Alterations in the regulation of lipolysis in brown and white adipose tissue in the diabetic state.
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Adenosine↗
Biomedical subjects
E D Saggerson
Publications and source records attributed to E D Saggerson.
Measurements of glycolytic flux rate in brown adipocytes. Effects of insulin, noradrenaline and streptozotocin diabetes.
Glycolytic flux was estimated in brown adipocytes by [3-3H]-glucose detritiation. Without insulin the process was slightly stimulated by noradrenaline or palmitate. Insulin stimulated glucose detritiation by 4-fold. Noradrenaline stimulated the process in the presence of insulin and synergism between these hormones was observed. Palmitate did not stimulate glucose detritiation in the presence of insulin suggesting that the effect of noradrenaline is not secondary to stimulation of lipolysis. With insulin, cells from streptozotocin-diabetic rats showed lower rates of glucose detritiation. Extracts from these cells also had lower maximum activities of phosphofructokinase.
Adipose Tissue, Brown↗
Changes in activities of some enzymes of glycerolipid synthesis in brown adipose tissue of cold-acclimated rats.
1. Measurements were made of the activities of the following enzymes of glycerolipid synthesis in homogenates of interscapsular brown adipose tissue obtained from rats subjected to a 4 degrees C environment for time periods of 6 h up to 12 days: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT) and Mg2+-dependent phosphatidate phosphohydrolase (PPH). 2. Relative to tissue DNA content, the activities of mitochondrial GPAT, MGPAT and Mg2+-dependent PPH were significantly increased after 1 day of exposure to cold, and continued to increase thereafter. By contrast, FAS and microsomal GPAT activities were unchanged relative to tissue DNA. 3. The time profile of the increase in MGPAT activity correlated well with a concomitant increase in the microsomal marker NADP+-cytochrome c reductase. Changes in mitochondrial GPAT and in Mg2+-dependent PPH activities were larger in amplitude than that of MGPAT. 4. It is proposed that these selective changes in enzyme activity may be associated with the onset of brown-adipose-tissue hyperplasia or possibly with an increase in triacylglycerol synthesis during cold-acclimation.
1-Acylglycerol-3-Phosphate O-Acyltransferase↗
Changes in brown-adipose-tissue mitochondrial processes in streptozotocin-diabetes.
Diabetic rats were used as a source of brown-adipose-tissue mitochondria 2 days after a single subcutaneous injection of streptozotocin (100 mg/kg). Diabetes caused an 80% decrease in carnitine-dependent oxidation of palmitoyl-CoA and a 50-60% decrease in overt carnitine palmitoyltransferase activity. An additional lesion in brown-adipose-tissue mitochondrial oxidative capacity was also indicated, since diabetes increased by 30-50% the rate of oxidation under uncoupled conditions of several respiratory substrates (i.e. malate + palmitoylcarnitine, malate + pyruvate, succinate, NNN'N'-tetramethyl-p-phenylenediamine + ascorbate). This decrease in mitochondrial function was accompanied by an approx. 30% decrease in the abundance of cytochromes (a + a3) and total cytochromes b.
Adipose Tissue, Brown↗
Lipogenesis in rat brown adipocytes. Effects of insulin and noradrenaline, contributions from glucose and lactate as precursors and comparisons with white adipocytes.
