Serum lipidomics reveals early differential effects of gastric bypass compared with banding on phospholipids and sphingolipids independent of differences in weight loss.

BACKGROUND/OBJECTIVES: Circulating phospholipids and sphingolipids are implicated in obesity-related comorbidities such as insulin resistance and cardiovascular disease. How bariatric surgery affects these important lipid markers is poorly understood. We sought to determine whether Roux-en-Y gastric bypass (RYGB), which is associated with greater metabolic improvement, differentially affects the phosphosphingolipidome compared with adjustable gastric banding (AGB). SUBJECTS/METHODS: Fasting sera were available from 59 obese women (body mass index range 37-51 kg m-2; n=37 RYGB and 22 AGB) before surgery, then at 1 (21 RYGB, 12 AGB) and 3 months follow-up (19 RYGB, 12 AGB). HPLC-MS/MS was used to quantify 131 lipids from nine structural classes. DXA measurements and laboratory parameters were also obtained. The associations between lipids and clinical measurements were studied with P-values adjusted for the false discovery rate (FDR). RESULTS: Both surgical procedures rapidly induced weight loss and improved clinical profiles, with RYGB producing better improvements in fat mass, and serum total cholesterol, low-density lipoprotein-cholesterol (LDL-C) and orosomucoid (FDR <10%). Ninety-three (of 131) lipids were altered by surgery-the majority decreasing-with 29 lipids differentially affected by RYGB during the study period. The differential effect of the surgeries remained statistically significant for 20 of these lipids after adjusting for differences in weight loss between surgery types. The RYGB signature consisted of phosphatidylcholine species not exceeding 36 carbons, and ceramides and sphingomyelins containing C22 to C25 fatty acids. RYGB also led to a sustained increase in unsaturated ceramide and sphingomyelin species. The RYGB-specific lipid changes were associated with decreases in body weight, total and LDL-C, orosomucoid and increased HOMA-S (FDR <10%). CONCLUSIONS: Concomitant with greater metabolic improvement, RYGB induced early and sustained changes in phosphatidylcholines, sphingomyelins and ceramides that were independent of greater weight loss. These data suggest that RYGB may specifically alter sphingolipid metabolism, which, in part, could explain the better metabolic outcomes of this surgical procedure.

Caspase-2, a novel lipid sensor under the control of sterol regulatory element binding protein 2.

Caspases play important roles in apoptotic cell death and in some other functions, such as cytokine maturation, inflammation, or differentiation. We show here that the 5'-flanking region of the human CASP-2 gene contains three functional response elements for sterol regulatory element binding proteins (SREBPs), proteins that mediate the transcriptional activation of genes involved in cholesterol, triacylglycerol, and fatty acid synthesis. Exposure of several human cell lines to statins, lipid-lowering drugs that drive SREBP proteolytic activation, induced the CASP-2 gene to an extent similar to that for known targets of SREBP proteins. Adenoviral vector-mediated transfer of active SREBP-2 also induced expression of the CASP-2 gene and the caspase-2 protein and increased the cholesterol and triacylglycerol cellular content. These rises in lipids were strongly impaired following small interfering RNA-mediated silencing of the CASP-2 gene. Taken together, our results identify the human CASP-2 gene as a member of the SREBP-responsive gene battery that senses lipid levels in cells and raise the possibility that caspase-2 participates in the control of cholesterol and triacylglycerol levels.

ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.

The transcription of genes encoding proteins involved in the hepatic synthesis of lipids from glucose is strongly stimulated by carbohydrate feeding. It is now well established that in the liver, glucose is the main activator of the expression of this group of genes, with insulin having only a permissive role. While ADD1/SREBP-1 has been implicated in lipogenic gene expression through temporal association with food intake and ectopic gain-of-function experiments, no genetic evidence for a requirement for this factor in glucose-mediated gene expression has been established. We show here that the transcription of ADD1/SREBP-1c in primary cultures of hepatocytes is controlled positively by insulin and negatively by glucagon and cyclic AMP, establishing a link between this transcription factor and carbohydrate availability. Using adenovirus-mediated transfection of a powerful dominant negative form of ADD1/SREBP-1c in rat hepatocytes, we demonstrate that this factor is absolutely necessary for the stimulation by glucose of L-pyruvate kinase, fatty acid synthase, S14, and acetyl coenzyme A carboxylase gene expression. These results demonstrate that ADD1/SREBP-1c plays a crucial role in mediating the expression of lipogenic genes induced by glucose and insulin.

