essentially identical basal and insulin-mediated glucose uptake in skeletal muscle. By 8 weeks of age, however, the obese rats no longer exhibit such intake. Plasma insulin concentrations in the normal fed state show only small increases up to 4 weeks, with a rapid rise to a marked hyperinsulinemia thereafter, with an age at half-development of 5.5 weeks. Plasma triacylglycerol concentrations in fed obese rats are elevated at 3 weeks and rise rapidly thereafter. The triacylglycerol content of skeletal muscle is significantly elevated in the obese rats at 4 weeks of age. Histological examination of Oil Red O-stained muscle tissue and transmission electron microscopy shows the presence of intracellular lipid droplets. Treatment with the potent triacylglycerol-lowering agent MEDICA 16 (beta,beta'-tetramethylhexadecanedioic acid) from 6 weeks of age reduces plasma lipids markedly, but it reduces body weight and insulin resistance only modestly. In contrast, treatment with MEDICA 16 from the time of weaning at 3 weeks of age results in the normalization of food intake and body weight to over 8 weeks of age. The development of hyperinsulinemia is also delayed until 8.5 weeks of age, and insulin levels remain strongly reduced. Plasma triacylglycerol concentrations remain at the same level as in lean rats, and neither an elevated muscle triacylglycerol content nor intracellular lipid droplets are found at 4 weeks of age. The results indicate that insulin resistance develops in the young animals and is not directly due to a genetically determined defect in insulin metabolism. The mechanism of induction instead appears to be related to an exaggerated triacylglycerol metabolism.

Extracellular matrix components secreted by microvascular endothelial cells stimulate preadipocyte differentiation in vitro.

Paracrine interaction between preadipocytes and microvascular endothelial cells may play a role in the regulation of adipose tissue growth. We report here a study of the effect of extracellular matrix factors secreted by microvascular endothelial cells, derived from adipose tissue, on preadipocyte differentiation in primary culture. Extracellular matrix components (EC) were prepared by differential centrifugation of medium conditioned by microvascular endothelial cells (CM). Preadipocyte differentiation was assessed by enumerating cells containing Oil-Red-O-stainable neutral lipids and by assaying cellular triacylglycerol (TG) content and glycerol-3-phosphate dehydrogenase (GPDH) specific activity. Both supernatant (containing soluble components) and pelleted (containing large complexes of EC) fractions of CM stimulated preadipocyte differentiation. When the supernatant fraction was used, the proportion of cells containing visible lipid droplets was 29% +/- 3% of total preadipocytes in the presence of extracellular complexes, as compared with 6% +/- 1% under control conditions. This differentiation induction was associated with fourfold increases in TG content and GPDH specific activity. Neither the supernatant nor the pelleted fraction of EC affected the maximal differentiation induced by hormonal stimulation in serum-supplemented or serum-free media. The major EC, fibronectin, laminin, and collagen IV, had no effect on differentiation when added individually to culture medium. Collection of CM under hyperglycemic (18 mmol/L glucose) compared with control (6 mmol/L glucose) conditions reduced the stimulatory effect of extracellular complexes by twofold, suggesting decreased or altered production by endothelial cells. The present findings demonstrate that microvascular endothelial cells release EC that promote preadipocyte differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid metabolites of the lipoxygenase as well as the cyclooxygenase pathway may be involved in regulating preadipocyte differentiation.

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.

The effect of vagotomy on the increase in food intake induced by the cholecystokinin antagonist, proglumide.

Cholecystokinin, secreted when ingested food enters the duodenum, may act as a satiety factor. Injection of proglumide, a specific antagonist of cholecystokinin, induced an increase in food intake. The satiety effect of administered cholecystokinin is abolished by bilateral subdiaphragmatic vagotomy. If endogenous and exogenous cholecystokinin act via the same mechanism, then vagotomy should abolish the proglumide-induced increase in food intake. Proglumide was used to block the satiety effect of a food preload in sham-operated and vagotomized rats. Proglumide induced an increase in food intake in sham-operated rats confirming earlier results. No change in meal size was observed in vagotomized rats following proglumide injection. These results suggest that vagotomy abolishes the effect of endogenous cholecystokinin on food intake. However, evidence of dumping in vagotomized rats prevents the interpretation of the data as a direct vagal involvement in endogenous CCK-induced satiety.

