Hamster Fat Cells Research Articles

Site-specific effects of sympathectomy on the adrenergic control of lipolysis in hamster fat cells.

Regional variations in the response of adipose tissue to lipolytic stimuli have been suggested to be involved in the development of visceral adiposity-related morbidity and mortality. Moreover, studies in humans and in laboratory rodents such as hamsters have shown that the response of adipocytes to catecholamines depends on their anatomical origin. The aim of the present study was to investigate the relative involvement of the adrenal medulla and of the sympathetic nervous system on regional differences in the adrenergic control of lipolysis in isolated adipocytes from inguinal and epididymal adipose tissues. For this purpose, we carried out adrenal demedullation or chemical sympathectomy in hamsters. The results confirmed that epididymal adipocytes were significantly more responsive to to a beta-adrenergic stimulation than inguinal adipocytes (p < or = 0.05). This site specificity could originate at a step distal to receptors since tissues exhibited a similar number of binding sites for [125I]cyanopindolol. No significant regional differences were observed in the alpha2-adrenergic antilipolytic response, with the exception of the clonidine EC50. A 14-day sympathectomy significantly increased the beta-adrenergic lipolytic response only in inguinal adipocytes (p < or = 0.05), and increased the alpha2-adrenergic response only in epididymal adipocytes (p < or = 0.05). On the other hand, adrenal demedullation had no effect on both adrenergic pathways. These results suggest that the sympathetic tone of adipose tissues could be involved in the alpha2- and beta-adrenergic site-specific response in hamster fat cells. The 33% increase of the beta-response in inguinal fat cells and the 38% increase of the alpha2-response in epididymal fat cells also suggest that the sympathetic pathway favors the lipolytic activation of the epididymal adipose tissue.

Regulation of white adipocyte guanine nucleotide binding proteins Gsα and [formula omitted] by testosterone in vivo : Influence of regional fat distribution

The influence of the androgenic status on the steady-state amounts of Giα 1–2 and Gsα subunits was compared in hamster fat cell membranes from the femoral subcutaneous (FSC) and epididymal (EP) adipose tissues, using immunoblotting experiments. In sham-operated hamsters, Giα 1–2 and Gsα steady-state amounts found in FSC fat cells were 38% and 40% reduced, respectively, as compared to EP adipocytes. In EP fat cells, castration induced a down-regulation of both Giα 1–2 (−39%) and Gsα (−33%), whereas testosterone replacement restored Gsα, but not Giα 1–2 levels, to control values. In contrast, these G protein α-subunits were insensitive to the androgenic status in FSC fat cells. These data provide the first evidence that the androgenic status can modulate the expression of both the Giα 1–2 and Gsα subunits of the fat cell adenylate cyclase regulatory Gi and Gs proteins and that this modulation depends on the anatomical origin of these cells.

Evidence that Testosterone Modulatesin Vivothe Adenylate Cyclase Activity in Fat Cells*

In male hamster fat cell membranes, the alpha 2-adrenoreceptor-mediated inhibitory response of adenylate cyclase was almost completely suppressed by castration and was restored to control values after testosterone treatment, whereas the cyclase inhibitory response to nicotinic acid was insensitive to androgenic status. Basal and forskolin-, guanylylimidodiphosphate- and isoproterenol-stimulated cyclase activities were decreased by 30-40% after castration and restored to control values after testosterone treatment. In addition, Mn2+ + forskolin-stimulated activity in the presence or absence of GDP beta S was lower (-30%) after castration and normalized after testosterone treatment. Finally, the effects of testosterone described above were completely abolished when the potent androgen receptor antagonist RU 23908 was administered together with testosterone. These results indicate that both the inhibitory and stimulatory responses of adenylate cyclase are promoted by testosterone through an androgen receptor-dependent mechanism; promotion of the inhibitory response concerns specifically the alpha 2-receptor-mediated pathway, whereas promotion of the stimulatory response appears unspecific and mainly due to increased activity of the cyclase catalytic subunit.