Pioglitazone induces in vivo adipocyte differentiation in the obese Zucker fa/fa rat.

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.

Sequence of rat lipoprotein lipase-encoding cDNA.

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.

Human alpha 2A-adrenergic receptor gene expressed in transgenic mouse adipose tissue under the control of its regulatory elements.

Catecholamines regulate white adipose tissue function and development by acting through beta- and alpha2-adrenergic receptors (ARs). Human adipocytes express mainly alpha 2A- but few or no beta 3-ARs while the reverse is true for rodent adipocytes. Our aim was to generate a mouse model with a human-like alpha2/beta-adrenergic balance in adipose tissue by creating transgenic mice harbouring the human alpha 2A-AR gene under the control of its own regulatory elements in a combined mouse beta 3-AR-/- and human beta 3-AR+/+ background. Transgenic mice exhibit functional human alpha 2A-ARs only in white fat cells. Interestingly, as in humans, subcutaneous adipocytes expressed higher levels of alpha2-AR than perigonadal fat cells, which are associated with a better antilipolytic response to epinephrine. High-fat-diet-induced obesity was observed in transgenic mice in the absence of fat cell size modifications. In addition, analysis of gene expression related to lipid metabolism in isolated adipocytes suggested reduced lipid mobilization and no changes in lipid storage capacity of transgenic mice fed a high-fat diet. Finally, the development of adipose tissue in these mice was not associated with significant modifications of glucose and insulin blood levels. Thus, these transgenic mice constitute an original model of diet-induced obesity for in vivo physiological and pharmacological studies with respect to the alpha2/beta-AR balance in adipose tissue.

[New insights into adipose cell biology]. / Le tissu adipeux: nouveaux aspects.

During the last past Years, obesity had become a major public health problem, and new aspects of fat cells biology have been unraveled. First, since the discovery of leptin, adipocytes have been recognized as true endocrine cells secreting a variety of factors in a regulated manner. The role of these factors on the development of obesity-associated metabolic complications is becoming increasingly clear. Also, the process of fat cell differentiation has been uncovered, leading to the possibility of efficient targeting protein expression in adipose tIssue. Finally, lines of transgenic mice have been created, some of which are totally resistant to obesity. These models led to the identification of new potential adipose targets for the treatment of obesity.

A new look at adipocyte lipid droplets: towards a role in the sensing of triacylglycerol stores?

Lipid droplets have been considered for a long time as inert intracytoplasmic deposits formed within cells under various conditions. Recently, new tools and new approaches have been used to visualize and study these intracellular structures. This revealed new aspects of lipid droplets biology and pointed out their organized structure and dynamic composition. In adipocytes, the specialized cell type for the storage of energy as fat, lipid droplets are particularly well-developed organelles and exhibit unique properties. Also discussed in this paper is the view that lipid droplets, through specific candidate constituents, can play a role in sensing the level of their lipid stores by adipocytes.

ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species.

Fatty acid synthase (FAS), a key lipogenic enzyme, is expressed in the two major sites of fatty acid production in the body, that is, the liver and the adipose tissue. Surprisingly, the relative contribution of these sites to lipogenesis is highly variable among species. For example, besides the situation in rodents, where liver and fat are equally active, lipogenesis in some mammals such as the pig occurs principally in adipose tissue, whereas in avian species, the liver is the main lipogenic site. We addressed the question concerning the factors determining the site of fatty acid synthesis. We show that the expression of adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein (ADD-1/SREBP-1) mRNA, but not SREBP-2, is linked to FAS protein content or activity in adipose tissues and livers of pig, chicken, and rabbit. Tissue differences in ADD-1/SREBP-1 mRNA expression between species were paralleled by commensurate variations in the nuclear concentration of SREBP-1 protein. Moreover, overexpression of ADD-1/SREBP-1 by adenoviral gene transfer induces FAS in chicken adipocytes, where lipogenesis is normally low. Conversely, the expression of a dominant negative form of ADD-1/SREBP-1 in pig adipocytes downregulates FAS expression. These results reinforce the role of ADD-1/SREBP-1 as a key regulator of lipogenesis, by extending its importance to nonrodent mammals and birds. Furthermore, they establish that differential expression of ADD-1/SREBP-1 is a key determinant of the site of fatty acid synthesis in the body.-Gondret, F., P. Ferré, and I. Dugail. ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. J. Lipid Res. 2001. 42: 106;-113.