1. Brown adipocytes were isolated from the interscapular depot of male rats maintained at approx. 21 degrees C. In some experiments parallel studies were made with white adipocytes from the epididymal depot. 2. Insulin increased and noradrenaline decreased [U-14C]glucose incorporation into fatty acids by brown adipocytes. Brown adipocytes differed from white adipocytes in that exogenous fatty acid (palmitate) substantially decreased fatty acid synthesis from glucose. Both noradrenaline and insulin increased lactate + pyruvate formation by brown adipocytes. Brown adipocytes converted a greater proportion of metabolized glucose into lactate + pyruvate and a smaller proportion into fatty acids than did white adipocytes. 3. In brown adipocytes, when fatty acid synthesis from [U-14C]glucose was decreased by noradrenaline or palmitate, incorporation of 3H2O into fatty acids was also decreased to an extent which would not support proposals for extensive recycling into fatty acid synthesis of acetyl-CoA derived from fatty acid oxidation. 4. In the absence of glucose, [U-14C]lactate was a poor substrate for lipogenesis in brown adipocytes, but its use was facilitated by glucose. When brown adipocytes were incubated with 1 mM-lactate + 5 mM-glucose, lactate-derived carbon generally provided at least 50% of the precursor for fatty acid synthesis. 5. Both insulin and noradrenaline increased [U-14C]glucose conversion into CO2 by brown adipocytes (incubated in the presence of lactate) and, in combination, stimulation of glucose oxidation by these two agents showed synergism. Rates of 14CO2 formation from glucose by brown adipocytes were relatively small compared with maximum rates of oxygen consumption by these cells, suggesting that glucose is unlikely to be a major substrate for thermogenesis. 6. Brown adipocytes from 6-week-old rats had considerably lower maximum rates of fatty acid synthesis, relative to cell DNA content, than white adipocytes. By contrast, rates of fatty acid synthesis from 3H2O in vivo were similar in the interscapular and epididymal fat depots. Expressed relative to activities of fatty acid synthase or ATP citrate lyase, however, brown adipocytes synthesized fatty acids as effectively as did white adipocytes. It is suggested that the cells most active in fatty acid synthesis in the brown adipose tissue are not recovered fully in the adipocyte fraction during cell isolation. Differences in rates of fatty acid synthesis between brown and white adipocytes were less apparent at 10 weeks of age.
Adipose Tissue↗
A tissue-specific increase in lipogenesis in rat brown adipose tissue in hypothyroidism.
1. Rats were made hypothyroid by feeding them with propylthiouracil together with a low-iodine diet for 4 weeks. 2. [U-14C]Glucose conversion into fatty acids was substantially enhanced in brown adipocytes isolated from hypothyroid rats. Incorporation of 3H2O into fatty acids in vivo was enhanced in hypothyroidism in interscapular brown fat, but not in epididymal white fat or in liver. Hypothyroidism increased the activities of fatty acid synthase and ATP citrate lyase in brown, but not in white, adipocytes. 3. Glycolytic flux in brown adipocytes, quantified by [3-3H]glucose detritiation, was increased by hypothyroidism. This change was accompanied by increased maximum activity of phosphofructokinase. In white adipocytes a large increase in phosphofructokinase maximum activity was observed in hypothyroidism, but this change was accompanied by only small increases in the rate of glucose detritiation by incubated cells. It is suggested that in the brown adipocyte the overall conversion of glucose into fatty acids is enhanced in thyroid deficiency, but that this change is muted in the white adipocyte, possibly because of limitation of glucose transport. 4. Fatty acid synthesis in brown adipocytes from hypothyroid animals was considerably less sensitive to inhibition by exogenous fatty acids than is the process in cells from euthyroid animals. Consequently, the effect of hypothyroidism to enhance lipogenesis is amplified in the presence of physiological concentrations of fatty acid.
Adipose Tissue↗
Comparison of triacylglycerol synthesis in rat brown and white adipocytes. Effects of hypothyroidism and streptozotocin-diabetes on enzyme activities and metabolic fluxes.