Imidazolinic radioligands for the identification of hamster adipocyte α2-adrenoceptors

Imidazolinic radioligands ([ 3H]UK 14304, [ 3H]idazoxan and [ 3H]RX 821002) were used for the identification of α 2-adrenoceptors on hamster fat cell membranes since there are limitations to the use of [ 3H]yohimbine and [ 3H]clonidine, which suggest α 2-adrenoceptor heterogeneity. Biological assays (lipolysis measurements) were performed on isolated fat cells and binding studies were carried out on fat cell membranes. The imidazolinic derivative, UK 14304, was a full agonist as compared to clonidine. Idazoxan and RX 821002 (2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline), a recently developed α 2-antagonist, were more potent α 2-antagonists than yohimbine in this fat cell model. [ 3H]UK 14304 was the most suitable agent for the quantification of the ‘high-affinity state’ α 2-adrenoceptors in binding studies since it did not exhibit the sensitivity to the composition of the buffer shown by [ 3H]clonidine. Although it is a potent α 2-antagonist, [ 3H]idazoxan had major limitations for use in the identification of α 2- adrenoceptors in this cell model since it also bound to ‘non-adrenaline displaceable’ binding sites which were revealed when imidazolinic derivatives (phentolamine) were used instead of adrenaline to determine the non-specific binding. We demonstrated that [ 3H]RX 821002 was a more suitable radioligand than [ 3H]yohimbine for labelling hamster fat cell α 2-adrenoceptors (K D = 1.0 ± 0.1 nM, B max = 776 ± 60 fmol/mg protein). Moreover, since it exhibited low affinity for ‘imidazoline-preferring sites’, it represents a valuable ligand even in tissues possessing such binding sites. We suggest that [ 3H]RX 821002 can be used to identify α 2-adrenoceptors in various tissues when these sites cannot be labelled with [ 3H]yohimbine.

Evidence that the lipolytic defect induced by estradiol-treatment in hamster adipocytes is related to an estrogen receptor-mediated defect in the adenylate cyclase catalytic subunit but not in N S

This study demonstrates that estradiol-treatment (10 μg per day × 5 days) does not impair the level of N S, the adenylate cyclase stimulatory regulatory protein, in hamster fat cell membranes. In addition, this report shows that the defective cyclic AMP response induced in intact adipocytes by the estradiol-treatment is either unaltered by the administration of α-bromocryptine or abolished by tamoxifen- or 4-hydroxyta-moxifen-treatment. It can thus be concluded that the reduced lipolytic response found in hamster fat cells after estradiol-treatment is related only to an estradiol-receptor-mediated defect in adenylate cyclase catalytic subunit activity which is independent from increased prolactin secretion.

Modulation of glucose transport in hamster adipocytes by insulin and by?- and? 2-adrenoceptor agonists

Glucose transport in hamster adipocytes and its modulation by insulin and isoprenaline was characterized with the aid of the non-metabolizable hexose 3-0-methylglucose. Insulin stimulated the initial uptake rates by an increase in Vmax of the transport without any detectable change in Km. The hormone concentration producing half maximal stimulation was identical to that required in rat adipocytes. However, hamster adipocytes were much less responsive to insulin (3-fold stimulation as compared to a 12-fold stimulation in rat fat cells), and maximal transport rates were 10-fold lower than that observed in rat adipocytes. Accordingly, the number of glucose transporters, as assessed by glucose-inhibitable cytochalasin-B binding, was considerably lower in plasma membranes of hamster adipocytes. Moreover, no transporters were detected in the low-density microsomes which in insulin-sensitive cell types represent the intracellular pool of recruitable glucose transporters. The relative insulin resistance of the hamster fat cells may therefore be due to a depleted pool of intracellular glucose transporters. In the presence of adenosine, the beta-adrenoceptor agonist isoprenaline produced a moderate stimulation of the basal transport rate which was antagonized by the alpha 2-agonist clonidine. If adenosine deaminase was added in order to remove endogenous adenosine, isoprenaline inhibited the insulin-stimulated transport by 50%. In contrast to the stimulatory effects of insulin and isoproterenol, the inhibitory effect of the catecholamine was reversed by cooling the cells to 22 degrees. Glucagon produced a comparable inhibition, suggesting that the inhibitory effect was mediated by adenylate cyclase or its regulatory subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies of prostaglandin E 2, phenylisopropyladenosine, clonidine and propranolol on lipolysis stimulated by methylisobutylxanthine and isoprenaline in hamster fat cells