Adipocyte cholesterol balance in obesity.

Adipose tissue is specialized in the storage of energy in the form of triacylglycerol. Within the fat cell, triacylglycerols are found in a well-defined structural compartment called the lipid droplet, which occupies the vast majority of the fat cell volume. However, many other lipids are present in the lipid droplet. These include sterols, carotenoids, cholecalciferol and lipophilic toxic pollutants of the environment such as dioxins and tocopherols. The topic of this article is the role of fat cell cholesterol in adipose tissue physiology and its potential implication in pathological states such as obesity.

Impaired fat storage capacity in adipocyte precursors of I versus C57BL mice.

I mouse strain displays adipocyte hypoplasia responsible for smaller fat pad size compared with C57BL mice. We investigated possible alterations in the proliferation and/or differentiation capacity of preadipocytes from the stroma-vascular fraction of adipose tissue in the I mouse strain. Control C57BL and I mice were studied at 8 weeks of age, and both adipose and stromal cells were isolated from epididymal and inguinal adipose tissue localizations. Results showed that the lower epididymal adipose mass in I mice was accompanied by a decrease in stromal cell number compared with C57BL mice. In inguinal fat pads, total cell number in the stroma-vascular fraction was unmodified; lipoprotein lipase activity significantly increased in stromal cells from I mice compared with control mice. In this depot, further characterization of cells from the stroma-vascular fraction by separation of cells according to density showed an increased number of preadipocytes in the I mouse whole stromal cell population. These preadipocytes seemed unable to undergo terminal maturation, thus leading to a decrease in the number of mature adipocytes. These results indicated that resistance to fat accumulation in I mice is characterized by site-dependent impairment of both the proliferative rate and the differentiation capacity of adipocyte precursors.

Genetic regulation of fatty acid synthetase expression in adipose tissue: overtranscription of the gene in genetically obese rats.

We have investigated the molecular mechanism of the overactivity of fatty acid synthetase (FAS) in adipose tissue from the genetically obese Zucker rat. Purified FAS from lean and obese rat adipose tissues displayed kinetics constants, molecular weight, and immunological properties that were identical. Western blot analysis revealed that FAS overactivity in obese versus lean rat adipose tissue was paralleled by a proportionate increase in FAS mass, i.e., 4-fold increase in suckling normoinsulinemic 16-day-old pups and 25-fold in weaned hyperinsulinemic 30-day-old rats. The determination of absolute FAS mass disclosed that FAS was quantitatively a major protein in obese rat adipose tissue accounting for 13% of cytosolic proteins versus 2% in lean rat at 30 days of age. FAS hyperabundance could be ascribed to an increased relative rate of FAS synthesis that was 6-fold higher in obese than in lean rat adipose tissue. Northern blot analysis demonstrated that FAS mRNA levels in obese rats were increased 4- and 14-fold over those of lean rats at 16 and 30 days of age, respectively, in very close proportion to the 3- and 15-fold increases in FAS gene transcription rates revealed by nuclear run-on assays. Southern analysis of genomic DNA did not allow for detecting amplification or any major structural changes in the FAS gene. It is concluded that FAS overactivity, shown here to be a life-long and general feature of all adipose tissue sites in the obese rat, arises primarily from FAS gene overtranscription.

Differential effects of retinoic acid upon early and late events in adipose conversion of 3T3 preadipocytes.