The cholecystokinin antagonist, proglumide, increases food intake in the rat.

Cholecystokinin, secreted in response to ingested food entering the duodenum, may play a role in limiting food intake. Inhibition of cholecystokinin should therefore induce an increase in food intake. Proglumide, a specific antagonist of cholecystokinin was used to block the satiety effect of a food preload in rats. A significant increase in food intake was obtained following proglumide injection, thus supporting the hypothesis that cholecystokinin, released by food in the duodenum, acts as a short-term satiety factor.

Proglumide, a cholecystokinin antagonist, increases gastric emptying in rats.

Injection of cholecystokinin (CCK) reduces food intake and delays gastric emptying. We have previously shown that endogenous CCK also reduces food intake. This may be achieved by a delay in gastric emptying. We investigated the role of CCK in gastric emptying by inhibiting the actions of CCK released by a meal, using a CCK antagonist, proglumide. We postulated that inhibition of CCK should induce an increase in gastric emptying. Gastric emptying was determined in rats by a marker dilution technique using direct gastric intubation. Proglumide (150 mg/kg) significantly accelerated emptying of liquid food by 12.8% (P less than 0.005, n = 12) when injected intraperitoneally following a food preload. Proglumide injected before feeding was ineffective. Oral proglumide, which inhibited gastrin-stimulated acid secretion, was also ineffective. We concluded that proglumide increased gastric emptying by acting on a factor released by the preload, and since proglumide is a specific antagonist, this factor was probably CCK. Therefore CCK may play a physiological role in the regulation of gastric emptying.

Regulation of new fat cell formation in rats: the role of dietary fats.

Factors that stimulate formation of new adipocytes during development of obesity are yet to be identified. We examined whether diet acts directly on preadipocytes to stimulate replication and differentiation or indirectly by interacting with adipocytes to release or modify local growth factors. Male Sprague-Dawley rats were fed chow or diets high in starch (HST), saturated (HFS) or polyunsaturated (HFP) fats until 5-7 months of age. We found that, compared to other diets, HFS induced acceleration of replication of preadipocytes in primary culture (doubling time of retroperitoneal-derived preadipocytes: HFS 17 +/- 1 versus chow 32 +/- 6 and HFP 29 +/- 3 h, P < 0.05). HFS stimulated greater expansion of retroperitoneal fat than HFP even when caloric intake was equal and increased adipocyte number threefold. Preadipocyte pool size in inguinal and retroperitoneal fat pads changed relative to fat pad weight in rats fed all diets compared to chow, suggesting that the balance between the number of cells capable of replicating and those terminally differentiated was perturbed. Differentiation of preadipocytes and release of adipocyte growth factors in vitro were unaffected by diet. We concluded that dietary saturated fats induced expansion of adipose tissue mass more effectively than polyunsaturated fats and that this may, in part, be achieved by acceleration of preadipocyte replication.

Endogenous and exogenous cholecystokinin may reduce food intake by different mechanisms.

To determine whether exogenous cholecystokinin (CCK) and endogenous CCK evoke different gastrointestinal motor responses, we investigated the motility induced by CCK by use of standard manometric methods. Injection (ip) of 500 ng/kg CCK caused immediate profound gastric inhibition and duodenal phasic excitation that did not resemble the postprandial pattern. Similar profound gastric inhibition has been associated with nausea. The contribution of endogenous CCK to the fed pattern of motility was investigated using proglumide; intraperitoneal injection of the antagonist before feeding caused no change, but injection before a second meal induced a decrease (P less than 0.025, n = 6) in gastric pressure with no accompanying duodenal change. This suggests that CCK causes an increase in gastric pressure that could result from a delay in gastric emptying. In support of this hypothesis, we have previously demonstrated, by use of proglumide, that endogenous CCK delays gastric emptying. Therefore exogenous CCK may reduce food intake by evoking an abnormal gastrointestinal motor pattern that may induce malaise, whereas endogenous CCK may decrease food intake by delaying gastric emptying, thus prolonging gastric satiety signals.

Exogenous triacylglycerol inhibits insulin-stimulated glucose transport in L6 muscle cells in vitro.

