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The exposure of brown adipocytes to extracellular ATP leads to exocytosis of intracellular membranes and increases the purinergic receptor mediated membrane trafficking. There is also a purinergic stimulated increase in the cell surface due to adipocyte vesicle-mediated secretion. The ATP exposure also increases the total cell membrane capacitance and the cytosolic calcium (Ca) levels. Adrenergic activation mobilizes calcium without influencing the amount of membrane trafficking. This indicates that the increase in Ca content is not solely responsible for the ATP-induced cellular responses.

The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH ([pH.sub.i]) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results in a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of [Na.sup.+]/[H.sup.+] exchange in adipocytes does not prevent the changes in phi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in [pH.sub.i] occur in adipocytes in response to the passive diffusion of unionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in phi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.

The influence of the extracellular matrix (ECM) and ECM components on preadipocyte development was examined in primary cultures of adipose tissue stromal-vascular (S-V) cells. Extracellular matrix derived from Engelbreth-Holm-Swarm (EHS) cells or tumors enhanced several aspects of adipogenesis in vitro. In comparison to uncoated and fibronectin substrata, EHS-ECM substratum markedly increased attachment, spreading, and hypertrophy of preadipocytes while antagonizing spreading of non-preadipocytes. In addition, adipocyte number increased (P < .05) on these substrata despite no increase in total cell number: this resulted in a greater (P < .05) proportion of preadipocytes. These effects of EHS-ECM were also observed with laminin substrata per se, whereas types I and IV collagen and fibronectin had no influence. In contrast to all other substrata, adipocyte number decreased and total cell number increased 2.5-fold on ECM derived from corneal endothelial cells; this resulted in the lowest proportion of preadipocytes. Challenging cultures with adipogenic media (+serum) did not counter the inhibitory influence of corneal endothelial ECM, whereas dexamethasone partially neutralized the inhibitory influence of this ECM. These studies clearly show that source or type of the ECM dictated the influence of ECM substrata on preadipocyte development in primary S-V cultures. However, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Key Words: Adipocytes, Extracellular Matrix, Differentiation, Tissue Culture

To study the effects of dietary fish oil on insulin-stimulated glucose metabolism in adipocytes of insulin-resistant rats (rats fed 50% sucrose and 30% fat), eighteen 5-wk-old Sprague-Dawley rats were fed, for 6 wk, a diet containing 30% fat as either fish oil (FO) or a mixture of vegetable and animal oils [control oils (CO)]. A third reference group was fed a standard diet (62% corn starch and 13% fat). At the end of the 6-wk period, the two experimental groups had comparable plasma glucose concentrations that were higher than that found in the reference group. FO feeding corrected the hyperinsulinemia of the experimental rats (P [less than] 0.05) to reach values in the reference group. Plasma triacylglycerol (P [less than] 0.01 ) and cholesterol (P [less than] 0.001) concentrations were also lower in rats fed FO than in those fed CO. The body weights of FO-fed rats were similar to that of CO-fed rats, but epididymal adipose tissue weight was lower (P [less than] 0.01). Adipocytes of FO-fed rats, compared with those of CO-fed rats, had high insulin-stimulated glucose transport (P [less than] 0.05), oxidation (P [less than] 0.001) and incorporation into total lipids (P [less than] 0.05). The incorporation of (n-3) polyunsaturated fatty acids in adipocyte membrane phospholipids was higher in FO-fed rats than in those fed CO (P [less than] 0.0001). Insulin action was positively correlated with the fatty acid unsaturation index in membrane phospholipids. Thus dietary fish oil has beneficial effects on insulinemia, plasma lipids and insulin-stimulated glucose metabolism in insulin-resistant slightly diabetic rats. J. Nutr. 126: 1951-1958, 1996. INDEXING KEY WORDS: sucrose feeding; insulin action; rats; adipocytes; (n-3) polyunsaturated fatty acids

The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover [approximately equal to]15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located > 10.5 kb upstream of the initiator ATG codon. Three kilo-bases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5[prime] sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBP[Alpha]. Mutation of the single C/EBP[Alpha] site in this region abolished inducibility of the promoter by C/EBP[Alpha] in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.

