Rabbit Adipocytes Research Articles

Pioglitazone reduces tumor necrosis factor-α serum concentration and mRNA expression of adipose tissue in hypercholesterolemic rabbits

Background Tumor necrosis factor-α (TNF-α) is an inflammatory cytokine involved in atherogenesis. Adipose tissue is an important source of endogenous TNF-α production. Pioglitazone, a member of the thiazolidinediones (TZDs), has anti-inflammatory and anti-atherogenic properties, while underlying mechanism has not been fully elucidated. The aim of this study was to evaluate the effect of pioglitazone on TNF-α serum concentration and mRNA expressions of subcutaneous adipose tissue in hypercholesterolemic rabbits. Methods Ten rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into two groups: (1) high cholesterol group (n = 5): maintained high cholesterol diet for 4 weeks; (2) pioglitazone group (n = 5): the same cholesterol diet plus pioglitazone (3 mg/kg/day) for 4 weeks. Control group (n = 5) was fed with normal diet for 12 weeks. Subcutaneous adipose tissue was collected for RNA analysis. The direct effect of pioglitazone on TNF-α release was assayed in primary rabbit adipocytes. TNF-α levels in serum and adipocytes culture supernatant were measured by ELISA. RT-PCR was used to evaluate TNF-α mRNA expressions in adipose tissue and adipocytes. Results Compared with control group, rabbits fed with high cholesterol diet showed significantly higher levels of serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and TNF-α. Though having no effect on serum glucose level and lipid profile, pioglitazone administration significantly reduced circulating TNF-α concentrations, which were positively correlated with TNF-α mRNA expressions of adipose tissue (r = 0.53, P < 0.01). Pioglitazone dose-dependently inhibited lipopolysaccharide (LPS)-induced TNF-α secretion and mRNA expression in cultured adipocytes. Conclusion Pioglitazone significantly reduced serum TNF-α level in hypercholesterolemic rabbits independent of its metabolic actions, which may at least partly be due to its direct inhibition of TNF-α expression and secretion of adipocytes. This may help to explain the mechanism by which pioglitazone exert anti-atherosclerotic effects.

Effect of niacin on adipocyte leptin in hypercholesterolemic rabbits

Leptin may play an important role in the development of atherosclerosis. Several transcription genes [including peroxisome proliferator-activated receptor gamma (PPARgamma) and CD36] involved in lipid and glucose metabolism and inflammatory processes may correlate to leptin expression. The aim of this study was to investigate the effect of niacin on serum leptin levels in hypercholesterolemic rabbits and the expression of leptin, PPARgamma, and CD36 in adipocytes from hypercholesterolemic rabbits. Eighteen rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into two groups: (a) high-cholesterol group (n=6), which is maintained on high-cholesterol diet for 6 weeks, and (b) niacin group (n=6), which receives the same cholesterol diet plus niacin (200 mg/kg/day) for 6 weeks. The control group (n=6) was fed with normal diet for 14 weeks. Subcutaneous adipose was collected for RNA analysis. The direct effect of niacin on leptin release was assayed in hypercholesterolemic rabbit adipocytes. Leptin levels in serum and adipocyte culture supernatant were measured via enzyme-linked immunosorbent assay. RT-PCR was used to evaluate leptin, PPARgamma, and CD36 mRNA expression in adipose and adipocytes. Compared with the control group, rabbits fed with high-cholesterol diets showed higher levels of serum total cholesterol, low-density lipoprotein cholesterol, and leptin, all of which were significantly reduced by niacin treatment. After 6 weeks of treatment with niacin, the leptin level was significantly decreased by 21.8% (6.87+/-1.58 vs. 8.79+/-1.45, P<.05) and leptin mRNA expression of adipose was significantly lower in rabbits treated with niacin than in those fed with high-cholesterol diet continuously (0.58+/-0.11 vs. 0.73+/-0.15, P<.05). Niacin dose-dependently inhibited leptin secretion and increased CD36 and PPARgamma expression in cultured adipocytes. The reduction of leptin mRNA expression of hypercholesterolemic rabbits by niacin was negatively correlated with the up-regulation of PPARgamma and CD36 mRNA expression by niacin (r=-.69 and r=-.63, respectively, P<.01). Niacin can reduce serum level and adipose mRNA expression of leptin and up-regulate PPARgamma and CD36 mRNA expression in hypercholesterolemic rabbits.