This study tested the hypothesis using cultured L6 myocytes that insulin resistance in muscle may be the consequence of triacylglycerol accretion in the tissue itself. Exposure of L6 myocytes to triacylglycerol for 4 hours resulted in significant transfer of lipid into the cells compared to control cells treated for only 5 min. Insulin-stimulated 2-deoxyglucose uptake in L6 myocytes was reduced when the cells were preloaded with triacylglycerol. Insulin-independent and insulin-stimulated 2-deoxyglucose uptake were inhibited by cytochalasin B, indicating that both were transporter-mediated. Diacylglycerol mimicked insulin action by increasing 2-deoxyglucose uptake and this was also reduced by triacylglycerol preloading, suggesting that the effect was not mediated at the insulin receptor. Thus, triacylglycerol may exert a direct effect on muscle cell insulin sensitivity possibly at the level of diacylglycerol second messenger pathway.

Hepatic and adipose tissue lipogenic enzyme mRNA levels are suppressed by high fat diets in the rat.

Small changes in lipogenic enzyme activity induced by dietary fats of different composition may, over the long term, have significant impact on the development of obesity. We have investigated the effect of high fat diets (45% of calories as fat) on abundance of mRNA encoding fatty acid synthetase (FAS) and glycerophosphate dehydrogenase (GPDH) in male Sprague-Dawley rats. When caloric intake was equal, the relative amount of hepatic FAS mRNA was greater in rats fed a saturated compared to a polyunsaturated fat diet. This difference could not be attributed to diet-induced changes in plasma insulin concentration. However, both fat diets suppressed hepatic FAS mRNA compared to a sucrose diet. Close correlation between FAS specific activity and the relative amount of mRNA suggested that regulation was mainly at a pre-translational level. Adipose tissue FAS mRNA was suppressed by the two fat diets equally while GPDH mRNA was unaffected by dietary composition. Retroperitoneal fat pads were significantly larger in rats fed saturated compared to those fed polyunsaturated fat for 26 weeks. We concluded that dietary saturated fats fail to suppress hepatic de novo lipogenesis as effectively as polyunsaturated fats, which may have implications for the prevention of obesity in humans.

Paradoxically slow preadipocyte replication and differentiation in corpulent rats.

We have investigated the in vitro rate of replication and differentiation of preadipocytes derived from lean (+/+) and obese (cp/cp) male JCR:LA-corpulent (cp) rats in an attempt to identify mechanisms that regulate adipose tissue growth. Cp/cp rats were twofold heavier than age-matched lean rats by 9-10 mo. Cp/cp-derived preadipocytes demonstrated an inherently slower rate of replication than +/+ preadipocytes (population doubling time: cp/cp 52.3 +/- 9.6 h vs. +/+ 19.7 +/- 1.6 h), although the preadipocyte pool in the cp/cp was significantly greater. Cp/cp preadipocytes were resistant to hormonally induced differentiation (19.9 +/- 9.4% of cells accumulated lipid) but differentiated when cocultured with mature adipocytes to the same extent as preadipocytes derived from Sprague-Dawley (SD) rats (cp/cp 48.4 +/- 15.2% vs. SD 52.2 +/- 11.9%). In contrast, SD preadipocytes did not differentiate in response to mature adipocytes from +/+ rats (13.8 +/- 5.2%). Our observations suggest that preadipocyte replication and maturation may not be controlled in a coordinated manner.

Overexpression of the obese gene in the genetically obese JCR:LA-corpulent rat.

Expression of the obese (ob) gene in JCR:LA-cp rats was examined. A 360 bp fragment of the conserved region of the gene was obtained by RT-PCR using total RNA isolated from adipose tissues of Sprague-Dawley (SD), JCR:LA-cp obese and lean rats. The three gene fragments were sequenced and shown to be identical. They were over 90% identical to the mouse ob gene sequence. The amplified fragments encode for 120 amino acids and have a glutamine residue at position +49. The gene was shown to be expressed only in adipose tissues, both white and brown. A ten-fold increase in ob mRNA was detected in white adipose tissues of obese animals compared to the lean ones of the JCR-LA:cp strain of rat. Ob gene was expressed in adipocytes and preadipocytes from the obese rat whereas in the lean and SD rats, ob gene expression was found in adipocytes only. No ob mRNA was detected in preadipocytes from the lean or SD rats, indicating a differentiation or maturation-dependent expression in normal rats.

Influence of paracrine factors on preadipocyte replication and differentiation.

