Proliferation of unilocular fat cells in the primary culture

Mature white fat cells (unilocular fat cells) have generally been considered to be in terminal differentiation and, hence, to have no proliferative ability. A new method, referred to as "ceiling culture," has been devised in our laboratory to culture unilocular fat cells in vitro. Under such culture conditions, the fat cells continue to exhibit specific functions of lipid metabolism and proliferate extensively. Intracytoplasmic lipid droplets did not inhibit division of the cells. There were two modes of proliferation of unilocular fat cells: "loculus-dividing" cell division, in which the single loculus of fat in the dividing cell was broken down into multiple droplets and distributed evenly between the daughter cells, and "loculus-preserving" cell division, in which the loculus in the dividing cell was minimally broken down and inherited with its shape preserved by one of the daughter cells with the other getting only a small number of fine lipid droplets. Such findings suggest that unilocular fat cells in mature fat tissue in vivo are probably capable of proliferation in such modes under some conditions.     

Unilocular fat cells in three-dimensional collagen gel matrix culture

Three-dimensional culture with collagen gel, developed recently for the in vitro study of some mammalian cells in a more physiological condition than a monolayer culture, was applied for a biological study of unilocular fat cells. Successfully embedded in the gel, the unilocular fat cells were shown to be able to keep their cellular function and actively proliferate. These findings confirm that unilocular fat cells do undergo proliferation under in vitro conditions as demonstrated in monolayer culture.     

Correlation of lipolytic and lipogenic actions of synthetic peptides with structure

The effects of various synthetic peptides on basal and ACTH-stimulated lipolysis in fat cells were examined. Glu-Arg-Gly-Phe-Phe-Phe possessed lipolytic activity and increased ACTH-stimulated lipolysis at concentrations higher than 0.5 mumol/ml. Glu-Arg-Gly-Phe-Phe-Tyr did not cause any release of FFA from fat cells. Glu-Arg-Gly-Leu-Leu-Leu had no lipolytic activity but inhibited ACTH-stimulated lipolysis at concentrations higher than 0.25 mumol/ml. Glu-Arg-Gly-Leu-Leu-Leu also inhibited epinephrine-stimulated lipolysis. The effects of the peptides on basal and insulin-stimulated lipogenesis in fat cells were examined. Glu-Arg-Gly-Phe-Phe-Tyr increased both basal and insulin-stimulated lipogenesis. A tripeptide, Glu-Arg-Gly, inhibited both basal and insulin-stimulated lipogenesis. Glu-Arg-Gly-Leu-Leu-Leu had no effect on either basal or insulin-stimulated lipogenesis. Glu-Arg-Gly-Phe-Phe-Phe and ACTH, which elicit FFA release from fat cells, also stimulated formation of [14C]triglyceride from [14C]glucose.     

Dynamic changes in lipid droplet-associated proteins in the “browning” of white adipose tissues

The morphological and functional differences between lipid droplets (LDs) in brown (BAT) and white (WAT) adipose tissues will largely be determined by their associated proteins. Analysing mRNA expression in mice fat depots we have found that most LD protein genes are expressed at higher levels in BAT, with the greatest differences observed for Cidea and Plin5. Prolonged cold exposure, which induces the appearance of brown-like adipocytes in mice WAT depots, was accompanied with the potentiation of the lipolytic machinery, with changes in ATGL, CGI-58 and G0S2 gene expression. However the major change detected in WAT was the enhancement of Cidea mRNA. Together with the increase in Cidec, it indicates that LD enlargement through LD–LD transference of fat is an important process during WAT browning. To study the dynamics of this phenotypic change, we have applied 4D confocal microscopy in differentiated 3T3-L1 cells under sustained β-adrenergic stimulation. Under these conditions the cells experienced a LD remodelling cycle, with progressive reduction on the LD size by lipolysis, followed by the formation of new LDs, which were subjected to an enlargement process, likely to be CIDE-triggered, until the cell returned to the basal state. This transformation would be triggered by the activation of a thermogenic futile cycle of lipolysis/lipogenesis and could facilitate the molecular mechanism for the unilocular to multilocular transformation during WAT browning. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Lipid metabolism and adipokine levels in fatty acid-binding protein null and transgenic mice