Effect of niacin on LXRα and PPARγ expression and HDL-induced cholesterol efflux in adipocytes of hypercholesterolemic rabbits

Background Adipose tissue contains a large amount of cholesterol and performs a buffer function for circulating cholesterol. Liver X receptors (LXR) α and peroxisome proliferator-activated receptor γ (PPARγ) might play a significant role in adipocyte cholesterol metabolism through mediation of cholesterol efflux. The aim of this study was to evaluate the effect of niacin on LXRα and PPARγ expression and HDL-induced cholesterol efflux in adipocytes from hypercholesterolemic rabbits. Methods Twelve rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into two groups: (1) high cholesterol group ( n = 6): maintained high cholesterol diet for 6 weeks; (2) niacin group ( n = 6): the same cholesterol diet plus niacin (200 mg/kg/d) for 6 weeks. Control group ( n = 6) was fed with normal diet for 14 weeks. Subcutaneous adipose was collected for adipocyte culture. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate adipocytes LXRα mRNA expressions. Cholesterol efflux rate was determined through measuring release of radioactivity from 3H-cholesterol prelabeled cells into medium containing high-density lipoprotein (HDL). The direct effect of niacin on LXRα and PPARγ mRNA expression in primary rabbit adipocytes was assayed. Results High cholesterol diet resulted in decreased LXRα mRNA expressions and reduced HDL-induced cholesterol efflux rate in adipocytes. Six weeks of niacin treatment significantly enhanced the cholesterol efflux from adipocytes, which was related to the increased mRNA expressions of LXRα ( r = 0.71, P < 0.05). In in vitro study, niacin dose-dependently stimulated LXRα and PPARγ mRNA expression in cultured adipocytes. And various doses of niacin-induced cholesterol efflux was positive correlation with LXRα and PPARγ mRNA expression ( r = 0.83 P < 0.01; r = 0.76 P < 0.05; respectively). Conclusion Niacin can up-regulate LXRα and PPARγ mRNA expression and promote the HDL-induced cholesterol efflux in adipocytes from hypercholesterolemic rabbits.

Avaliação da gordura orbitária de coelhos após enucleação e evisceração

To evaluate rabbit adipocytes of the orbital fat tissue after enucleation and evisceration. Twenty-tree specimens from 23 rabbits aged 42 days which had undergone socket surgery (evisceration and enucleation) were evaluated. The animals were sacrificed on the 30th, 90th and 180th postoperative day. The orbital fat tissue was prepared for light microscope evaluation (magnification: 200x) and analyzed by IpWin 32 software program. The number of cells in each field and the area of each adipocyte were used to calculate the cell median area. The data were compared between the groups (enucleation and evisceration) and submitted to statistical analysis by a non-parametrical test. There were no statistical differences when considering the mean area of adipocytes between enucleation or evisceration or time of sacrifice. The volume decrease in the anophthalmic cavity is not related to orbital fat atrophy. The genesis of orbital volume reduction might result from the spacial distribution and interrelationships of the orbital soft tissue rather than changes in volume of adipocytes.

Effect of atorvastatin on SR-BI expression and HDL-induced cholesterol efflux in adipocytes of hypercholesterolemic rabbits