The antilipolytic effects of prostaglandin E 2 (PGE 2), phenylisopropyladenosine (PIA), clonidine (alpha-2 adrenoceptor agonist) and propranolol (beta- adrenoceptor antagonist) on lipolysis stimulated by methylisobutylxanthine (MIX) (2 × 10 −4M) and isoprenaline (5 × 10 −7M) were compared in hamster fat cells. PGE 2, PIA and clonidine inhibited markedly lipolysis stimulated by MIX, and slightly that stimulated by isoprenaline. Whilst propranolol inhibited markedly lipolysis stimulated by isoprenaline and slightly that stimulated by MIX. These results indicate that the inhibitory activity of PGE 2, adenosine and alpha-2-adrenergic receptor stimulation depends upon the presence of MIX, whereas that of the beta-adrenoceptor blockade does not.

Prostaglandins, alpha-adrenergic receptors and lipolysis in hamster fat cells

In hamster fat cells, PGE 2, PGI 1 and clonidine ( α 2-adrenoceptor agonist) were shown to inhibit markedly lipolysis stimulated by methylisobutylxanthine (MIX) (2 × 10 −4M) and slightly that provoked by isoprenaline (5 × 10 −7M). The similar inhibitory actions of the α 2-adrenoceptor agonist clonidine and PGE 2 on lipolysis stimulated by MIX led us to examine possible reversal of the antilipolytic effect of PGE 2 by yohimbine ( α 2-adrenoceptor antagonist). However, the absence of an effect of yohimbine on PGE 2, antilipolytic action indicated that the PGE 2 receptor was different from the α 2-adrenoceptor of hamster fat cells. In these studies, PGE 2 was shown to be a more potent antilipolytic agent that PGI 2 (IC 50 values 2 × 10 −9 and > 10 -4M respectively).

Activities and some properties of 5′nucleotidase in isolated hamster fat cells

1. 1. Kinetic parameters and the possible cellular location of 5′nucleotidase were studied in isolated hamster fat cells. 2. 2. Measurements of the enzyme were performed using 5′IMP instead of 5′AMP as substrate. 3. 3. The apparent V max and K m values obtained support the view that two forms of 5′nucleotidase, having different affinities, are present in hamster fat cells. The major form was located in the outer surface of plasma membrane and the other one inside the cell. 4. 4. The effects of ADP, adenosine and inosine on the enzyme were studied. ADP and adenosine were competitive inhibitors. The enzyme was not modified by inosine.

Comparative effects of RX 781094, mianserin, yohimbine, rauwolscine and prazosin in reversing clonidine inhibition of mix-stimulated lipolysis in hamster isolated white fat cells

Different alpha-adrenergic receptor antagonists: RX 781094, yohimbine and rauwolscine (selective alpha 2-adrenergic antagonists); mianserin (tetracyclic anti-depressant, antagonist at alpha 2-presynaptic autoreceptors) and prazosin (selective alpha 1-adrenergic antagonist) were used at concentrations ranging from 10(-7) to 10(-4) M, to reverse clonidine inhibition of MIX-stimulated lipolysis in the hamster fat cell. In this adipose tissue (like human but unlike rat) there co-exist prolipolytic beta-adrenoceptors and antilipolytic alpha-adrenoceptors. Although no effects were observed with prazosin, RX 781094 was ten times more potent than yohimbine or its isomer rauwolscine in reversing clonidine inhibition of the MIX-stimulated lipolysis. Mianserin was an effective blocker only from a concentration of 10(-4) M, consistent with its relative lack of specificity for alpha 2-adrenoceptors cited elsewhere. Overall these results confirm the utility of this model for testing compounds presumed to act at alpha 2-adrenoceptors.