Adipocytes from several mammalian species have predominant [[Beta].sub.3]-adrenergic receptors ([[Beta].sub.3]-AR). Attempts to classify porcine adipocyte [Beta]-adrenergic receptors ([Beta]-AR) into subtypes have not been successful. Selectivity of agonists and antagonists for stimulation of lipolysis and for ligand binding is considerably more restrictive than for the classic rat and guinea pig [Beta]-AR subtypes. The unique pattern for activity of agonists and antagonists in porcine [Beta]-AR precludes analogy to classic receptors and consequently there is no conclusive evidence regarding porcine [Beta]-AR subtypes. Porcine adipocyte membranes were used in ligand binding experiments designed specifically to demonstrate [[Beta].sub.3]-AR. Equilibrium saturation curves with dihydroalprenolol, CGP 12,177, or iodocyanopindolol indicated saturation at low concentrations with a single binding site. Equilibrium competitive ligand binding with iodocyanopindolol as radioligand and isoproterenol or propranolol as competitive ligands indicated both ligands totally inhibited radioligand binding; propranolol was more potent than isoproterenol. Nonradioactive CGP 12,177 also competed with iodocyanopindolol. Ligand binding experiments provided no evidence of a low-affinity [Beta]-AR (binding at high concentrations of ligand), the [[Beta].sub.3]-AR. Positive evidences of a [[Beta].sub.3]-AR were that CGP 12,177, a [[Beta].sub.1]- and [[Beta].sub.2]-adrenergic receptor antagonist but a [[Beta].sub.3]-AR agonist, partially stimulated porcine adipocyte lipolysis. Furthermore, transcripts for a [[Beta].sub.3]-AR, as well as a [[Beta].sub.1]- and [[Beta].sub.2]-AR, have been demonstrated in RNA from porcine adipocytes in other studies. The [Beta]-AR subtypes expressed and functional in porcine adipocytes remain unknown. The multiple ligand binding sites cannot be attributed to classic [Beta]-AR subtypes. The porcine [Beta]-AR may be a single unique receptor to impart atypical binding properties, or, more likely, multiple subtypes, each different enough from classic subtypes to impart the unique properties observed. Key Words: Pigs, Ligand Binding, Lipolysis, Adrenergic Receptors, Adipocytes

Adipocytes are stimulated to produce lactate through the alpha-adrenergic receptors. This was proven by subjecting insulin-sensitive epididymal adipocytes to either cold temperatures or glucose perfusion. Glucose-treated adipocytes produced a large amount of lactate but did not change their lipolytic rate. This response points to the reduced sensitivity of their beta-adrenergic receptors which had no effect on lactate production.

Adipocyte lipid metabolism is primarily regulated by insulin and the catecholamines norepinephrine and epinephrine. Stimulation of the [Beta]-adrenergic receptors ([Beta]-AR) by catecholamines causes an increase in the rates of adipocyte lipid degradation and a decrease in the rates of lipid synthesis. These catabolic effects are in opposition to insulin, which causes net anabolic effects. Because most of the postnatal development of adipose tissue mass in pigs results from hypertrophy of adipocytes (rapid in first few weeks of life) caused by increased net synthesis of triacylglycerol, there is interest in the modulation of [Beta]-AR in adipocytes of growing pigs. The [Beta]-AR are characterized by measuring ligand binding to the receptor to ascertain the affinity of the ligand for the receptor and the receptor number. We found the affinity of the receptor did not vary with animal age (10, 28, and 75 d), with adipose tissue depot site, or in adipocytes of protein-deficient pigs. The [Beta]-AR in obese pigs tended to have greater affinity than those in crossbred pigs of the same age and weight. The [Beta]-AR number was not different when expressed per milligram of adipocyte membrane protein in pigs of different age, in obesity, in different adipose tissue depots, or during protein deficiency. The number expressed per cell or per unit adipocyte surface area did not differ between depots or during protein deficiency. The number per cell tended to be greater in the larger cells from 75-d-old pigs than in the smaller cells from 10- and 28-d pigs. It was greatest in obese pigs with the largest adipocytes. Under the various experimental conditions (age, obesity, depot, protein deficiency), the membrane fatty acid composition was greatly different, but in most cases there was no modulation of [Beta]-AR affinity. Key Words: Pigs, Adipocytes, [Beta]-Adrenergic Receptors, [Beta]-Adrenergic Agonists

Molecular dynamics simulations were conducted for adipocyte lipid-binding protein by utilizing the apo and holo forms bound with stearic and oleic acid. The charged holo forms showed more electrostatic binding energy and more stable hydrogen bonds compared to the neutral forms. However, fatty acid behavior was barely affected by electrostatics. Instead, fatty acid reactions were related to the absence or presence of an unsaturated bond within the acyl chain.