Fatty acid-binding proteins (FABPs) facilitate the diffusion of fatty acids within cellular cytoplasm. Compared with C57Bl/6J mice maintained on a high-fat diet, adipose-FABP (A-FABP) null mice exhibit increased fat mass, decreased lipolysis, increased muscle glucose oxidation, and attenuated insulin resistance, whereas overexpression of epithelial-FABP (E-FABP) in adipose tissue results in decreased fat mass, increased lipolysis, and potentiated insulin resistance. To identify the mechanisms that underlie these processes, real-time PCR analyses indicate that the expression of hormone-sensitive lipase is reduced, while perilipin A is increased in A-FABP/aP2 null mice relative to E-FABP overexpressing mice. In contrast, de novo lipogenesis and expression of genes encoding lipoprotein lipase, CD36, long-chain acyl-CoA synthetase 5, and diacylglycerol acyltransferase are increased in A-FABP/aP2 null mice relative to E-FABP transgenic animals. Consistent with an increase in de novo lipogenesis, there was an increase in adipose C16:0 and C16:1 acyl-CoA pools. There were no changes in serum free fatty acids between genotypes. Serum levels of resistin were decreased in the E-FABP transgenic mice, whereas serum and tissue adiponectin were increased in A-FABP/aP2 null mice and decreased in E-FABP transgenic animals; leptin expression was unaffected. These results suggest that the balance between lipolysis and lipogenesis in adipocytes is remodeled in the FABP null and transgenic mice and is accompanied by the reprogramming of adipokine expression in fat cells and overall changes in plasma adipokines.     

Tools for the identification of bioactives impacting the metabolic syndrome: screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell-based assays

Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis.     

PERILIPIN-dependent control of lipid droplet structure and fat storage in Drosophila

Lipid droplets are intracellular organelles enriched in adipose tissue that govern the body fat stores of animals. In mammals, members of the evolutionarily conserved PERILIPIN protein family are associated with the lipid droplet surface and participate in lipid homeostasis. Here, we show that Drosophila mutants lacking the PERILIPIN PLIN1 are hyperphagic and suffer from adult-onset obesity. PLIN1 is a central and Janus-faced component of fat metabolism. It provides barrier function to storage lipid breakdown and acts as a key factor of stimulated lipolysis by modulating the access of proteins to the lipid droplet surface. It also shapes lipid droplet structure, transforming unilocular into multilocular fat cells. We generated flies devoid of all PERILIPIN family members and show that they exhibit impaired yet functional body fat regulation. Our data reveal the existence of a basal and possibly ancient lipid homeostasis system.     

Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity

Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.     

Regulation of lipid metabolism by dipyridamole and adenosine antagonists in rat adipocytes

1. Acute effects of dipyridamole, an inhibitor of adenosine transport, direct activators of adenylate cyclase and thirteen adenosine antagonist analogs on fatty acid synthesis have been examined in terms of the control of [1-14C]acetate incorporation into labeled fatty acids in the presence of glucose. 2. This monosaccharide acts as a stimulator of lipogenesis by generating NADPH for the lipid synthesis. 3. The relationship between lipogenesis and lipolysis was compared with a variety of adenylate cyclase stimulators. 4. The data obtained reveals that dipyridamole potentiated the inhibitory or stimulatory effects of isoproterenol and forskolin on lipogenesis and on lipolysis, respectively. 5. In these cases the data show that it exists an inverse relationship between lipogenesis and lipolysis. 6. Dipyridamole and methylxanthine analogs only moderately affect the rate of lipolysis whereas its effects are more potent on lipogenesis and lend further support to the hypothesis that dipyridamole antagonize adenosine actions as well as methyl xanthines. 7. These results suggest that dipyridamole and adenosine antagonists alter lipogenesis independently of the lipolytic process and that it exists an inverse relationship between lipogenesis and lipolysis under some conditions whereas there are not under others.     

