scholarly journals Attenuation of Gs α coupling efficiency in brown-adipose-tissue plasma membranes from cold-acclimated hamsters

1993 ◽  
Vol 295 (3) ◽  
pp. 655-661 ◽  
Author(s):  
P Svoboda ◽  
L Unelius ◽  
B Cannon ◽  
J Nedergaard
Keyword(s):  
Adipose Tissue ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Coupling Efficiency ◽  
Ouabain Binding ◽  
Beta Adrenergic ◽  
Brown Adipose ◽  
Density Band ◽  
Gs Protein ◽  
Gs Alpha

In order to localize site(s) of beta-adrenergic desensitization found in brown adipocytes from cold-acclimated animals, total brown-adipose-tissue homogenates (postnuclear supernatant) were obtained from control or cold-acclimated hamsters and were fractionated on discontinuous sucrose gradients. A low-density band (cytosolic proteins) and a high-density band (mitochondria) were obtained; in the middle fractions only low levels of protein were recovered. However, these fractions displayed a high level of specific [3H]ouabain binding, indicating that they represented fractions enriched in plasma membranes. The level of [3H]ouabain binding was significantly higher in plasma membranes from cold-acclimated animals, indicating an increased density of Na,K-ATPase units. The maximal activity of adenylate cyclase, as estimated with forskolin, was not changed by cold acclimation. However, the levels of cyclase activity observed after Gs-protein-mediated activation (with guanosine 5′-[gamma-thio]triphosphate, isoprenaline, both of these, or fluoride) were decreased, indicating a decreased coupling efficiency. Notably, a significant decrease was observed in the functional activity of the Gs protein, as directly measured by estimation of the ability of cholate extracts of brown-fat plasma membranes to reconstitute Gs-protein-mediated stimulation of adenylate cyclase in cyc- membranes. Further, a functionally significant decrease (to 72%) was observed in the ratio between the amount of functional Gs proteins and adenylate cyclase units. The total content of Gs alpha protein was decreased to the same extent as the coupling efficiency of the membranes, indicating that a lower content of functionally equivalent Gs alpha molecules could explain the decreased coupling. It could therefore be concluded that a decrease in Gs-protein-mediated coupling efficiency, owing to a decrease in the amount of Gs alpha, is at least one site of beta-adrenergic desensitization in cold-acclimated animals. This may, at least in part, explain that desensitization takes place despite the fact that the beta 3-adrenoceptor itself apparently lacks some of the sites known to be involved in the desensitization process in other beta-adrenergic receptors.

scholarly journals The decrease in the short variant of gsalpha protein is associated with an increase in [3H]CGP12177 binding, [3H]ouabain binding and Na, K-ATPase activity in brown adipose tissue plasma membranes of cold-acclimated hamsters

1999 ◽  
Vol 22 (1) ◽  
pp. 55-64 ◽  
Author(s):  
L Bourova ◽  
J Novotn ◽  
P Svoboda
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Brown Adipose Tissue ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Immunoblot Analysis ◽  
Ouabain Binding ◽  
Brown Adipose ◽  
Sds Page

Sucrose density gradient purified plasma membranes isolated from brown adipose tissue of cold-acclimated hamsters (4-10 weeks at 0-4 degreesC) were analysed for the content of the short (GsalphaS) and long (GsalphaL) variants of Gsalpha protein (the alpha subunit of the stimulatory G protein) and compared with the membranes isolated from control animals. The relative ratio between the two variants (GsalphaS/GsalphaL) decreased from 0.48 to 0.24 (P<0.01). This result, obtained by electrophoretic resolution of membrane proteins by standard SDS-PAGE and an immunoblot analysis with an antiserum oriented against an internal sequence of Gsalpha, was verified by resolution on urea-containing gels and an antiserum oriented against the C-terminus decapeptide of Gsalpha. Under these conditions, the GsalphaS/GsalphaL ratio was decreased from 0.41 to 0.31 (P<0.05). The total amount of both isoforms (GsalphaS plus GsalphaL) decreased to 83% (P<0.05) or 68% (P<0.01) by standard or urea SDS-PAGE respectively. These data demonstrate that cold-acclimation of hamster brown adipose tissue is associated with preferential decrease in the plasma membrane density of the short variant of the Gsalpha protein.%This decrease was paralleled by an increase in the other plasma membrane constituents, [3H]CGP12177 binding sites, [3H]ouabain binding sites and Na,K-ATPase activity to 147%, 212% and 191% respectively.