Anti-lipolytic action of prazosin in hamster fat cells

Using isolated white fat cells of the hamster, we studied the possible inhibitory effects on lipolysis of the preferentially selective alpha-1-adrenoceptor blocking and anti-hypertensive agent prazosin. Parallel studies were undertaken with the preferentially alpha-2-adrenoceptor blocking agent yohimbine. Neither of the compounds had any significant effects on basal (non-stimulated) lipolysis. When lipolysis was stimulated by noradrenaline or isoprenaline, however, prazosin showed significant anti-lipolytic effects from a concentration of 10(-6) M. Lipolysis stimulated by methylisobutylxanthine was antagonized by prazosin only at a concentration of 10(-4) M, and dibutyrylcyclic adenosine 3'5' monophosphate-stimulated lipolysis was not inhibited at all. It is suggested that prazosin inhibits lipolysis by preventing adenyl cyclase activation by an indirect mechanism not identifiable with an alpha-2-adrenergic function but associated possibly with the formation of a local inhibitory mediator. These observations are compatible with a reduction of the outflow of free fatty acids from adipose stores in patients undergoing prazosin therapy and thus a breaking of the "vicious circle" leading to elevated plasma lipoproteins and may explain, in part at least, the reduction in plasma lipid levels observed during prazosin treatment in man.

Direct action of pertussis toxin in isolated hamster fat cells

Incubation of pieces adipose tissue in the presence of pertussis toxin (10–100 μg/ml) resulted in blockade in the isolated fat cells of the action of agents that inhibit adenylate cyclase through receptor-mediated GTP-dependent process ( α 2-adrenergic amines, prostaglandins, Ri-adenosine, niotic acid) and enhancement of the action of isoproterenol. There was a lag period of ∼ 22 h for the action of the toxin.

Effect of pertussis toxin on the hormonal regulation of cyclic AMP levels in hamster fat cells

Pertussis toxin was purified approx. 1800-fold from pertussis vaccine. Administration of as little as 1 μg of toxin/100 g body weight to hamsters markedly decreased the sensitivity of their adipocytes to agents that inhibit adenylate cyclase through receptor-mediated, GTP-dependent mechanisms such as α 2-adrenergic amines, prostaglandins, phenylisopropyladenosine and nicotinic acid. On the contrary, the inhibitory effect of 2′,5′-dideoxyadenosine on cyclic AMP accumulation was not affected by the toxin. Activation of adenylate cyclase by isoproterenol, ACTH or forskolin was not diminished by the toxin but the maximum cyclic AMP accumulation was consistently increased. Furthermore, the dose-response curves for ACTH and forskolin were clearly shifted to the left in adipocytes from toxin-treated hamsters as compared to control adipocytes. It is concluded that pertussis toxin blocks the transfer of inhibitory information from the receptors to adenylate cyclase.

Lack of functional antilipolytic α-adrenoceptor in rat fat cell: Comparison with hamster adipocyte

1. The small population of [ 3H]clonidine binding sites in rat fat cell membranes do not have the characteristics of typical α 2-adrenoceptors. 2. Clonidine (an α 2-adrenoceptor agonist) has no antilipolytic effect on rat fat cells stimulated by theophylline. 3. In contrast to the rat, [ 3H]clonidine labels an α 2-adrenoceptor in hamster fat cell membranes and clonidine exerts a strong antilipolytic effect on theophylline-stimulated lipolysis.

Demonstration of alpha-2-adrenoceptor antagonist (yohimbine-like) activity of cirazoline using a hamster fat cell preparation

Summary Lipolysis in hamster isolated adipocytes stimulated by 2 × 10 −4 M methylisobutylxanthine (MIX) was inhibited over 75% by clonidine (1 × 10 −6 M). This inhibition was completely reversed by the alpha-2-adrenoceptor antagonist yohimbine, almost completely by cirazoline (reversal beginning at 5 × 10 −6 M) and not at all by prazosin (upto 10 −4 M). These results clearly demonstrate the alpha-2-adrenoceptor blocking action of cirazoline (considered until recently a specific alpha-1-agonist) and the utility of this fat cell method as a simple screening procedure in the search for compounds working through alpha-2-adrenoceptors which may be potentially useful in the study or treatment of hypertension or depression.