Mouse 3T3-L1 cells differentiate into fatladen adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the [beta] and members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the [alpha] form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP[alpha], and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP[beta] and C/EBP[delta], is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated Fk506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs-cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein a, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.

Insulin-sensitive adipose and hepatoma cells contain a phosphooligosaccharide which behaves like insulin in changing the phosphorylation process of proteins. This phosphooligosaccharide is produced in response to stimulation by insulin. It is present in the caveolae containing plasma membranes. The phosphooligosaccharide is short lived, is present in the form of two species which have different charges but are interconvertible and contains a free amino group.

A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by [approximately equal to] 85%) the leptin mRNA level of adipose tissue of lean (ob/ + or +/+) mice but had no effect on the [approximately equal to] 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-fur refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.

Mouse obese gene (ob) RNA is found in all adipose tissue depots, and high levels are found in mice with hypothalamus lesions as well as mice mutant at the db locus. This indicates that the hypothalamus and the db gene are downstream of the ob gene in the adipose tissue regulatory pathway. It also appears that the db locus encodes the ob receptor.

Experimental data to define the role of L-Arginine (L-Arg) in the glucose metabolism in differentiated 3T3-LI adipocytes in culture indicates that the L-Arg's metabolic degradation is unrelated to the citric acid cycle activity in the process to enhance insulin-stimulated glycogen synthesis. L-Arg is found to have no effect on 2-deoxyglucose uptake or basal glycogen synthesis. The response to insulin is not altered by L-lysine, but the effect of L-Arg is attenuated by L-lysine.

Adipocytes express Rab3A, a GTP-binding protein mainly expressed in brain and neuroendocrine cells. In fat cells, Rab3A protein is bound to membranes and appears to promote regulated exocytosis. These proteins in adipocytes are not associated with synaptic and synaptic-like vesicles as is evident in neuronal and endocrine cells.

A study of the subcellular distribution of caveolin in rat adipocytes shows that caveolin regulates access for fusion with GLUT4-containing vesicles due to its structural segregation from GLUT4. Unlike GLUT4, caveolin is present in the plasma membrane. Immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody shows that these vesicles have no detectable caveoline.

a defect in phosphatidylinositol 3-kinase could take part in the establishment of insulin resistance in both adipose and muscle tissues in obese mice. The alterations at IRS-1 phosphorylation level appear to play an important role in insulin resistance. Developement of insulin resistance could differ depending on the tissue. There is an alteration in insulin activation of PIK in vitro of both, muscle and isolated adipocytes of young and old obese in antiphosphotyrosine and anti-IRS-1 immunoprecipitates despite unchanged total PIK activity. The quantitative aspect of defect is more prominent in old than in young.

White and brown adipocytes can identify excessive energy stores and generate a signal that restricts food intake and maintains the 'set point' of body fat. The adipocytes express tumor necrosis factor alpha whose dysregulation correlates to the obese state and hence insulin resistance. The ob ob is the recessive autosomal recessive mutation that causes the obesity in mice, with the ob gene encoding a 4.5 kb mRNA. A human homolog of the rat ob gene is seen in human fat, with the protein encoding a signal sequence that indicates the secreted form of the protein.

Overexpression of the peroxisome proliferator-activated receptor gamma 2 (PPAR-gamma-2) in fibroblast cells using retroviral vectors results in the activation of adipose differentiation in the fibroblasts by PPAR-gamma-2. The second transcription factor, CCAATT enhancer-binding protein alpha (C/EBP-alpha) incorporated during adipogenesis promotes the activation of adipogenesis by PPAR-gamma-2. The physiological effects of PPAR-gamma-2 in the fibroblasts indicates a correlation between the systemic lipid metabolism and adipocyte differentiation through PPAR-gamma-2.