Regional adipose tissue growth may be modulated by paracrine factors that influence preadipocyte replication and/or differentiation. To investigate this hypothesis, we have studied the effects of culture media conditioned by adipose microvascular endothelial cells, preadipocytes, or mature fat cells, on rat preadipocyte replication and differentiation in vitro. Endothelial cell-conditioned medium (ECCM) stimulated preadipocyte replication while medium conditioned by mature fat or preadipocytes had little effect. ECCM contained heat and trypsin sensitive polypeptides, with molecular masses in the 18-35 kDa range. Mature fat-conditioned medium, but not medium enriched with triacylglycerols, induced differentiation in about 50 percent of preadipocytes. This effect was greatly reduced in cells derived from genetically obese JCR:LA-corpulent (cp) rats compared to those derived from lean JCR:LA-cp or Sprague-Dawley rats. The present studies demonstrate the presence of paracrine factors which may play a role in regulating regional adipose tissue growth.

A novel method for studying preadipocyte differentiation in vitro.

In vitro differentiation of rat preadipocytes has typically been induced in medium supplemented with pharmacological concentrations of hormonal mixtures. These conditions probably do not reflect the milieu within adipose tissue in vivo. We have developed a new method for inducing differentiation of preadipocytes using culture medium which has been conditioned by isolated adipocytes (ACM). In the presence of ACM, 70%-80% of test preadipocytes contained lipid inclusions compared to < 5% of control. When differentiation was assessed by assay of glycerol-3-phosphate dehydrogenase activity, ACM activity was shown to be reproducible and the consistency of response to ACM by different pools of preadipocytes was comparable to that induced by standard differentiation procedures. We have also demonstrated that the adipogenic activity of ACM may not depend on prostaglandin secretion by adipocytes. We propose that use of paracrine factors produced by components of adipose tissue provides a new approach to preadipocyte differentiation induction which may more closely reproduce the adipose tissue environment.

Long-term regulation of leptin expression is correlated with adipocyte number in obese rats.

OBJECTIVE: To investigate long-term regulation of leptin expression in adipose tissues of obese JCR:LA-corpulent rats, which have been shown to overexpress leptin. DESIGN: Manipulation of adipose tissue growth in obese rats by dietary restriction. INTERVENTIONS: Weanling female obese rats were maintained on 1 of 3 diets until 8 months old. One group was allowed to feed ad libitum, the second was pair-fed with lean rats, and the third had food intake restricted to maintain weights equal to those of age-matched lean rats. OUTCOME MEASURES: Body and fat pad weights, leptin messenger RNA (mRNA) levels, and size and number of adipocytes in retroperitoneal fat pads. RESULTS: Adipose tissue mass was increased 6-fold in the obese rats compared with the lean ones, despite equal body weight and intake restriction that was sufficient to impair growth. Although leptin mRNA level was down-regulated by intake restriction, it was still twice as elevated in the obese rats as in the lean ones, and was highly correlated with specific fat pad mass and adipocyte number, but not with size. CONCLUSIONS: These data suggest that leptin expression is correlated with adipocyte number within a fat pad, and that there is inappropriate hepatic de novo synthesis and storage of triacylglycerols in obese rats. A role for leptin in nutrient partitioning is proposed.

Fatty acid synthase and adipsin mRNA levels in obese and lean JCR:LA-cp rats: effect of diet.

In Sprague-Dawley rats, fatty acid synthase (FAS) activity is suppressed by dietary fat. To test the hypothesis that a defect in regulation of de novo fatty acid synthesis exists in massive obesity, we investigated the effect of diet on FAS mRNA levels in genetically obese JCR:LA-corpulent (cp) rats. We also determined levels of mRNA encoding adipsin, a fat cell-derived protein possibly associated with lipid metabolism. Hepatic FAS mRNA levels were elevated five-fold in obese compared to lean cp rats and were unsuppressed by dietary fat. Dietary sucrose increased FAS mRNA levels in lean cp rats, but, in contrast to Sprague-Dawley rats, little deposition of lipid resulted. Adipsin mRNA levels were fivefold lower in obese cp and Sprague-Dawley rats than in lean cp rats and were unaffected by diet. We conclude that exaggerated de novo fatty acid synthesis may play a major role in the pathogenesis of obesity in obese JCR:LA-corpulent rats.