Catecholamine binding to the α-adrenergic receptors of hamster adipocytes evidence that guanine nucleotides regulate this binding to the α2-receptor subtype

The binding characteristics of the α-component of (−)-[ 3H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, reaching equilibrium in 20 min at 25°C. Dissociation of specific binding by 10 μM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [ 3H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity ( D = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity ( K D = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands of competed with [ 3H]norepinephrine binding in the following order of potency: (−)-norepinephrine>(−)-epinephrine>>(+)-norepinephrine>(−)-isoproterenol. Displacement by the selective α-adrenergic drugs prazosin, clonidine and yohimbine yielded biphasic curves consistent with binding of [ 3H]norepinephrine to both α 1- (14–22%) and α 2- (78–86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [ 3H]dihydroergocryptine, it severely reduced the binding affinity of (−)-epinephrine, (−)-norepinephrine and the selective α 2-agonist, clonidine. The inhibitory effects of clonidine and of the α-component of (−)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [ 3H]norepinephrine and [ 3H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the α-inhibitory effect of norepinephrine on cyclic AMP production. These data led to concluded that [ 3H]norepinephrine can be successfully used to study the entire α-adrenergic receptor population of hamster fat cells and that the predominant α 2 -receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.

A postsynaptic α, 2-receptor: The alpha-adrenergic receptor of hamster white fat cells

The effects of selected alpha-agonists and alpha-antagonists on theophylline-induces lipolysis were investigate in isolated hamster white fat cells, alpha 2-Agonists (tramazoline, clonidine) inhibited theophylline-induced lipolysis while in alpha 1-agonist (methoxamine) was without any effect. The inhibitory effect of alpha 2-agonists was suppressed by yohimbine (alpha 2-antagonist), whereas alpha 1-antagonists were inefficient. This result implies that the alpha-adrenergic receptor of hamster fat cells is of the alpha 2-type, although postsynaptically.

Role of alpha 1 adrenoceptors in the turnover of phosphatidylinositol and of alpha 2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin⪢phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha 1 adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ⪢prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha 2 adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha 1 and alpha 2 adrenoceptors but about 90% of the binding sites were alpha 2. These data support the hypothesis that alpha 2 effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha 1 adrenoceptor activation.

Inhibition of lipolysis in hamster adipocytes with selective alpha-adrenergic stimuli. Functional characterization of the alpha-receptor.

This communication shows the relative potencies of the alpha-agonists clonidine, methoxamine, methyl norepinephrine and phenylephrine in producing inhibition of lipolysis. At cell densities greater than 15 mg cell/ml lipolysis activated by either 1-methyl-3-isobutyl xanthine or adenosine deaminase was inhibited by alpha-adrenergic stimuli with a rank order of potency of clonidine greater than methoxamine greater than methyl norepinephrine; phenylephrine produced a further stimulation of lipolysis. At the same cell density isoproterenol-accelerated lipolysis was inhibited by alpha-adrenergic stimuli with a rank order of potency of phenylephrine greater than methoxamine greater than clonidine greater than methyl norepinephrine. When the density of fat cells was reduced to less than 5 mg/ml, clonidine was a more effective inhibitor of isoproterenol-activated lipolysis thatn phenylephrine. Lipolysis that was activated by dibutyryl cyclic AMP, ACTH or cholera enterotoxin was not reduced by any alpha-adrenergic agent. Under conditions when clonidine failed to inhibit catecholamine-activated lipolysis (i.e., at cell densities greater than 15 mg/ml), it failed to antagonize the antilipolytic activity of phenylephrine. The antilipolytic activities of clonidine and phenylephrine were most effectively antagonized by the blocking drugs phentolamine and yohimbine; in contrast, phenoxybenzamine and prazosin were less effective blockers. These data indicate that the alpha-adrenergic receptor on hamster fat cells is similar to presynaptic alpha-adrenergic receptors. The data further suggest the possibility that phenylephrine may exert its action through a separate alpha-adrenergic receptor mechanism.

Inhibition of hamster fat cell adenylate cyclase by prostaglandin E 1 and epinephrine: Requirement for GTP and sodium ions

Inhibition of hamster fat cell adenylate cyclase by prostaglandin E 1 and epinephrine: Requirement for GTP and sodium ions