Docosahexaenoic acid regulates serum amyloid A protein to promote lipolysis through down regulation of perilipin

Docosahexaenoic acid (DHA) increases lipolysis and decreases lipogenesis through several pathways. DHA also enhances the expression of serum amyloid A protein (SAA), a possible lipid metabolism related gene. The question of whether DHA regulates the expression of SAA to affect lipid metabolism and increase lipolysis needs to be demonstrated in human adipocytes. We designed experiments to determine the role of SAA in regulating lipid metabolism in HepG2 cells using microarray technology. In human hepatocytes, recombinant human SAA1 (hSAA1) inhibited the expression of genes related to lipogenesis and promoted the expression of those involved in lipolysis. When human breast adipocytes were treated with hSAA1 or DHA in vitro, the expression of peroxisome proliferator-activated receptor γ and other lipogenic genes was decreased, whereas the expression of several lipolytic genes was increased. Glycerol release was increased by both SAA and DHA treatments, suggesting that they increased lipolytic activity in human adipocytes. The expression of perilipin, a lipid droplet-protective protein, was decreased, and hormone-sensitive lipase was increased by both of hSAA1 and DHA treatment. We speculate that the mechanism of lipolysis by DHA or SAA is at least partially the result of increased expression of hormone-sensitive lipase and decreased expression of perilipin. Whereas DHA treatment increased expression of hSAA1 in human adipocytes, the DHA-mediated reduction in expression of lipogenesis genes and enhancement of lipolysis may be through the activity of hSAA1. These results may be useful in developing new approaches to reduce body fat deposition.     

Proliferation and differentiation of progeny of ovine unilocular fat cells (adipofibroblasts)

Summary The responsiveness of progeny of sheep-derived unilocular fat cells (adipofibroblasts) to dexamethasone, insulin, insulinlike growth factor I (IGF-I), growth hormone (GH), and basic fibroblast growth factor (FGF) was determined in a clonal culture system. Primary cultures of mature adipocytes were obtained from intermuscular adipose tissue (semimembranosus/semitendinosus seam depot) of sheep by ceiling culture techniques. Following degeneration of unilocular fat droplets and re-establishment of fibroblasticlike adipofibroblasts, all adipofibroblasts adhering to upper flask surfaces were collected and isolated away from fibroblasts (which had no multilocular vesicles) by Percoll^? gradient centrifugation. Progeny derived from a single adipofibroblast were isolated and tested for the ability to proliferate, differentiate, and accumulate lipids. Stock cultures of adipofibroblasts reached confluence in 5 d and were induced to differentiate from 7 to 9 d with dexamethasone-methyl isobutylxanthine-insulin (DMI). Incubation with insulin, IGF-I, GH, or FGF prior to confluence followed by induction with DMI produced no direct (priming) effect on subsequent differentiation. When substituted individually in place of DMI during the 2 d differentiation/induction period, all factors induced differentiation of cultured adipofibroblasts as determined by lipogenesis (P<.05) and lipoprotein lipase activity (P<.05). Thus, isolated adipofibroblasts from sheep muscle may be induced by hormones and growth factors to display mature adipocyte morphology in cell culture. Further definition of the adipofibroblast culture system may aid in the identification of mechanisms regulating adipocyte development in sheep skeletal muscle, as well as in the study of intercommunication between fat and muscle cells. With technical assistance from B. Mathison.     

Partial Inhibition of Adipose Tissue Lipolysis Improves Glucose Metabolism and Insulin Sensitivity Without Alteration of Fat Mass

When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet–fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.     

No difference in lipolysis or glucose transport of subcutaneous fat cells between moderate-fat and low-fat hypocaloric diets in obese women.

The objective of the present study was to evaluate the effect of two different diets on lipolysis and lipogenesis in subcutaneous fat cells from obese women. In a ten-week nutritional intervention study, forty women were randomly assigned to a hypoenergetic-2,514 kJ (- 600 kcal/day) diet of either moderate-fat/moderate-carbohydrate or low-fat/high-carbohydrate content. Body weight was equally reduced by approximately 7.5 % in both diet groups (p = 0.58). A subcutaneous adipose tissue biopsy was obtained for subsequent measurement of triglyceride breakdown (lipolysis) using drugs active at different steps of the lipolytic signaling cascade, and lipid synthesis (glucose transport) before and after intervention. No difference was found between the two diet groups at the maximum rate of either lipolysis or adrenoceptor sensitivity (p-values: 0.14 - 0.97). Inhibition of lipolysis by insulin was also similar in both diet groups before and after intervention. Finally, insulin-stimulated glucose transport did not show any changes that could be attributed to the type of diet. In conclusion, our data suggest that macronutrient diet composition has no major influence on glucose transport or mobilization of triglycerides in human subcutaneous fat cells of obese women.     