Background Adipose tissue contains a large amount of cholesterol and performs a buffer function for circulating cholesterol. Scavenger receptor class B type I (SR-BI) might play a significant role in adipocytes cholesterol metabolism through mediation of cholesterol efflux. We evaluated the effect of atorvastatin on SR-BI expression and HDL-induced cholesterol efflux in adipocytes from hypercholesterolemic rabbits. Methods Sixteen rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into 2 groups: (1) maintained on a high cholesterol diet for 6 weeks ( n = 8); (2) the same cholesterol diet plus atorvastatin (2.5 mg/kg/day) for 6 weeks ( n = 8). Control group ( n = 5) was fed with normal diet for 14 weeks. Subcutaneous adipose was collected for adipocyte culture. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate adipocytes SR-BI mRNA expression. Cholesterol efflux rate was determined through measuring release of radioactivity from 3H-cholesterol prelabeled cells into medium containing high-density lipoprotein (HDL). The direct effect of atorvastatin on SR-BI mRNA expression in primary rabbit adipocytes was assayed. Results High-cholesterol diet decreased SR-BI mRNA expression and reduced HDL-induced cholesterol efflux rate in adipocytes. Six weeks of atorvastatin treatment significantly enhanced the cholesterol efflux from adipocytes, which was related to the increased mRNA expression of SR-BI ( r = 0.58, P < 0.05). Adipocytes SR-BI mRNA expression were negatively correlated with the serum total cholesterol levels at the end of the study ( r = − 0.46, P < 0.05). Atorvastatin dose-dependently stimulated SR-BI mRNA expression in cultured adipocytes. Conclusion Atorvastatin can up-regulate SR-BI mRNA expression and promote the HDL-induced cholesterol efflux in adipocytes from hypercholesterolemic rabbits possibly through lowering serum cholesterol levels and directly stimulating SR-BI mRNA expression.

Effect of Atorvastatin on Tumor Necrosis Factor α Serum Concentration and mRNA Expression of Adipose in Hypercholesterolemic Rabbits

Tumor necrosis factor alpha (TNFalpha) is an inflammatory cytokine involved in atherogenesis. Adipose tissue is an important source of endogenous TNFalpha production. The aim of this study was to evaluate the effect of atorvastatin on TNFalpha serum concentration and mRNA expressions of subcutaneous adipose in hypercholesterolemic rabbits. Sixteen rabbits fed with a high-cholesterol diet for 8 weeks were randomly divided into 2 groups: (1) the high-cholesterol group (n=8) was maintained on a high-cholesterol diet for 6 weeks; (2) the atorvastatin group (n=8) had the same high-cholesterol diet plus atorvastatin (2.5 mg/kg/d) for 6 weeks. A control group (n=5) was fed with a normal diet for 14 weeks. Subcutaneous adipose was collected for mRNA analysis. Additionally, the direct effect of atorvastatin on TNFalpha release and mRNA expression was assayed in primary rabbit adipocytes. TNFalpha levels in serum and adipocyte culture supernatant were measured by ELISA. RT-PCR was used to evaluate TNFalpha mRNA expression in adipose and adipocytes. Serum TNFalpha concentration was significantly associated with serum total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) (both P<0.01). Compared with the control group, rabbits fed with a high-cholesterol diet showed higher levels of TNFalpha serum concentration and mRNA expression of adipose, both of which were significantly reduced by atorvastatin treatment (both P<0.05). TNFalpha mRNA expressions of adipose were significantly correlated with circulating TNFalpha levels among the 3 groups (r=0.51, P<0.05). Atorvastatin dose-dependently inhibited lipopolysaccharide (LPS)-induced TNFalpha secretion and mRNA expression in cultured adipocytes. In conclusion, atorvastatin can directly inhibit TNFalpha expression and secretion in adipocytes. Atorvastatin reduced TNFalpha serum concentration in hypercholesterolemic rabbits, which might be because of its cholesterol-lowering effect and direct inhibition of TNFalpha expression in adipose.

Atorvastatin reduces serum leptin concentration in hypercholesterolemic rabbits

Background Leptin may play an important role in the development of atherosclerosis. We evaluated the effect of atorvastatin on leptin secretion in vivo and in vitro. Methods Sixteen rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into 2 groups: (1) high cholesterol diet for 6 weeks ( n = 8), and (2) the same cholesterol diet plus atorvastatin (2.5 mg/kg/day) for 6 weeks ( n = 8). A control group ( n = 5) was fed with normal diet for 14 weeks. Subcutaneous adipose was collected for RNA analysis. The direct effect of atorvastatin on leptin release was assayed in primary rabbit adipocytes. Leptin levels in serum and adipocytes culture supernatant were measured by ELISA. RT–PCR was used to evaluate leptin mRNA expressions in adipose and adipocytes. Results Compared with control group, rabbits fed with high cholesterol diet showed higher levels of serum total cholesterol, LDL cholesterol and leptin, all of which were significantly reduced by atorvastatin treatment. Leptin mRNA expression of adipose was significant lower in rabbits treated with atorvastatin than those fed with high cholesterol diet continuously (0.81 ± 0.31 vs. 1.23 ± 0.36, P < 0.05). Atorvastatin dose-dependently inhibited leptin secretion and mRNA expression in cultured adipocytes. Conclusion Atorvastatin can inhibit leptin release and mRNA expression, and reduces serum leptin level in hypercholesterolemic rabbits.

