Fractions Of Fat Cells Research Articles

Adipose‐Specific Expression, Developmental and Nutritional Regulation of the Gene‐Encoding Retinol‐Binding Protein 7 in Pigs

Modulation of expression of adipose tissue-specific transcripts has been known to regulate adipogenesis and lipid metabolism. Recently, adipose-specific expression patterns and developmental regulation of the gene-encoding retinol-binding protein 7 (RBP7) was identified. However, its expression in adipose tissue of the porcine species has yet to be explored. In this study, adipose tissue-specific expression of porcine RBP7 was identified and conservation of the fatty acid-binding domains and evolutionary relationship of the RBP7 gene were verified comparatively across mammalian species. Our in vitro and in vivo analysis of gene expression revealed that RBP7 expression was significantly high in fat cell fraction compared to stromal vascular cells (p < 0.05) and increased during development (p < 0.05). The level of RBP7 expression was upregulated during a 24-h short-term fasting intervention and restored 6 h after refeeding (p < 0.05). Taken together, these studies provide insights into the role of RBP7 in adipose tissue of pigs during development and nutritional intervention and pave the way for future studies on the regulation of retinol homeostasis in porcine adipose tissue.

Steroid-Associated Hip Joint Collapse in Bipedal Emus

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients.

Porcine G0/G1 Switch Gene 2 (G0S2) Expression is Regulated During Adipogenesis and Short‐Term In‐Vivo Nutritional Interventions

Adipose triglyceride lipase (ATGL), catalyzing the initial step of hydrolysis of triacylglycerol (TAG) in adipocytes, has been known to be inhibited by G0/G1 switch gene 2 (G0S2). In this study, we report the porcine G0S2 cDNA and amino acid sequences as well as the expression level of porcine G0S2. The porcine G0S2 mRNA was abundantly expressed in adipose tissue and liver among various tissues. In adipose tissue, porcine G0S2 expression was 16-fold higher in the fat cell fraction than the stromal vascular fraction. The G0S2 level increased significantly during adipogenesis in vitro and in vivo. These data indicate that G0S2 expression is closely associated with lipid accumulation and adipogenesis. Considering G0S2 as an inhibitor of cell proliferation, the relatively low levels of G0S2 in preadipocytes and adipose tissues of fetal and neonatal pigs compared to adipocytes and adipose tissues of adult pigs may allow the fast cell proliferation rates. Further studies showed that a short-term 24-h fast down-regulated G0S2 expression and increased ATGL expression in adipose tissue; however, a long-term calorie restriction for 8days had no influence on the level of G0S2 but increased ATGL expression. Therefore, porcine G0S2, which is both a negative regulator of ATGL-mediated lipolysis and cell proliferation in adipose tissue, can be down-regulated in vivo by a short-term 24-h fast followed by increased ATGL-mediated lipolysis.

Biochemical Assessment of Cellular Damage after Adipocyte Harvest

Free fat transplantation for soft tissue augmentation yields variable results, which may be related to the technique of fat harvest. To compare the viability of adipocytes harvested by liposuction (sal) or by excision (exc), fat harvested by both techniques from seven lipectomy patients was analyzed by glycerol-3-phosphate dehydrogenase (G3PDH) enzyme assay. Leakage of this lipogenic enzyme through the plasma membrane is a potential indicator of fat cell damage. Preliminary experiments showed this assay to be sensitive and specific for adipocyte G3PDH activity. Treatment of fat tissue with collagenase H resulted in complete release of the component fat cells for analysis with less loss of G3PDH activity, compared to other collagenase preparations. Each sample was digested and separated into three compartments: mature adipocytes–floating layer (F), acellular supernatant (S), and stromal pellet (P). Samples from each compartment were assayed for G3PDH activity, normalized to DNA content, and represented as a percentage of the whole (F + S + P). Within the subgroups, the fat cell fraction of the liposuction samples (Fsal) showed statistically more activity than the excised samples (Fexc) by paired Student'sttest (P= 0.004). The supernatant (representing leaked G3PDH) and pellet fractions of excised samples revealed more G3PDH activity than the same fractions from liposuctioned tissue; the former (Sexc) to a significant degree (P= 0.036). Using this assay, the results indicate that liposuction fat harvest does not result in increased fat cell damage compared to fat harvested by excision.