Effects of insulin on lipolysis and lipogenesis in hamster white adipocytes with high sensitivity to hormones

A modified procedure for preparation of hamster adipocytes by collagenase digestion under carefully controlled conditions has been developed. The adipocytes were 4- to 8-fold more sensitive to catecholamine stimulation of lipolysis than cells prepared by a commonly used method (Hittelman, K.J., Wu, C.F. and Butcher, R.W. (1973) Biochim. Biophys. Acta 304, 188-196) and also more sensitive to the anti-lipolytic action of insulin. The effects of insulin on lipogenesis, measured as [3H]glucose conversion to cell lipids, and on catecholamine-stimulated lipolysis were compared under identical conditions with the same cell batch. Isoprenaline-stimulated lipolysis was found to be half-maximally inhibited by an insulin concentration 8-fold lower than that stimulating lipogenesis to a corresponding extent (half-maximal effects at insulin concentrations of 40 vs. 300 pM). A similar difference was found when cells had been stimulated with adrenaline instead of isoprenaline.     

Effects of dietary fat and adipose tissue location on insulin action in young boar adipocytes

1. Regulation of lipogenesis and lipolysis by insulin was studied on adipocytes isolated from 100 kg Large white male pigs. Two adipose tissues were studied: subcutaneous and perirenal. Animals were fed either a control low fat diet or a diet containing 14.7% sunflower seed oil. 2. The cell diameter was higher in the group fed the sunflower diet. 3. De novo lipogenesis was decreased for each adipose tissue in the group fed the sunflower diet. The perirenal site had a higher lipogenic activity than subcutaneous site whatever the diet. 4. Insulin did not significantly stimulate lipogenesis but had an important antilipolytic effect on stimulated lipolysis by isoproterenol. 5. The antilipolytic action of insulin was higher in perirenal adipocytes with the control diet. With the sunflower diet, the decrease was about 54.4% for subcutaneous adipocytes, whereas the inhibition was decreased in perirenal adipocytes. Addition of theophylline reversed the antilipolytic action of insulin. 6. Insulin binding was not affected neither by the dietary fat nor by the adipose tissue location. 7. Absence of de novo lipogenesis stimulation by insulin was not due to an impairment in insulin binding. 8. The different effects of dietary fat and adipose tissue location on the antilipolytic action of insulin could not be explained by a modification of insulin binding but rather by a latter event, probably at a post-insulin binding stage.     

Biomodulator-mediated susceptibility of endogenous lipid droplets from rat adipocytes to hormone-sensitive lipase

The amount of fatty acid release by a fat cell homogenate without pretreatment with epinephrine was found to be slightly more than that released from fat cells by epinephrine, suggesting that fat cells contain high lipolytic activity even in the absence of lipolytic agents. Fat cells contain high hormone-sensitive lipase activity (1383 mumole free fatty acids/g/hr) in the absence of epinephrine, and addition of epinephrine to the cells did not increase the activity, significantly. Like epinephrine, DBcAMP and/or theophylline also elicited marked release of glycerol from fat cells without activating the hormone-sensitive lipase activity. However, although fat cells contain a large amount of hormone-sensitive lipase, lipolysis was negligible in the absence of these lipolytic agents. These results suggest that lipolytic agents such as epinephrine, DBcAMP, and theophylline induce lipolysis in fat cells through some mechanism other than activation of hormone-sensitive lipase and that in the absence of lipolytic agents, some system in fat cells inhibits lipolysis of endogenous lipid droplets by hormone-sensitive lipase. The lipid droplets in fat cells consist mainly of triglyceride with phospholipids, cholesterol, carbohydrate, and protein as minor constituents. The phospholipid fraction was found to consist of 75% phosphatidylcholine and 25% phosphatidylethanolamine. Of the minor constituents of endogenous lipid droplets, only phosphatidylcholine strongly inhibited hormone-sensitive lipase activity in a [3H]triolein emulsion. These results suggest that phosphatidylcholine in endogenous lipid droplets may be responsible for inhibition of hormone-sensitive lipase. Then, a cell-free system was established in which epinephrine, DBcAMP, and theophylline stimulated lipolysis of endogenous lipid droplets from fat cells by lipase solution. In this system, these lipolytic agents did not induce lipolysis in the absence of added lipase. Lipolysis in the mixture of the endogenous lipid droplets and lipase solution was accelerated by phospholipase C with concomitant loss of epinephrine-induced lipolysis. After pretreatment of the endogenous lipid droplets with phospholipase C, these lipolytic agents no longer induced lipolysis. Pretreatment of the endogenous lipid droplets with phospholipase C reduced their phospholipid content with the formation of phosphorylcholine, but did not affect their triglyceride and cholesterol contents. Treatment of the endogenous lipid droplets with phospholipase D did not affect lipolysis in the cell-free system. These results suggest that phosphatidylcholine in the endogenous lipid droplets may inhibit their lipolysis by hormone-sensitive lipase in fat cells and also be involved in the mechanisms of the stimulatory effects of epinephrine, DBcAMP, and theophylline on lipolysis.     