When confluent 3T3-F442A cell cultures (Day 0) were grown for 3 days in fetal calf serum-supplemented medium containing isobutyl methyl xanthine and dexamethasone (induction phase) and then shifted to serum-free hormone-defined medium (expression phase), they spontaneously exhibited a sharp rise in lipoprotein lipase activity (LPL); at the peak (Day 7) the LPL activity was about 25 times higher than in control cultures and was further enhanced by insulin. Although this expression of LPL activity was spontaneous, the emergence of glycerophosphate dehydrogenase (G3PDH) activity was completely dependent upon insulin as well as upon the expression of the differentiated phenotype. In committed cells, insulin elicited sustained DNA synthesis associated with limited cell proliferation. The addition of retinoic acid during the phase of expression inhibited insulin-dependent terminal differentiation (i.e., the emergence of G3PDH activity and acquisition of the differentiated phenotype). In addition, retinoic acid counteracted the stimulating effect of insulin upon LPL activity, but affected neither the mitotic process nor the spontaneous emergence of LPL activity. When added during the phase of induction, it prevented the overall process of adipogenic differentiation. Thus, the use of retinoic acid can indicate independent control of the mitogenic and lipogenic effects of insulin following commitment to adipogenic differentiation.

[Role of adipocyte precursors in the initiation of nutritional obesity before weaning]. / Rôle des précurseurs d'adipocytes dans l'installation d'une obésité nutritionnelle avant le sevrage.

The stroma vascular fraction of adipose tissue partly consists of adipose precursor cells which can convert into adipocytes in vitro. The aim of this study was to investigate the possible contribution of cells from the stroma vascular compartment to the initiation of obesity induced by overfeeding during the early neonatal weeks in rats. Overfeeding during the suckling period was obtained by reducing the litter size. The inguinal adipose tissue of overfed rats raised in litters of 4 pups each was overdeveloped compared to that of controls raised in litters of 8 pups each, and the difference between the two groups became significant as early as 10 days of age. At this age, adipose tissue enlargement was only due to adipocyte hypertrophy; afterwards, hyperplasia of the mature fat cells contributed to the overdevelopment of adipose tissue in 15-day old overfed rats. The cell number in the stroma vascular fraction increased in the overfed group as early as 10 days of age and thus preceded the onset of mature fat cell hyperplasia. The developmental pattern of lipoprotein lipase, glycerol-3-phosphate dehydrogenase, glycerol-acyl-transferase and acyl-CoA ligase activities in stromal cells did not depend on litter size, but specific enzyme activities wee increased in 10-day old overfed rats compared to the controls. These results indicate that early overfeeding induced cell proliferation in the stroma vascular compartment and also induced the enzyme activities involved in adipose conversion to increase in these cells. This strongly suggests that precursor cell differentiation was greater in overfed rats than in control rats.

A method for separating cultured preadipocytes according to their density: application to stromal cells from overfed suckling rats.

The stroma vascular fraction of adipose tissue consists of a heterogeneous cell population; not all the cells in this compartment undergo adipose conversion in primary culture. A density gradient centrifugation procedure was used to separate cultured cells on the basis of their triglyceride content. This method was applied to both stroma vascular cells from rat adipose tissue and to a 3T3 F442A preadipose cell line as a reference. Comparison of the results obtained from these two cell types suggests that this separation procedure can lead to a quantification of adipose differentiation in the heterogeneous stromal cell population. Separation procedures were applied to cultured stromal cells derived from young rats during the onset of nutritional obesity induced by overfeeding in early life. Results show that early overfeeding induced an increase in the stromal cell differentiation capacity which is expressed in vitro.

Role of adipocyte precursors in the onset of obesity induced by overfeeding in suckling rats.