Developmental aspects of the Adipose tissue renin‐angiotensin system: Therapeutic implications

AbstractAdipose tissue possesses considerable growth potential, and excess adipose tissue mass is associated with noninsulin dependent diabetes mellitus (NIDDM). Adipose tissue is a significant source of angiotensinogen, the precursor of angiotensin II (All), a vasoactive peptide with in vitro mitogenic effects. The renin‐angiotensin system remains only partially identified in adipose tissue, however, and knowledge of whether All has a physiologic role in this tissue is dependent in part upon other components of the system being characterized. We have chosen to further investigate aspects of the renin‐angiotensin system in separate adipose tissue depots of rats during growth and development of the tissue. Either epididymal or retroperitoneal adipose tissue was examined in young lean, or adult obese male Sprague‐Dawley rats. Adipose tissue was subjected to collagenase digestion, yielding separate vascular and fat cell fractions. Each fraction was analyzed for angiotensin converting enzyme (ACE) activity and All receptor binding capacity. ACE activity was significantly greater when expressed per gram of adipose tissue in the young lean rats. Vascular and fat cell fractions exhibited All receptors of high affinity, with a greater density of receptors observed in the epididymal adipose tissue fractions. Incubation of adipocytes with exogenous All was associated with the release of prostaglandin into the medium, and analyses of adipocyte cytosol indicated the presence of angiotensinogen. Growing rats given oral losartan, an All receptor antagonist, once daily (15 mg/kg) for 2 weeks exhibited reductions in body weight gain, fat mass, fat cell volume, and All receptor number. These data further document the presence of a peripheral renin‐angiotensin system in anatomically distinct adipose tissue depots at different stages of growth. While the characterization of the effects of All upon adipose tissue development remains incomplete, future investigations with agents affecting the adipocyte renin‐angiotensin system could provide additional information on the role of this system in adipose tissue growth.

Effect of physical training on glucose transporters in fat cell fractions.

Physical training increases maximally insulin-stimulated glucose assimilation and 3-O-methylglucose transport in epididymal fat cells. In the present report, glucose-inhibitable cytochalasin B binding in subcellular fractions of epididymal adipocytes was measured to assess changes in number of glucose transporters induced by training. Groups of rats trained by swimming were compared to control groups of the same age, matched with respect to body weight by restricted feeding. It was found that in trained rats the number of glucose transporters in the low density microsome fractions from non-insulin-stimulated fat cells was larger than in untrained rats. In both groups of rats, insulin stimulation of adipocytes decreased the number of glucose transporters in low-density microsomes by about 60% and increased the number of glucose transporters in the plasma membrane fractions. The number of glucose transporters in the plasma membrane fractions from maximally insulin-stimulated fat cells was larger in trained rats than in control rats. [U-14C]Glucose incorporation into lipids varied in proportion to plasma membrane cytochalasin B binding per cell under all conditions tested. The results explain the enhancing effect of training on insulin responsiveness transport of hexose in fat cells.

Insulin-induced rapid decrease of a major protein in fat cell plasma membranes.

In order to increase our understanding of the mode of action of insulin in rat fat cells, we investigated the effect of insulin on protein concentrations in purified fat cell fractions using two-dimensional electrophoresis in combination with an ultrasensitive color silver stain technique. Incubation of fat cells with insulin caused a 90% decrease in the plasma membrane concentration of a major plasma membrane protein with a molecular mass of 90 kDa. The insulin effect was dose-dependent with a half-maximal effect at 9.5 microunits/ml, and time-dependent with a t 1/2 of less than 20 s. Insulin-like growth factor I, orthovanadate, and lanthanum mimicked the effect of insulin. Likewise, fractionation of adipocytes in the presence of divalent cation chelating agents caused a similar reduction in the concentration of the 90 kDa protein, and it was possible to overcome the effects of the chelating agents by adding equivalent amounts of calcium. This suggests the involvement of calcium. The 90 kDa protein was also found in low and high density microsomes, but it was not affected in those fractions by either insulin or chelators. It is suggested from the study that the movement of a 90 kDa protein in fat cell plasma membranes probably represents part of the transmission system in the mechanism of insulin action in rat adipocytes.

Development of brown fat cells in monolayer culture: I. Morphological and biochemical distinction from white fat cells in culture

The ability of cells from the stromal-vascular fraction of rat brown adipose tissue to develop into adipocytes in primary cell cultures was investigated. Comparison was made with precursor cells isolated by the same procedure from the white adipose tissue of the same animals and cultured in parallel under identical conditions. The culture procedure used allowed the cells isolated from both tissues to rapidly proliferate and differentiate. During the first week in culture the brown fat cells grew to confluence and accumulated fat in a multilocular way. During the second week, further fat was accumulated, but the cells remained multilocular. Analysis of the parallel white fat cell cultures revealed clear differences between the two adipocyte types, although the rates of cell growth were identical. Measurement of the size of the cellular lipid inclusions as a function of the time in culture indicated a much higher number of fat droplets larger than 30 μm in the white adipocytes. Moreover, after isolation of pelleting fractions of both cultured cell types, comparative functional analysis of their mitochondria by oxygen consumption measurement, as well as direct cytochrome- c-oxidase determinations, showed a significantly higher amount of mitochondria in the brown fat cell fractions than in the white fat cell fractions. It was concluded that mature brown fat contains precursor cells which can proliferate and develop into adipocytes in monolayer cell culture and which have inherent characteristics distinct from those of white fat precursor cells.