An expression cloning technique and a cDNA library from 3T3-L1 adipocytes help determine a cDNA that enhances long chain fatty acid (LCFA) uptake when expressed in cultured cells. This cDNA binds integrally with membranes and codes for a 646 amino acid fatty acid transport protein (FATP) with six membrane-spanning regions. The localization of FATP on the plasma membrane of 3T3-L1 adipocytes, as revealed by immunocytochemistry and subcellular fractionation, indicates that FATP is a plasma membrane transporter for LCFAs.

Stromal-vascular (S-V) cells isolated from adipose tissue of newborn pigs (NBPC) and mature pigs (MPC) by collagenase digestion were used to evaluate differences in preadipocyte culture and development. Cells were seeded at a density of 3 x [10.sup.4] cells/[cm.sup.2] on six-well (35-mm) tissue culture plates in 3 mL of DMEM/HAM's F12 medium plus 10% fetal calf serum and cultured at 37 [degrees] C under a humidified atmosphere of 95% air:5% C[O.sub.2] for 24 h. Cells were then washed thoroughly in DMEM/HAM's F12 medium without fetal calf serum and maintained in serum free (SF) medium or SF medium supplemented with 2.5% newborn pig serum (NBPS) or mature pig serum (MPS) for 12 d. After 1 d, more NBPC adhered to the culture plates, as indicated by DNA values. After 12 d, protein per culture well was not significantly different, but DNA concentration per well remained higher (P [is less than] .05) in cultures of NBPC than in the MPC cultured in the same medium, indicating fewer MPC. Protein:DNA ratios were higher (P [is less than] .05) in cultures of MPC regardless of the medium, reflecting larger cell size. More cells containing fat deposits were seen with NBPC in all conditions in comparison with MPC, and more fat was deposited in NBPC in SF than in SF plus NBPS or MPS. The NBPC had higher (P [is less than] .05) sn-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) per protein than MPC regardless of the medium. For both cell types, GPDH activity in either serum was less than activity of cells grown in SF. This study demonstrated that adipose S-V cells from NBPC attach to the plate more efficiently, maintain a smaller cell size, and exhibit more differentiation when using morphological criteria and GPDH activity/milligram of protein. The results also indicated that the intrinsic activity of the S-V cells, rather than serum-borne factors, are responsible for successful adipose tissue culture and development.

A study of membrane association of lipoprotein lipase and cAMP-binding ectoprotein in rat adipocytes reveals that glycosylphosphatidylinositol (GPI)-proteins are not released from the cell surface upon lipolytic cleavage because of association through a receptor which recognizes the terminal inositol(cyclic) monophosphate epitope of the GPI-phospholipase C (PLC)-cleaved GPI moiety. PI-PLC-cleaved and released lipoprotein lipase LPL or Gcel reassociates with isolated plasma membranes of rat adipocytes. Removal or masking of the terminal inositol(cyclic) monophosphate moiety of hydrophilic Gcel and LPL inhibits reassociation.

Experimental studies of the brown fat cells to determine whether the coexisting beta1-, beta3- and beta2- adrenoceptors contribute to the thermogenic response of the cells reveal that despite the coexistence, beta1- and beta2-adrenoceptors do not mediate thermogenic response and that only beta3-adrenoreceptor mediates thermiogenesis. BRL-37344, CGP-12177 and norepinephrine function through the beta3-adrenoreceptor to cause thermiogenesis. Beta-antagonists inhibit the CGP-12177- and BRL-37344-induced thermiogenesis in cells.

Experiments examining the influence of sex steroids on the alpha2-adrenoreceptor (AR) expression in male hamsters reveal that testosterone increases alpha2-AR expression during the adipose conversion process. The upregulation is androgen-dependent, indicating that androgen directly affects fat cells to mediate the upregulation of alpha2-AR expression. The upregulation is not seen in female hamsters and aromatization processes do not mediate the upregulation.