Rats were overfed during the suckling period by litter size manipulation in order to investigate the possible contribution of preadipocytes from the stroma-vascular compartment of adipose tissue to the development of obesity. Rats raised in litters of four pups were overfed; for normal feeding we assigned eight pups per litter. As early as 10 d of age, overfed rats became fatter than controls, and showed an increase in both plasma insulin and triacylglycerol levels. At this age, adipose tissue overdevelopment arose only from adipocyte hypertrophy, since hyperplasia occurred only at 15 d of age. Concurrently, compared to normal feeding, overfeeding led to significantly higher activities of lipoprotein lipase (LPL), glycerophosphate dehydrogenase (GPDH) and glycerophosphate acyltransferase (GPAT) in mature fat cells; 10-d-old overfed pups exhibited a higher stromal cell number. Further separation of this heterogeneous fraction by density gradient centrifugation showed a higher preadipocyte number as compared to that of controls. In stromal cells, LPL, GPDH, GPAT and acyl CoA ligase activities were detected during the suckling period. As compared to controls, overfeeding induced an increase in both LPL and GPDH activities in 10-d-old pups. Results indicate that overfeeding in early life induced an excess of fat storage capacity through a simultaneous increase in proliferation and differentiation rates of adipocyte precursors.

[Development of esterification capacity of fatty acids in the adipocytes of the rat before weaning: the effect of milk overfeeding]. / Evolution de la capacité d'estérification des acides gras dans les adipocytes de rat avant le sevrage: effet de la suralimentation lactée.

During the suckling period, lipid storage in rat adipose tissue arises from exogenous triglycerides, as milk contains a large amount of lipid. In the present work, we studied the effect of overfeeding during the suckling period on the developmental pattern of the fatty acid esterification pathway in fat cells. Early overfeeding was induced by restricting litter size; at 2 days of age, the pups were randomly distributed to form litters of 4 (overfed group) or 8 (controls). During the suckling period the activity of lipoprotein lipase (LPL), glycerophosphate dehydrogenase (G3PDH), glycerophosphate acyl transferase (GPAT) and acyl CoA ligase were measured in isolated fat cells from the inguinal adipose tissue. As early as 10 days of age, plasma triglyceride levels were increased in overfed pups as compared to the controls and, in these animals, the adipose tissue was overdeveloped, mainly due to fat cell hypertrophy. At that age, overfeeding induced a significant increase in LPL and GPAT activities, but acyl CoA ligase and GPDH were not modified in overfed pups. Fat cell hypertrophy appeared to be responsible for the increased GPAT activity in the overfed pups. On the contrary, the effect of overfeeding on LPL activity was not only related to fat cell enlargement, as the enzyme activity continued to increase in the overfed rats when expressed per unit of cell surface area. These results show that overfeeding during the suckling period rapidly induced an increase in the fat storage capacity of the adipose tissue. This suggests that circulating triglyceride levels could regulate directly or through hormonal control the activity of the fatty acid esterification pathway in fat cells.

Progesterone stimulates adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c gene expression. potential mechanism for the lipogenic effect of progesterone in adipose tissue.

Fatty acid synthase (FAS), a nutritionally regulated lipogenic enzyme, is transcriptionally controlled by ADD1/SREBP1c (adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c), through insulin-mediated stimulation of ADD1/SREBP1c expression. Progesterone exerts lipogenic effects on adipocytes, and FAS is highly induced in breast tumor cell lines upon progesterone treatment. We show here that progesterone up-regulates ADD1/SREBP1c expression in the MCF7 breast cancer cell line and the primary cultured preadipocyte from rat parametrial adipose tissue. In MCF7, progesterone induced ADD1/SREBP1c and Metallothionein II (a well known progesterone-regulated gene) mRNAs, with comparable potency. In preadipocytes, progesterone increased ADD1/SREBP1c mRNA dose-dependently, but not SREBP1a or SREBP2. Run-on experiments demonstrated that progesterone action on ADD1/SREBP1c was primarily at the transcriptional level. The membrane-bound and mature nuclear forms of ADD1/SREBP1 protein accumulated in preadipocytes cultured with progesterone, and FAS induction could be abolished by adenovirus-mediated overexpression of a dominant negative form of ADD1/SREBP1 in these cells. Finally, in the presence of insulin, progesterone was unable to up-regulate ADD1/SREBP1c mRNA in preadipocytes, whereas its effect was restored after 24 h of insulin deprivation. Together these results demonstrate that ADD1/SREBP1c is controlled by progesterone, which, like insulin, acts by increasing ADD1/SREBP1c gene transcription. This provides a potential mechanism for the lipogenic actions of progesterone on adipose tissue.

Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats.

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.