Adenylyl cyclase inhibitory pathway is differentially modified in rat white and brown fat by high-energy diets

1997 ◽  
Vol 272 (6) ◽  
pp. E1043-E1049 ◽  
Author(s):  
Y. Kenan ◽  
M. Levinson ◽  
M. Pines ◽  
M. Naim
Keyword(s):  
Adipose Tissue ◽  
Adenylyl Cyclase ◽  
High Energy ◽  
Inhibitory Pathway ◽  
Balanced Diet ◽  
Brown Adipose ◽  
Gs Alpha ◽  
Phenylisopropyl Adenosine ◽  
Camp Levels ◽  
Camp Formation

Incubation of white adipose tissue (WAT) adipocytes from rats fed a high-energy diet (Exp group) with antilipolytic Gi-coupled adenylyl cyclase inhibitory agonists, nicotinic acid (Nic) and N8-(L-2-phenylisopropyl)adenosine (PIA), resulted in lower cellular adenosine 3',5'-cyclic monophosphate (cAMP) levels than in stimulated adipocytes from rats fed a nutritionally balanced diet (Con group). In contrast to WAT, incubation of brown adipose tissue (BAT) adipocytes with Nic yielded higher cAMP levels in the Exp vs. Con rats. In both WAT and BAT adipocytes, pertussis toxin treatment abolished the differences in Nic- and PIA-inhibited cAMP formation between Exp and Con animals. Immunoblotting of adipocyte membranes indicated a lower content of Gi alpha but not Gs alpha in BAT membranes of Exp vs. Con animals after 6 and 10 wk of feeding. No such differences were found in the Gs alpha or Gi alpha contents of WAT membranes. Thus the inhibitory pathway of adenylyl cyclase is proposed to be sensitized in WAT and desensitized in BAT of rats fed high-energy diets. These modifications in sensitivity are in line with reduced cAMP and lipolysis in WAT and increased cAMP and thermogenesis in BAT during obesity.

Refeeding and insulin increase lipoprotein lipase activity in rat brown adipose tissue

1989 ◽  
Vol 256 (5) ◽  
pp. E645-E650 ◽  
Author(s):  
C. M. Carneheim ◽  
S. E. Alexson
Keyword(s):  
Enzyme Activity ◽  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Lipoprotein Lipase ◽  
Lipase Activity ◽  
Starvation Period ◽  
Lipoprotein Lipase Activity ◽  
Mrna Synthesis ◽  
Beta Adrenergic ◽  
Brown Adipose

Induction of lipoprotein lipase activity in brown adipose tissue (BAT) in response to cold stress has earlier been shown to be regulated by a beta-adrenergic mechanism and to be dependent on mRNA synthesis. In the present study, we have investigated the acute effects of refeeding after a short starvation period and the hormonal mechanism underlying the observed effects. Refeeding was found to rapidly increase tissue wet weight and lipoprotein lipase activity. The increase in enzyme activity could be blocked by the RNA synthesis inhibitor actinomycin D, indicating a gene activation. beta-Adrenergic blockade had no effect on this elevation of enzyme activity, but the increase could be mimicked by insulin injection. The results suggest that BAT contains two different pathways for regulation of lipoprotein lipase activity, both involving mRNA synthesis.

cAMP metabolism and lipolysis in brown adipocytes of hamsters consuming a cafeteria diet

1985 ◽  
Vol 248 (2) ◽  
pp. E224-E229
Author(s):  
R. J. Schimmel ◽  
L. McCarthy ◽  
K. K. McMahon
Keyword(s):  
Adenylate Cyclase ◽  
Sympathetic Activity ◽  
Brown Fat ◽  
Adenylate Cyclase System ◽  
Brown Adipocytes ◽  
Beta Adrenergic ◽  
Fat Depots ◽  
Brown Adipose ◽  
Camp Levels ◽  
In Cells