Stimulation of beta1 and or beta2-adrenoceptors is responsible for the lipolytic effect of catecholamines in baboon macaque and human white fat cells while beta3-adrenoceptor controls lipolysis in dogs. Lipolysis and binding studies facilitate the characterization of beta adrenoceptor subtypes observed in white fat cells. Selective beta1 and beta2-adrenoreceptor antagonists prove the vital role of beta-adrenoreceptors in the lipolytic effect of norepinephrine.

Rat adipocytes exhibit a posttranslational process that diminishes the GLUT-4 degeneration rate when the cellular GLUT-4 amount is decreased by a pretranslational deficiency. The posttranslational process decreases the rate of GLUT-4 degradation, which is a first-order process, in both fasting and streptozotocin-induced diabetic rates. Decrease in GLUT-4 levels causes insulin-resistance in these adipocytes.

Study of the lipolytic effects of the distally acting cyclic AMP analogue on rat fat cells in epididymal and perirenal fat pads of mature and senescent Fischer 344 rats indicates the pathway for lipolysis. An adenosine regulated system is used to measure the lipolytic response to epinephrine. Anatomic site of origin of the fat cells influences the age effects on lipolysis. Several mechanisms in various fat depots are responsible for the decreased lipolytic responsiveness of fat cells to epinephrine during senescence.

Tyrosine kinase-deficient mutant insulin receptors, expressed in high concentrations in transfected rat adipose cells, do not mediate GLUT4 translocation in the basal cell surface and shift the insulin dose-response curve. These properties assign the insulin receptor tyrosine kinase activity to insulin-stimulated glucose transport in the rat adipose cells and elucidate the insulin signal transduction pathway involved in the glucose transport.

Certain reversible transcription-dependent mechanisms induce adipocyte differentiation to suppress selectively the serum-inducibility of c-jun and junB. This suppression effectively reduces the ability of the cell to grow when subjected to a 10% fetal bovine serum treatment. The suppression of junB and c-jun inducibility is not dependent on c-fos or c-myc expression.

A study was conducted to determine which fatty acids in plasma membranes of adipose tissue from ruminants are changed when the diet is supplemented with unsaturated fatty acids and to determine the effect of the fat supplement on adipocyte metabolism. Ten sheep were randomly assigned to two isonitrogenous diets containing either no added fat (control) or 5 g soybean oil/100 g diet. Perirenal fat was removed at slaughter, adipocytes isolated by collagenase digestion, and plasma membranes prepared by centrifugation on a Percoll gradient. Feeding soybean oil to the sheep increased (P < 0.05) linoleic acid (18:2(n-6)) concentration in subcutaneous fat and isolated adipocytes, suggesting partial escape of dietary unsaturated fatty acids from ruminal biohydrogenation. Soybean oil consumption also decreased (P < 0.05) concentrations of myristic acid, arachidonic acid (20:4(n-6)) and anteiso 17:0 in plasma membranes, but increased (P < 0.05) trans 18:1. Lipogenesis was not affected by diet, but lipolysis tended to be greater (P = 0.07) in sheep fed the soybean oil-containing diet than in those fed the control diet. In ruminants, fatty acids of ruminal origin, namely trans intermediates of biohydrogenation or branched-chain fatty acids of microbial lipid, may account for as much change in the composition of plasma membranes and in cellular metabolism as do the small quantities of unsaturated fatty acids in the diet that escape biohydrogenation.

Lipolysis, though species-dependent and stimulated by different hormones, is regulated by antilipolytic modulators such as catecholamines, neuropeptide Y, adenosine and prostaglandin E,. Total inhibition of lipolysis activation in nine mammalian species was possible with prostaglandin E1 (PGE1). A constitutive system related to paracrine mediators and a regulatory system with catecholamines and NPY, including other neuroendocrine messengers, caused inhibitory regulation of lipolysis in white adipocytes.

Blunted adenosine 3',5'-cyclic monophosphate (cAMP) responses related to aging in obese Sprague-Dawley rats resulted in modified regulation of fatty acid synthase (FAS) and lipoprotein lipase (LPL) gene. Isoproterenol activated cAMP deposit in isolated adipocytes quickly decreased the expression of the mRNAs for LPL and FAS. The expression of FAS and LPL mRNAs in cells from older rats is not restricted by isoproterenol.

