scholarly journals Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity

1976 ◽  
Vol 154 (1) ◽  
pp. 209-223 ◽  
Author(s):  
D L Severson ◽  
R M Denton ◽  
B J Bridges ◽  
P J Randle
Keyword(s):  
Isotopic Exchange ◽  
Calcium Content ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Total Calcium ◽  
Epididymal Fat ◽  
Mitochondrial Fractions ◽  
Fat Pads

1. Isolated fat-cells and intact epididymal fat-pads were incubated in medium containing 45+ and the incorporation of 45Ca into mitochondrial and extramitochondrial fractions was studied. Redistribution of 45Ca between these fractions was essentially prevented by the addition of EGTA [ethanedioxybis(ethylamine)tetra-acetate] and Ruthenium Red to the sucrose-based extraction medium. 2. Incorporation of 45Ca into mitochondrial fractions of both fat-cells and fat-pads was found to be complete within 2-5 min, suggesting that mitochondria contain a pool of calcium in rapid isotopic exchange with extracellular +. This pool was about 20 times larger in mitochondria within fat-cells than within fat-pads. In fat-cells, 45Ca incorporation into the mitochondrial fraction accounted for about 34% of the total 45Ca incorporation into cells after 20 min and about 50% of the total mitochondrial calcium content measured by atomic absorption; values in fat-pads were about 7 and 20% respectively.

scholarly journals The effect of insulin on the washout of [45Ca]calcium from adipocytes and soleus muscle of the rat

1977 ◽  
Vol 164 (1) ◽  
pp. 251-255 ◽  
Author(s):  
T Clausen ◽  
B R Martin
Keyword(s):  
Soleus Muscle ◽  
Rat Plasma ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Epididymal Fat ◽  
Fat Pads

Insulin stimulates the washout of 45Ca from preloaded isolated fat-cells, whole epididymal fat-pads and isolated soleus muscles of the rat. This effect occurs within 10 min after the addition of the hormone, and it can be detected at concentrations down to those measured in rat plasma. When K+ is omitted from the washout medium, the effect on soleus muscles is more pronounced and increases with the time of exposure.

scholarly journals Evidence that insulin activates casein kinase 2 in rat epididymal fat-cells and that this may result in the increased phosphorylation of an acid-soluble 22 kDa protein

1991 ◽  
Vol 279 (2) ◽  
pp. 545-551 ◽  
Author(s):  
T A Diggle ◽  
C Schmitz-Peiffer ◽  
A C Borthwick ◽  
G I Welsh ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Peptide Substrate ◽  
Heat Stable ◽  
Epididymal Fat ◽  
Rat Adipose Tissue ◽  
Fat Pads

Casein kinase 2 activity as measured by phosphorylation of the peptide substrate Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu is increased by about 50% in extracts from insulin-treated epididymal fat-pads or isolated fat-cells after purification by Mono Q chromatography. Insulin acts to increase the Vmax. of the kinase. An acid-soluble protein with an apparent subunit molecular mass of about 22 kDa appears to be a substrate for casein kinase 2. The protein possesses a number of properties in common with the acid-soluble heat-stable 22 kDa protein which exhibits increased phosphorylation in rat adipose tissue exposed to insulin.

scholarly journals Relationships between the exchange of calcium and phosphate in isolated fat-cells

1975 ◽  
Vol 152 (1) ◽  
pp. 121-129 ◽  
Author(s):  
B R Martin ◽  
T Clausen ◽  
J Gliemann
Keyword(s):  
Incubation Medium ◽  
Radioactive Isotope ◽  
Slow Phase ◽  
Detectable Effect ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Rapid Phase ◽  
45Ca Uptake ◽  
Epididymal Fat ◽  
Fat Pads

1. The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+ proceeds with an initial rapid phase of about 1 min duration, followed by a slower subsequent accumulation. In contrast with the rapid phase, the slow phase is inhibited by 2,4-dinitrophenol, warfarin, oligomycin and verapamil, shows saturation, and presumably represents transport across the plasma membrane. 3. The washout of 45Ca2+ from preloaded cells consists of a rapid (1 min) initial phase and a slow phase which is non-monoexponential, suggesting that the radioactive isotope is released from several cellular pools. 4. When Pi is omitted from the incubation medium, the slow phase of 45Ca uptake is almost abolished, and the washout of 45Ca from preloaded fat-cells is markedly accelerated. At elevated extracellular concentrations of Pi (2,4-6.2mM), the uptake of 45Ca is stimulated by 2-10-fold, and the release of the radioactive isotope from preloaded cells is inhibited. In whole epididymal fat-pads, variations in the extracellular concentration of Pi have no detectable effect on the uptake or the washout of 45Ca. 5. In isolated fat-cells, the accumulation of 32Pi is inhibited by 2,4-dinitrophenol or the omission of glucose from the incubation medium. In a Ca2+-depleted buffer, the uptake of 32Pi is diminished, and hyperosmolarity, which stimulates 45Ca uptake, also accelerates the accumulation of 32Pi. 6. It is concluded that in free fat-cells, the uptake and release of Ca2+ and Pi take place by closely interrelated processes, which are dependent on mitochondrial energy production.