Feeding animals cafeteria diets causes increased sympathetic activity to brown adipose tissue and this is believed to be responsible for the concomitant activation of thermogenesis. Because chronic catecholamine stimulation in other systems leads to a desensitization of beta-adrenergic receptors, we examined lipolysis and cAMP production in brown adipocytes of hamsters eating cafeteria diets for evidence of diminished beta-adrenergic responses. Basal cAMP levels were similar in chow- and cafeteria-fed hamsters. However, adipocytes from overfed animals formed less cAMP in response to isoproterenol than those of control animals. Isoproterenol-stimulated adenylate cyclase activity was similarly decreased in membrane preparations from cafeteria-fed hamsters. However, when the diterpene forskolin was used, equal amounts of cAMP were formed in cells and membrane preparations from control and overfed animals. In contrast to the reduced responses of the cAMP system to isoproterenol stimulation observed in overfed hamsters, isoproterenol-stimulated lipolysis was greater in cells from overfed animals than in cells from control animals. These results are consistent with a desensitization of the adenylate cyclase system in brown adipocytes occurring during chronic hyperphagia. Because eating cafeteria diets has been reported to increase sympathetic activity to brown fat depots, the apparent desensitization of brown adipocytes observed in this study may result from a persistent stimulation of the brown fat with norepinephrine in vivo. Our data also suggest the existence of mechanisms that preserve lipolysis in the face of low cAMP levels.

Alterations of adenylyl cyclase-linked G proteins in rat liver during aging

1996 ◽  
Vol 270 (1) ◽  
pp. E126-E132 ◽  
Author(s):  
A. T. Eakes ◽  
T. K. Hymer ◽  
M. J. Rosenthal ◽  
J. Moss ◽  
M. S. Katz
Keyword(s):  
Adenylyl Cyclase ◽  
Cholera Toxin ◽  
G Proteins ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
Adp Ribosylation ◽  
Old Rats ◽  
Gs Alpha

beta-Adrenergic stimulation of adenylyl cyclase in rat liver increases during aging. We examined whether this increase is related to alterations in the stimulatory and inhibitory G proteins (Gs and Gi) linked to adenylyl cyclase. Levels of immunoreactive alpha- and beta-subunits of Ga and Gi in liver plasma membranes from 6-, 12-, 18-, and 24-mo-old rats were unchanged with age, as was pertussis toxin-catalyzed [32P]ADP ribosylation of Gi alpha. Cholera toxin-catalyzed [32P]ADP ribosylation of Ga alpha and Gs bioactivity, assessed as reconstitution of adenylyl cyclase activity in S49 cyc- cell membranes, increased two- to threefold between 6 and 12-18 mo, and declined by 24 mo. Recombinant ADP ribosylation factor (ARF) enhanced cholera toxin labeling of Gs alpha at all ages, yet abolished the increase in toxin labeling at 12-18 mo. Auto-ADP ribosylation of the cholera toxin A1 peptide also increased transiently with age. Alteration of Gs alpha, as reflected by increased cholera toxin labeling and Gs bioactivity, may be involved in the regulation of beta-adrenergic-responsive adenylyl cyclase in rat liver during aging. Moreover, changes in endogenous ARF levels could contribute to age differences in cholera toxin labeling of Gs alpha.

Perinatal expression of adenylyl cyclase subtypes in rat brown adipose tissue

1996 ◽  
Vol 270 (4) ◽  
pp. R755-R760 ◽  
Author(s):  
A. Chaudhry ◽  
L. A. Muffler ◽  
R. Yao ◽  
J. G. Granneman
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Adenylyl Cyclase ◽  
Perinatal Period ◽  
Mrna Levels ◽  
Protection Assay ◽  
Brown Adipose ◽  
Nuclease Protection Assay ◽  
6 Hydroxydopamine ◽  
Gs Alpha

The ability of norepinephrine to stimulate adenylyl cyclase (AC) activity increases during the perinatal period in rat brown adipose tissue (BAT), and this increase is associated with changes in the activities of both GS alpha and AC. The purpose of this study was to determine which AC subtypes are present in neonatal BAT and to examine whether the perinatal increase in AC activity corresponds to an increase in the expression of a particular AC subtype. Analysis of AC mRNAs by nuclease protection assay demonstrated the presence of mRNAs encoding AC-III, AC-IV, AC-VI, and AC-IX in embryonic and postnatal BAT. Of the subtypes detected, only AC-III mRNA levels increased substantially during the perinatal period. The increase in AC-III expression was paralleled by an increase in isoproterenol-stimulated AC activity. Treatment of neonates was the sympathetic neurotoxin 6-hydroxydopamine abolished the perinatal increase in both AC activity and AC-III mRNA levels but had no effect on the expression of other AC subtypes. These results strongly indicate that the increase in AC activity during the perinatal period is due to an increase in the expression of AC-III.

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