Benzylamine exhibits insulin-like effects on glucose disposal, glucose transport, and fat cell lipolysis in rabbits and diabetic mice.

Benzylamine, a substrate of semicarbazide-sensitive amine oxidase (SSAO), stimulates glucose transport in rat adipocytes and improves glucose disposal in diabetic rats only in the presence of vanadate. These effects have been described to result from a synergism between the hydrogen peroxide formed during amine oxidation and vanadate, via the generation of pervanadate, a powerful insulin mimicker. However, it has also been reported that benzylamine alone can stimulate glucose uptake and inhibit lipolysis in human fat cells. In this work, we therefore investigated whether benzylamine on its own was able to induce both in vivo and in vitro insulin-like responses in animal models other than rat. In rabbits, the i.v. infusion of 7 micromol/kg benzylamine before a glucose tolerance test resulted in a net reduction of the hyperglycemic response without a change in insulin secretion. Benzylamine also improved glucose tolerance and reduced lipid mobilization in hyperglycemic/obese mice. In vitro, 0.1 mM benzylamine stimulated glucose transport and inhibited lipolysis in mouse and rabbit adipocytes. These effects were blocked by previous treatments with semicarbazide, a SSAO inhibitor. Levels of benzylamine oxidation were more elevated in mouse than in rabbit adipose tissues, whereas the reverse was observed for skeletal muscles. Finally, benzylamine was unable to stimulate insulin secretion by isolated pancreatic islets from both species and SSAO activity was hardly detectable in pancreas. Together, our results bring evidence that benzylamine on its own can improve glucose tolerance in rabbit and mouse, likely by stimulating glucose uptake via amine oxidase activation in insulin-sensitive tissues.

Effects of verapamil and elgodipine on isoprenaline-induced metabolic responses in rabbits

Verapamil (0.17 μg kg −1 min −1 intravenous, i.v.) but not elgodipine (35 ng kg −1 min −1) modestly enhanced the weak blood glucose increase induced by the i.v. infusion of isoprenaline (0.3 μg kg −1 min −1) in conscious rabbits. However, elgodipine but not verapamil suppressed the increase in circulating insulin evoked by the agonist. Both drugs enhanced the rise in plasma lactate mediated by isoprenaline but only elgodipine potentiated the lipolytic effect of the agonist. In isolated islets elgodipine (10 −6 M) blocked forskolin (10 −6 M)-induced insulin release. However, in rabbit adipocytes elgodipine potentiated both glycerol release and cAMP accumulation induced by isoprenaline (10 −8–10 −6 M). Excess K + (40–60 mM) did not alter basal lipolysis or the response to isoprenaline in either rabbit or mouse adipocytes. Therefore, Ca 2+ influx through L-type Ca 2+ channels does not seem to play a significant role in the lipolytic effect of isoprenaline. Metabolic alterations found with Ca 2+ channel antagonists were of minor intensity and probably devoid of pathological implications.

Rodent Insulin Receptors Are Immunologically Different from Other Mammalian Insulin Receptors