Fasting downregulates low-density lipoprotein (LDL) receptor expression in rat adipocytes through changes at transcriptional and posttranscriptional levels. The adipose tissue and liver in fasting rats regulate LDL receptor expression through different mechanisms. Adipose cells show very low levels of LDL receptor mRNA and proteins after two days of fasting while hepatic LDL receptor components remain unaffected.

The replicative ability of preadipocytes decreases with an increase in age, although they replicate or degenerate into fat cells in rats. Epididymal preadipocyte in rats rise with senescence, but perirenal numbers are unaltered as perirenal preadipocytes in vitro and perirenal preadipocytes degenerate into fat cells. The rate of degeneration of preadipocytes into fat cells can have an undesirable impact on the preadipocyte number.

Experiments aimed at detecting the prevalence of a haparin-sensitive binding protein (HRP)-116 on adipocyte surfaces and identifying its regulatory role during lipoprotein lipase (LPL) metabolism suggested the dependence of LPL binding to adipocytes on heparin-secreted LPL-binding protein. The postulate included a partial regulatory role for LPL-binding protein interaction during LPL activation within the adipose tissue. LPL binding to adipocytes is of two types, nondirective LPL binding to heparan sulfate proteoglycans and specific protein association with HRP-116.

Experiments with mouse embryo stem with an increased expression of G(i-alpha2) to study the possibility of the promotion of terminal differentiation to adipocytes reveal that under the influence of the cytomegalovirus promoter, the expression vector yields a 1.7-fold Q205L mutant G(i-alpha2) and a 2.2-fold wild-type G(i-alpha2) increase in the steady-state levels of these G-protein alpha-subunits. Elevation of G(i-alpha2) activity promotes adipogenic conversion.

The cytosol of adipose cells reveals that the concentrated adipocyte fatty acid binding protein (A-FABP) is a 15-kDa protein. A resonance energy transfer assay is used to control the transfer of fluorescent anthroyloxy ffa(AOffa) to small unilamellar phospholipid vesicles to judge the process of ffa transfer from A-FABP to model membranes. The AOffa transfer rate is not affected by the structural features of ffa, which increase aqueous solubility. The transfer of A-FABP to phospholipid vesicles does not happen through the aqueous phase. The concentration of the acceptor membranes is directly related to the rate of AOffa transfer from A-FABP.

This study examined the influence of dietary essential fatty acids on the cooperativity of isolated adipocytes and stromal-vascular cells in the biosynthesis of prostaglandins. Sprague-Dawley rats were fed either a diet rich in essential fatty acids (20% corn oil) or a diet poor in essential fatty acids (20% tallow) for 4 wk. Preparations containing primarily adipose cells (adipocytes) or stromal-vascular cells (nonfat cells) were obtained from epididymal fat pads by collagenase digestion and repeatedly washed. Prostaglandin production was evaluated in basal and epinephrine-stimulated media after incubation with either adipocytes or adipocytes + nonfat cells. Prostaglandin E2 and prostacyclin production by adipocytes + nonfat cells was dose-dependent with epinephrine stimulation in cells from rats fed both diets. Both prostaglandin E2 and glycerol release in response to epinephrine (10-100 micromol/L) stimulation from adipocytes or from adipocytes + nonfat cells were significantly higher for cells from corn oil-fed rats than for cells from tallow-fed rats. Regardless of dietary treatment, epinephrine-stimulated prostaglandin E2 and prostacyclin release from adipocytes + nonfat cells was much greater than from adipocytes. These results suggest that a diet high in essential fatty acids precipitates a higher prostaglandin E2 secretion and that nonfat cells potentiate the secretion of prostaglandin E2 and prostacyclin by adipocytes regardless of diet.

A cloned preadipocyte cell line, ST-13, was used to study the effects of fibronectin (FN) and FN fragments on adipocyte differentiation. Fibronectin, an element of the extracellular matrix, has been known to regulate cell differentiation including adipocyte conversion. Results showed that FN in solution and as matrices inhibit differentiation of ST-13 preadipocytes, which can be reversed with GRGDPS cell recognition peptide or anti-alpha5beta1. FN fragments, on the other hand, stimulate preadipocyte differentiation.