Evaluation of polyvinyl alcohol for fatty acid supplementation in adipose tissue explant culture

Cultures of adipose tissue explants are a valuable tool for studying the intracellular mechanisms involving hormones and nutrients. However, testing how fatty acids affect cells requires a carrier molecule; bovine serum albumin (BSA) has been used for this purpose. However, contaminants can alter the cellular response. Our objectives were to: 1) test BSA as a fatty acid carrier and 2) evaluate polyvinyl alcohol (PVA) as a replacement for BSA. Adipose tissue explants from nine pigs were cultured in medium 199 for 4, 12, 24, and 48 h, with the following treatments: control, PVA (100 mM PVA added) and PVA + pGH (100 mM PVA plus 0.1 mg/mL porcine growth hormone). After each culture period, explants were collected and assayed for lipogenesis. After 48 h in culture, explants were assayed for lipolysis. A preliminary study with different commercial sources and high concentrations showed that BSA affected lipogenic rates. On the other hand, there were no effects of PVA on lipid synthesis, while pGH (positive control) reduced glucose incorporation into lipids (P < 0.01) when compared to both control and PVA (P < 0.05). There was no difference between control and PVA for lipolysis rates. However, pGH increased lipolysis when compared to control (P < 0.01) and PVA (P < 0.05). We demonstrated that BSA can alter lipogenesis, which precludes its use as a carrier molecule. On the other hand, addition of PVA had no effect on lipolysis or lipogenesis. We suggest the use of PVA instead of BSA for adding bioactive fatty acids to cultures of adipose tissue.     

Effects of central or peripheral leptin administration on norepinephrine turnover in defined fat depots

Leptin preserves lean tissue but decreases adipose tissue by increasing lipolysis and/or inhibiting lipogenesis. The sympathetic nervous system (SNS) is a primary regulator of lipolysis, but it is not known if leptin increases norepinephrine turnover (NETO) in white adipose tissue. In this study, we examined the effect of leptin administered either as a chronic physiological dose (40 microg/day for 4 days from ip miniosmotic pumps) or as an acute injection in the third ventricle (1.5 microg injected two times daily for 2 days) on NETO and the size of brown and white fat depots in male Sprague Dawley rats. NETO was determined from the decline in tissue norepinephrine (NE) during 4 h following administration of the NE synthesis inhibitor alpha-methyl-para-tryrosine. The centrally injected leptin-treated animals demonstrated more dramatic reductions in food intake, body weight, and fat pad size and an increase in NETO compared with the peripherally infused animals. Neither route of leptin administration caused a uniform increase in NETO across all fat pads tested, and in both treatment conditions leptin decreased the size of certain fat pads independent of an increase in NETO. Similar discrepancies in white fat NETO were found for rats pair fed to leptin-treated animals. These results demonstrate that leptin acting either centrally or peripherally selectively increases sympathetic outflow to white fat depots and that a leptin-induced change in fat pad weight does not require an increase in NETO.