1. Adipocytes were isolated from the interscapular brown fat and the epididymal white fat of normal, streptozotocin-diabetic and hypothyroid rats. 2. Measurements were made of the maximum rate of triacylglycerol synthesis by monitoring the incorporation of [U-14C]glucose into acylglycerol glycerol in the presence of palmitate (1 mM) and insulin (4 nM) and of the activities of the following triacylglycerol-synthesizing enzymes: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), dihydroxyacetonephosphate acyltransferase (DHAPAT), monoacylglycerol phosphate acyltransferase (MGPAT), Mg2+-dependent phosphatidate phosphohydrolase (PPH) and diacylglycerol acyltransferase (DGAT). 3. FAS activity in brown adipocytes was predominantly localized in the mitochondrial fraction, whereas a microsomal localization of this enzyme predominated in white adipocytes. Subcellular distributions of the other enzyme activities in brown adipocytes were similar to those shown previously with white adipocytes [Saggerson, Carpenter, Cheng & Sooranna (1980) Biochem. J. 190, 183-189]. 4. Relative to cell DNA, brown adipocytes had lower activities of triacylglycerol-synthesizing enzymes and showed lower rates of metabolic flux into acylglycerols than did white adipocytes isolated from the same animals. 5. Diabetes decreased both metabolic flux into acylglycerols and the activities of triacylglycerol-synthesizing enzymes in white adipocytes. By contrast, although diabetes decreased metabolic flux into brown-adipocyte acylglycerols by 80%, there were no decreases in the activities of triacylglycerol-synthesizing enzymes, and the activity of PPH was significantly increased. 6. Hypothyroidism increased metabolic flux into acylglycerols in both cell types, and increased activities of all triacylglycerol-synthesizing enzymes in brown adipocytes. By contrast, in white adipocytes, although hypothyroidism increased the activities of FAS, microsomal GPAT and DGAT, this condition decreased the activities of mitochondrial GPAT and PPH. 7. It was calculated that the maximum capabilities for fatty acid oxidation and esterification are approximately equal in brown adipocytes. In white adipocytes esterification is predominant by approx. 100-fold. 8. Diabetes almost abolished incorporation of [U-14C]glucose into fatty acids in both adipocyte types. Hypothyroidism increased fatty acid synthesis in white and brown adipocytes by 50% and 1000% respectively.
Adipose Tissue↗
A novel assay for 5'-nucleotidase using 1,N6-etheno-AMP as substrate, and comments on the properties of the reaction product, ethenoadenosine.
A novel assay for 5'-nucleotidase is described in which 1,N6-etheno-AMP is converted into ethenoadenosine. The product ethenoadenosine is neither a substrate for nor an inhibitor of adenosine deaminase. Ethenoadenosine appears to have little effect at adenosine receptors on adipose-tissue cells.
5'-Nucleotidase↗
Factors influencing the altered thermogenic response of rat brown adipose tissue in streptozotocin-diabetes.
1. Adipocytes were isolated from the interscapular brown fat of male rats maintained at 21 degrees C. These animals were controls, streptozotocin-diabetics or 2-day insulin-treated diabetics. 2. With adipocytes from diabetic animals, maximum rates of noradrenaline-stimulated O2 uptake were decreased by 58%, and the Bmax. of [3H]GDP binding to mitochondria was decreased by 55%. Insulin administration reversed both of these changes. 3. Streptozotocin-diabetes increased basal lipolysis in adipocytes incubated with adenosine deaminase (1 unit/ml), decreased the EC50 (concn. giving 50% of maximum effect) for noradrenaline, but did not change the maximum rate of noradrenaline-stimulated lipolysis. Except for some small differences at very low concentrations (10-100 pM), diabetes or insulin treatment did not alter the sensitivity of noradrenaline-stimulated lipolysis or O2 uptake to the inhibitory effect of N6-phenylisopropyladenosine. It is therefore concluded that the lesion(s) in thermogenesis in diabetes are not attributable to any changes in lipolysis. 4. Blood flow through interscapular brown fat, measured by accumulation of [14C]DDT [14C-labelled 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] was increased by 2.3-fold 70 min after a single administration of insulin to diabetic rats. This treatment decreased blood flow through epididymal white fat by 58%. 5. Propranolol treatment of diabetic rats muted the ability of insulin treatment to increase the maximum rate of noradrenaline-stimulated O2 uptake, suggesting that this action of insulin may be a secondary one rather than a direct effect of the hormone on the adipocytes.
Adenosine Deaminase↗
Chemically induced hypothyroidism produces elevated amounts of the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) and the beta subunit common to all G-proteins.
Adipocytes of hypothyroid rats display an increased responsiveness to agents which function by inhibiting the production of cyclic AMP. Anti-peptide antisera which selectively recognise the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) detected a 40 kDa polypeptide in adipocyte plasma membranes of both euthyroid and hypothyroid rats. Amounts of the alpha subunit of Gi were elevated some 2-fold in the hypothyroid preparations in comparison with the euthyroid controls, when equal amounts of membrane protein of the two treatments were examined. As cells from the hypothyroid animals contained 2.7 times as much membrane protein as those from the control animals, the amounts of alpha subunit of Gi are elevated some 5.6-fold per cell in adipocytes of the hypothyroid animals compared with the euthyroid controls. Amounts of the 36 kDa beta subunit of G-proteins were also elevated in plasma membranes of adipocytes of hypothyroid animals, in this case by some 50% when compared on a protein basis. These results provide direct evidence for alterations in the amounts of the subunits of Gi caused by the hypothyroid state.