Four monoclonal antibodies (MA-5, MA-10, MA-20, and MA-51) and one polyclonal antibody (ARS-2) against human insulin receptor were used to immunoprecipitate the insulin receptor from several species which had been photolabeled with Nϵ B29-monoazidobenzoyl-[125I]iodoinsulin. All four monoclonal antibodies immunoprecipitated human insulin receptor from human placental membranes. MA-10 and MA-51, but not MA-5 or MA-20, immunoprecipitated insulin receptors from liver plasma membranes of rabbit, guinea pig, dog, cattle, pig, and chicken. None of the monoclonal antibodies immunoprecipitated insulin receptors of rat, mouse, hamster, or chinchilla. In contrast, all of the insulin receptors were immunoprecipitated by the polyclonal anti-insulin receptor antibody, ARS-2. MA-10 and MA-51 compared with [125I]iodoinsulin for binding to guinea pig and rabbit liver plasma membranes in a fashion similar to insulin, although less effectively. MA-51 also mimicked the action of insulin by stimulating lipogenesis and autophosphorylation of the insulin receptor β subunit in isolated rabbit adipocytes. The results suggest that insulin receptors of mammals, other than rodent, share with human insulin receptor the same epitope(s) recognized by MA-10 and MA-51. Rodent insulin receptors, with the exception of guinea pig, are different. We speculate that the difference lies in the amino acid sequence 485-599 of the α subunit of the insulin receptor.

Insulin inhibits lipolytic activity and degradation of β-lipotropin in rabbit adipose tissue

β-Lipotropin, a pituitary peptide, is a potent stimulator of lipolysis in rabbit adipose tissue in vitro and in vivo. Insulin inhibited the β-lipotropin (1–100 nM)-stimulated glycerol release from rabbit adipocytes and fat pads significantly at concentrations of 10 and 100 μM. Both these concentrations of insulin also decreased the degradation of β-lipotropin in intact adipose tissue to the same extent as the lipolytic activity. Furthermore, insulin reduced the degradation of β-lipotropin in rabbit adipose tissue homogenate. Like insulin, several lysosomotropic agents also decreased significantly the degradation and the lipolytic activity of β-lipotropin. On the other hand, insulin-like growth factor I in lower concentrations (1–100 nM) did not effect degradation and lipolytic activity of β-lipotropin in rabbit adipose tissue. Thus, a direct influence of insulin on lysosomal enzymes degrading β-lipotropin in rabbit adipose tissue can be suggested.

Processing of the lipid-mobilizing peptide β-lipotropin in rabbit adipose tissue

β-Lipotropin, a pituitary peptide, is a strong stimulator of lipolysis in rabbit adipose tissue. This polypeptide is shown to be degraded by intact fat pads, homogenized adipose tissue and adipocytes of the rabbit dependent on the amount of adipose tissue, time and the pH of the incubation medium. In subcellular fractions of rabbit adipocytes the proteolytic activity could be localized into the cytosol and the microsomal fraction. To obtain information about the processing of β-lipotropin in its target cell lipolysis and degradation of this polypeptide were investigated in the presence of inhibitors of distinct cellular mechanisms and in different physiological states such as obesity and starvation. Thus, the stronger lipolytic response in adipocytes from obese rabbits respectively animals fed ad libitum was accompanied by a significantly increased degradation in comparison to lean respectively starved rabbits. The six lysosomotropic agents (chloroquine, NH 4Cl, propranolol, quinacrine, acridine orange and tetracaine), the proteinase inhibitors α 2-macroglobulin and monodansylcadaverine, cellular ATP depletion by 2-deoxy- d-glucose and 2,4-dmitrophenol and the omission of Ca 2+ ions from the incubation medium inhibited dose-dependently the lipolytic activity as well as the degradation of β-lipotropin in intact and homogenized adipose tissue. Inhibitors of the cytoskeleton such as colchicine, cytochalasin B, vinblastine and concanavalin A also reduced lipolysis but only the degradation in intact adipose tissue. It can be concluded that after receptor-mediated uptake the cytoskeleton and lysosomal proteases are involved in the processing of β-lipotropin.

Discrimination between α2-adrenoceptors and [ 3H]idazoxan-labelled non-adrenergic sites in rabbit white fat cells