RELATIONSHIPS BETWEEN CALCIUM TRANSPORT AND THE LIPOLYTIC EFFECT OF SHEEP β-LIPOTROPIC HORMONE β-LPH) IN ADIPOSE TISSUE

1972 ◽  
Vol 69 (3) ◽  
pp. 507-516 ◽  
Author(s):  
M. Lis ◽  
C. Gilardeau ◽  
M. Chrétien
Keyword(s):  
Adipose Tissue ◽  
Intravenous Injection ◽  
Calcium Transport ◽  
Lipolytic Activity ◽  
Fat Cells ◽  
Tetraacetic Acid ◽  
Isolated Fat Cells ◽  
Fat Tissue ◽  
Adipose Cell ◽  
Epididymal Fat

ABSTRACT Intravenous injection of β-lipotropic hormone β-LPH) into rabbits caused a marked increase of Ca++ concentration in perirenal or epididymal fat tissue. β-LPH also increased the amount of Ca++ taken up during incubation of isolated fat cells. Incubation of fragments of rabbit fat tissue in presence of 45Ca and 3H mannitol indicated that Ca++ accumulated intra-cellularly after administration of β-LPH. In incubation media containing no Ca++, or containing Ca++ and the Ca++ sequestering agent EGTA (ethylenebis [oxyethylene nitrilo]-tetraacetic acid), β-LPH did not induce lipolysis. Addition of excess Ca++ to the EGTA containing medium restored lipolysis, whereas addition of EGTA to incubation mixtures containing Ca++ in which lipolysis in the presence of β-LPH was already proceeding stopped the lipolytic reaction. These results indicated that Ca++ is essential for lipolytic activity of β-LPH as it is for the lipolytic activity of ACTH and other structurally related peptides. Marked shift of Ca++ towards the adipose cell was correlated with β-LPH induced lipolysis.

scholarly journals Comparison of the effects of insulin and adrenergic agonists on the phosphorylation of an acid-soluble 22 kDa protein in rat epididymal fat-pads and isolated fat-cells

1992 ◽  
Vol 282 (3) ◽  
pp. 729-736 ◽  
Author(s):  
T A Diggle ◽  
R M Denton
Keyword(s):  
Cyclic Amp ◽  
Phorbol Esters ◽  
Protein Composition ◽  
Fat Cells ◽  
Adrenergic Agonists ◽  
Beta Adrenergic ◽  
Epididymal Fat ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Fat Pads

1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble 22 kDa protein and that increases in phosphorylation were also evident in cells exposed to adrenaline [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383; Belsham, Brownsey, Hughes & Denton (1980) Diabetologia 18, 307-312]. 2. The effects of adrenaline are shown to be brought about through beta-adrenergic receptors and to be mimicked by other agents which increase cell cyclic AMP concentrations. The maximum extent of phosphorylation is about 60% of that observed with insulin. Increased phosphorylation is also observed in fat-cells exposed to vasopressin, oxytocin and phorbol esters, but not to alpha-adrenergic agonists. 3. No changes in the phosphorylation of the protein are evident in epididymal fat-pads from fat-fed, starved or starved/refed animals, despite the large changes in protein composition of fat-cells which accompany these nutritional alterations. This suggests that the protein is not closely involved in lipogenesis or associated metabolic pathways, but rather that it may play a more general regulatory role. 4. The 22 kDa protein migrates as a doublet on SDS/PAGE even after purification to apparent homogeneity by sequential use of Mono Q chromatography, SDS/PAGE and h.p.l.c. The amino acid compositions of the two components are very similar and share features in common with a number of proteins, including inhibitor-1, inhibitor-2, dopamine- and cyclic-AMP-regulated phosphoprotein (DARPP-32), and G-substrate, which may be involved in the regulation of protein phosphatase activity. 5. Phosphopeptide mapping and phosphoamino acid analysis reveals that insulin increases the phosphorylation of two distinct peptides within the protein (in one peptide insulin increases the amount of phosphothreonine, whereas in the other the hormone increases the amounts of phosphothreonine and phosphoserine). Both components of the doublet exhibit similar changes in phosphorylation, and hence the differences in migration are not the result of differences in phosphorylation, as suggested previously [Blackshear, Nemenoff & Avruch (1983) Biochem. J. 214, 11-19]. The pattern of phosphorylation observed with the beta-adrenergic agonist isoprenaline was similar to that observed with insulin. 6. The possible role and regulation of the 22 kDa protein are discussed.

scholarly journals An effect of insulin on adipose-tissue adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1970 ◽  
Vol 120 (1) ◽  
pp. 187-193 ◽  
Author(s):  
E. G. Loten ◽  
J. G. T. Sneyd
Keyword(s):  
Adipose Tissue ◽  
Cyclic Amp ◽  
Cyclic Nucleotide ◽  
Fat Cells ◽  
Maximal Velocity ◽  
Isolated Fat Cells ◽  
Phosphodiesterase Activity ◽  
Cyclic Monophosphate ◽  
Cyclic Nucleotide Phosphodiesterases ◽  
Fat Pads

1. 3′:5′-Cyclic nucleotide phosphodiesterase activity was measured in homogenates prepared from epididymal fat-pads and isolated fat-cells incubated in the absence and presence of insulin. 2. Homogenates of insulin-treated tissues showed an increase in phosphodiesterase activity compared with controls. No effect of insulin was observed when the hormone was added directly to homogenates. 3. There was kinetic evidence for the presence of two 3′:5′-cyclic nucleotide phosphodiesterases in adipose tissue. Insulin raised the maximal velocity of the low-Km enzyme and lowered the Km of the higher-Km enzyme. 4. It is suggested that the effect of insulin on adipose tissue phosphodiesterase accounts for the ability of this hormone to lower cyclic-AMP concentration in the tissue.

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