Adipose Tissue↗
Enzymes involved in adenosine metabolism in rat white and brown adipocytes. Effects of streptozotocin-diabetes, hypothyroidism, age and sex differences.
1. Adipocytes were isolated from epididymal white fat and interscapular brown fat of male rats, and activities of 5'-nucleotidase, adenosine deaminase and adenosine kinase were measured in cell extracts. 2. 5'-Nucleotidase activity in white adipocytes was increased in streptozotocin-diabetes, decreased in hypothyroidism and increased with age. That activity in brown adipocytes was unchanged in diabetes, decreased in hypothyroidism and increased with age. 5'-Nucleotidase activity was higher in white adipocytes from female rats. 3. Adenosine deaminase activity in white adipocytes was increased in diabetes, decreased in hypothyroidism and increased with age. That activity in brown adipocytes was decreased in diabetes and hypothyroidism. 4. Adenosine kinase activity in both cell types was unchanged in diabetes or hypothyroidism, but increased with age.
5'-Nucleotidase↗
Effects of streptozotocin-diabetes and insulin administration in vivo or in vitro on the activities of five enzymes in the adipose-tissue triacylglycerol-synthesis pathway.
At 2 days after administration of streptozotocin (100 mg/kg), activities in rat epididymal fat-pads of the following enzymes were significantly decreased: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT) and Mg2+-dependent phosphatidate phosphohydrolase (PPH). There were no significant changes in diacylglycerol acyltransferase or Mg2+-independent PPH. Insulin administration to diabetic rats over 2 days restored activities of FAS, both forms of GPAT, MGPAT and Mg2+-dependent PPH. Significant restoration of all five activities was also seen 2 h after a single administration of insulin, but was not observed 45 min after insulin treatment. Insulin significantly increased all five enzyme activities when adipocytes from diabetic rats were incubated for 2 h with a mixture of glucose, lactate, pyruvate and amino acids.
Acyltransferases↗
Enzyme activities in regions of the hypothalamus.
The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].
Animals↗
Adipose-tissue Mg2+-dependent phosphatidate phosphohydrolase. Control of activity and subcellular distribution in vitro and in vivo.
The subcellular distribution of Mg2+-dependent phosphatidate phosphohydrolase in rat adipocytes between a soluble and a membrane-bound fraction was measured by using both centrifugal fractionation and a novel Millipore-filtration method. The relative proportion of the phosphohydrolase associated with the particulate fraction was increased on incubation of cells with noradrenaline or palmitate. Insulin on its own decreased the proportion of the phosphohydrolase that was particulate and abolished the effect of noradrenaline, but not that of palmitate. The effect of noradrenaline on phosphohydrolase distribution was rapid, the effect being maximal within 10 min. Noradrenaline exerted this effect with a similar concentration-dependence to its lipolytic effect. Inclusion of albumin in homogenization buffers decreased the proportion of the phosphohydrolase that was particulate, but did not abolish the effect of noradrenaline. There was limited correlation between the proportion of the phosphohydrolase that was particulate and the measured rate of triacylglycerol synthesis in adipocytes incubated under a variety of conditions. Starvation, streptozotocin-diabetes and hypothyroidism decreased the specific activities of the phosphohydrolase and glycerolphosphate acyltransferase in homogenates from epididymal fat-pads. Restoration of these activities in the diabetic state was seen after administration of insulin over 2 days or, in the short term, within 2 h after a single administration of insulin. Administration of thyroxine over 3 days caused restoration of these activities in the hypothyroid state. Starvation and diabetes increased the proportion of the phosphohydrolase found in the microsomal fraction. This change was not seen when albumin was present in homogenization buffers. The possible role of fatty acids as regulators of the intracellular translocation of the phosphohydrolase, together with the role of this enzyme in the regulation of triacylglycerol synthesis in adipose tissue, is discussed.
Adipose Tissue↗
Sensitivity of adipocyte lipolysis to stimulatory and inhibitory agonists in hypothyroidism and starvation.