Previous results indicated that the rabbit could represent a suitable model for investigations on the functional role of α 2-adrenoceptors in fat cells, but the characterization of these receptors was not resolved yet. In the present report, imidazoline compounds were used to attempt a better definition of rabbit adipocyte α 2-receptivity by means of lipolysis and binding studies. Lipolysis data showed that UK14304 is a full α 2-adrenoceptor agonist promoting a strong antilipolysis in rabbit fat cells. Moreover, the methoxy derivative of idazoxan, RX821002, is a more potent antagonist of UK14304-induced antilipolysis than idazoxan or yohimbine. Whereas [ 3H]yohimbine failed to bind at rabbit adipocyte α 2-adrenoceptors, [ 3H]UK14304 and [ 3]RX821002 are valuable tools to study this receptor. Analysis of binding data demonstrated that [ 3H]UK14304 labels the high-affinity-state receptor while [ 3H]RX821002 binds to the whole α 2-adrenergic population. Inhibition studies [ 3H]RX821002 and [ 3H]UK14304 binding by various compounds confirmed the α 2-adrenergic nature of the sitesl labelled by both radioligands. The other α 2-adrenoceptor radioligand, [ 3H]idazoxan, labelled binding sites which are insentitive to catecholamine. Competition studies of [ 3H]idazoxan binding with imidazoline derivatives revealed structure-activity relationships different from those of α 2-adrenoceptors. The most striking observation is that substitutions in the 2-position of idazoxan markedly reduce the affinity for the non-adrenergic sites, whereas the α 2-potency is increased or unchanged.

Studies on hormonal regulation of lipolysis and lipogenesis in fat cells of various mammalian species

1. 1. Corticotropin-stimulated lipolysis in adipocytes of rats, mice, hamsters, guinea pigs and rabbits. Melanotropins elicited high lipolytic activity only in guinea pig and rabbit adipocytes. Opiate peptides were active only in rabbit adipocytes. Pituitary and chorionic gonadotropins and somatotropin were lipolytic in guinea pig adipocytes. Other hormones tested including prolactin, somatostatin, substance P, neurotensin, angiotensin II, thyrotropin releasing hormone and pancreatic polypeptide were devoid of lipolytic activity in all of the adipocytes studied. 2. 2. In the rabbit adipocytes γ-melanotropin was lipolytic only at high doses. At these doses the peptide inhibited the lipolytic response to a high dose of corticotropin. 3. 3. Lipolysis stimulated by vasoactive intestinal peptide and epinephrine in rat adipocytes was antagonized by insulin. The lipolytic hormones corticotropin, epinephrine, vasoactive intestinal peptide and secretin suppressed basal and insulin-stimulated lipogenesis.

In vivo effects of a treatment with antibodies to adipocyte plasma membranes in the rabbit

Antibodies against rabbit adipocyte plasma membranes were injected in 6-week-old rabbits. Controls received normal IgG. Animals were killed 1, 2, 5 or 9 weeks after treatment. Body weight and food intake were reduced significantly until the 7th week for the live weight and the 5th week for the intake. Whatever the anatomical location considered, adipose tissue was markedly reduced: -75% for week 1 and -20% for week 9 respectively for the total adipose mass. Cell volume and enzymatic activities of G3PDH, LPL and LDH were highly decreased during the first 2 weeks after treatment. Simultaneously the plasma levels of triglycerides and plasma free fatty acids were increased. As shown by others in the rat, it is possible to induce a long-term fatness reduction in the rabbit by treatment with antibodies to adipocyte plasma membranes. The cytotoxic effects of antibodies have also been discussed.

Differentiation of rabbit adipocyte precursor cells in a serum-free medium.

A serum-free, hormone-supplemented medium containing insulin, transferrin, and triiodothyronine (ITT medium), able to support differentiation of rat adipose precursor cells, has been used to study the regulation of the development of adipocytes in the rabbit. Adipose conversion was assessed by the appearance of glycerol-3-phosphate dehydrogenase activity. Stromal-vascular cells from rabbit perirenal adipose tissue differentiated to a very low extent or not at all in ITT medium. Supplementation of ITT medium with growth hormone or fibroblast growth factor did not increase the proportion of differentiated cells. In contrast, rabbit stromal-vascular cells were able to differentiate in ITT medium supplemented with glucocorticoids (dexamethasone, corticosterone) whereas sex steroids (beta-estradiol, testosterone, progesterone) did not affect the differentiation process. In the presence of both dexamethasone and insulin, 20 to 50% of rabbit stromal-vascular cells differentiated into adipocytes within 2 wk of culture. The stimulatory actions of dexamethasone or insulin were dose-dependent. Insulin-like growth Factor-I (IGF-I), did not replace insulin under our culture conditions and had only a slight effect when added along with dexamethasone (100 nM) and insulin (1.7 nM). The results suggest that glucocorticoids, in association with insulin, may play an important role in the development of adipocytes from rabbit precursor cells.