Evidence that translocation of the glucose transport activity is the major mechanism of insulin action on glucose transport in fat cells.

The glucose transport activity associated with the plasma membrane-rich and Golgi-rich fractions of fat cells was determined after they were reconstituted into egg lecithin liposomes. When the two subcellular fractions were isolated under conditions that would minimize their cross-contamination, the transport activity in the plasma membrane-rich fraction was found to be increased 6.3- to 8.6-fold by insulin, which was added to cells before homogenization, and that the activity in the Golgi-rich fraction was reduced approximately to one-half. In this study, the glucose transport activity in the plasma membrane-rich fraction (either in the basal or plus insulin state) was solubilized, reconstituted, and assayed with an overall efficiency of 25-35%. Four agents known to have insulin-like effects on the glucose transport activity in intact fat cells (hydrogen peroxide, sodium vanadate, trypsin, and p-chloromercuriphenyl sulfonate) not only increased the transport activity in the plasma membrane-rich fraction, but also decreased the activity in the Golgi-rich fraction. The effect of hydrogen peroxide, unlike that of insulin, was not abolished when the insulin receptor was modified proteolytically. Upon administration of insulin to fat cells, and subsequent elimination of the hormone, the glucose transport activities associated with the plasma membrane-rich and Golgi-rich fractions were affected almost concomitantly towards opposite directions. It is proposed as a working hypothesis that translocation of the glucose transport system to the plasma membrane from the Golgi-rich fraction is the major, if not the sole, mechanism by which insulin stimulates glucose transport in fat cells.

Insulin Degradation by Isolated Fat Cells and their Subcellular Fractions

Isolated frt cells and purified subcellular fractions of fat cells have been shown to degrade insulin to biologically inactive trichloroacetic-acid-soluble fragments. Further study of this activity has revealed the following characteristics: 1 Most of the insulin-degrading enzymes are intracellular, inaccessible to insulin or trypsin when fat cells are intact. More that 90 per cent of the recovered activity is found in the high-speed supernatant (cytosol) when cell fractionation studies are performed. 2. The plasma membrane contains significant insulin-degradative capacity, as shown by tryptic digestion of intact cells and cell fractionation. 3. The pH optimum of the cell-membrane insulin-degrading site is more acid than that of the cytosol activity, but the tow enzyme systems are similar with regard to substrate specificity, response to metabolic inhibitors, and elution volume of degradation products on gel filtration. 4. The plasma-membrane-degrading activity differs from the specific insulin-binding site with regard to saturation kinetics, optimum temperature, substrate specificity, sensitivity to sulfhydryl-blocking agents, and trypsin snesitivity.

Regulation of DNA synthesis in fat cells and stromal elements from rat adipose tissue.

The incorporation of tritiated thymidine into the deoxyribonucleic acid (DNA) of adipose fat and stromal cells was followed under a variety of conditions. After in vitro incubation of adipose slices or up to 2 days after in vivo injection of the isotope, all DNA radioactivity was in the stromal cell fraction. From 2 to 15 days after thymidine injection total tissue DNA radioactivity was constant, while between 2 and 5 days after injection label in fat cell DNA increased markedly. Thus new labeled fat cells, initially collected in the stromal pool, required 2-5 days after completion of DNA synthesis to accumulate sufficient lipid to be harvested in the fat cell fraction. Fasting before thymidine injection practically abolished DNA synthesis in primordial fat cells and reduced less drastically formation of stromal elements. However fasting sufficient to deplete lipid stores by 50% neither destroyed mature fat cells nor impaired their capacity to reaccumulate fat with refeeding. Other studies evaluated the role of new fat cell formation in the process of lipid accretion accompanying refeeding. These experiments indicated that at least during the early phase of rapid weight gain, accumulation of fat was due to deposition of triglyceride in existing cells rather than to accelerated formation of new fat cells. Studies with hypophysectomized rats demonstrated that pituitary ablation variably affected stromal DNA synthesis and nearly abolished the formation and (or) maturation of primordial fat cells. In these animals growth hormone markedly enhanced thymidine incorporation into stromal DNA but had no effect on fat cell precursors. In intact animals the predominant effect of growth hormone was also on the stromal fraction, although an action of the hormone of lesser magnitude on fat cell precursors was also evident.