Differentiation of rabbit adipocyte precursors in primary culture

A primary culture system was used to study the adipose conversion of adipocyte precursors derived from the stromal-vascular fraction of perirenal adipose tissue of rabbit fetuses Differentiation was assessed by the development of glycerol-3-phosphate dehydrogenase, Acid:CoA ligase and lipoprotein lipase activities. Stromal-vascular cells were not able to differentiate when maintained in a medium supplemented with fetal calf serum or with rabbit serum. In contrast, differentiation was induced when the medium was supplemented with rabbit plasma. It also occurred when the growth phase was performed in serum provided that the serum was replaced by plasma when the cultures reached confluence. Supplementation of the culture medium with mesenteric lymph or chylomicrons as lipid sources greatly enhanced both lipid accumulation and the level of enzymatic markers of adipocyte differentiation. Following confluence in serum, cell proliferation ceased almost completely. In contrast, cells in the presence of plasma continued to proliferate, leading to a higher cell density at the time of adipocyte differentiation. These results suggest a positive effect of plasma on the post-confluent mitoses of susceptible cells. To our knowledge, it is the first time that such a difference between plasma and serum has been shown for the differentiation of adipocytes, using an homologous system. These studies also demonstrate that rabbit adipocyte precursors differentiating in primary culture show both similarities to and differences from the adipocytes of cell lines or cell precursors obtained from other animal species.

Degradation of β-lipotropin by subcellular fractions of rabbit adipocytes

Degradation of β-lipotropin by subcellular fractions of rabbit adipocytes

Conditional inhibition of forskolin-activated adenylate cyclase by guanosine diphosphate and its analog

Forskolin-activated adenylate cyclases (AC) in intact membranes, solubilized with Lubrol or eluted following adsorption on a forskolin-Sepharose column, were examined for inhibition by GDP and GDPβS. AC in intact membranes of rat or rabbit adipocytes was activated by 100 μ m forskolin and further potentiated by 10 μ m Gpp(NH)p in combination with either 230 μ m epinephrine or 50 mU · ml −1 ACTH. GDP (0–1 m m) or GDPβS (0–500 μ m) inhibited activation in a dose-dependent manner to a level similar to or slightly below that produced by 100 μ m forskolin alone. Forskolin at 100 μ m stimulated solubilized AC of rabbit adipocytes and rat liver membranes for 10 ± 4 to 160 ± 10 and from 26 ± 2 to 274 ± 21 pmol(mg · min) −1, respectively, in the absence of GDPβS; forskolin-activated activity decreased from 160 ± 10 to 157 ± 6 and from 274 ± 21 to 238 ± 14 pmol(mg · min) −1 in the presence of 500 μ m GDPβS. Forskolin-activated solubilized enzyme was further potentiated by 10 μ m Gpp(NH)p from 160 ± 10 to 289 ± 52 and from 274 ± 21 to 702 ± 50 pmol(mg · min) −1. GDPβS at 500 μ m inhibited 93 and 103% of the Gpp(NH)p-potentiated activity. AC of rat adipocytes eluted from forskolin-Sepharose affinity column with 500 m m NaCl and 100 μ m forskolin was not significantly activated by Gpp(NH)p nor inhibited by GDPβS. However, it was activated by forskolin. The lack of inhibition of unmodified forskolin-activated activity by GDP or GDPβS in contrast to the inhibition of Gpp(NH)p-activated enzyme or Gpp(NH)p-potentiated forskolin-activated enzyme may be a general phenomenon descriptive of the action of forskolin on AC. Furthermore, inhibition of forskolin-activated AC by GDP and its analog may be a useful index in analyzing the degree of guanine nucleotide potentiation of this enzyme.

A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources

We have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and β,γ-imidoguanosine 5′-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC 50 for forskolin was 10 μ m for rat liver, 15 μ m for rabbit and rat adipocytes and 17 μ m for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC 50 for forskolin diminished markedly to the range of 0.5 to 4.0 μ m. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.