The responsiveness of lipolysis to the stimulatory agonists noradrenaline, corticotropin and glucagon and to the inhibitory agonists N6-phenylisopropyladenosine, prostaglandin E1 and nicotinic acid was investigated with rat white adipocytes incubated with a high concentration of adenosine deaminase (1 unit/ml). The cells were obtained from fed or 48 h-starved euthyroid animals or from fed or starved animals rendered hypothyroid by 4 weeks of treatment with low-iodine diet and propylthiouracil. Hypothyroidism increased sensitivity to and efficacy of all three inhibitory agonists in their opposition of noradrenaline-stimulated lipolysis. Starvation decreased sensitivity to all three inhibitory agonists when opposing basal lipolysis. Hypothyroidism decreased sensitivity to noradrenaline, glucagon and corticotropin by 37-, 4- and 4-fold respectively and decreased the maximum response to these agonists by approx. 50%, 50% and 75% respectively. Starvation reversed decreases in maximum response to these agonists in hypothyroidism. Starvation in the euthyroid state increased sensitivity to glucagon and noradrenaline, but did not alter sensitivity to corticotropin. Cells from hypothyroid rats were relatively insensitive to Bordetella pertussis toxin, which substantially increased basal lipolysis in the euthyroid state.
Adenosine Deaminase↗
Effect of adenosine deaminase, N6-phenylisopropyladenosine and hypothyroidism on the responsiveness of rat brown adipocytes to noradrenaline.
Adenosine deaminase (1 unit/ml) potentiated the lipolytic action of noradrenaline in adipocytes isolated from brown adipose tissue of 1- and 6-week-old rats by decreasing the EC50 (concn. giving 50% of maximal effect) for noradrenaline by 3-4-fold. With cells from neonatal rabbit tissue, adenosine deaminase only had a small, non-significant, effect on the EC50 for noradrenaline. Lipolysis in rat brown adipocytes was inhibited by low concentrations of N6-phenylisopropyladenosine (PIA). Rabbit cells were far less sensitive to PIA. PIA, prostaglandin E1 and nicotinate all inhibited noradrenaline-stimulated respiration in rat brown adipocytes. Hypothyroidism diminished the maximum response of respiration and lipolysis to noradrenaline in rat cells and increased the EC50 for noradrenaline. Responsiveness of lipolysis to noradrenaline was particularly decreased in hypothyroidism and was partially restored by addition of adenosine deaminase. Lipolysis in cells from hypothyroid rats was more sensitive to the anti-lipolytic action of PIA. Bordetella pertussis toxin increased lipolysis in the presence of PIA, suggesting an involvement of the Ni guanine-nucleotide-binding protein in the control of brown-adipocyte metabolism.
Adenosine↗
Intertissue differences in the hysteretic behaviour of carnitine palmitoyltransferase in the presence of malonyl-CoA.
In the presence of malonyl-CoA, the overt form of carnitine palmitoyltransferase (CPT1) in mitochondria from rat liver, kidney cortex, heart, skeletal muscle and brown adipose tissue shows non-linear time courses, suggesting hysteretic behaviour. The pattern of this hysteresis is similar in heart, skeletal muscle and brown adipose tissue, but the hysteretic behaviour of the enzyme in these three tissues differs markedly from that seen in liver and kidney.
Acyl Coenzyme A↗
Carnitine palmitoyltransferase in liver and five extrahepatic tissues in the rat. Inhibition by DL-2-bromopalmitoyl-CoA and effect of hypothyroidism.
Mitochondria were isolated from rat adult liver, foetal liver, kidney cortex, heart, skeletal muscle and interscapular brown adipose tissue. DL-2-Bromopalmitoyl-CoA inhibited the overt form of carnitine palmitoyltransferase (CPT1) in heart, skeletal muscle and brown adipose tissue, with an IC50 value (concentration giving 50% inhibition) of 1.3-1.6 microM. By contrast, the IC50 value for inhibition of the kidney or adult liver enzyme was 0.08-0.1 microM. CPT1 in near-term foetal liver differed from that in adult liver in that the IC50 for inhibition by 2-bromopalmitoyl-CoA was 0.57 microM. It is suggested that there may be tissue-specific forms of the catalytic entity of CPT1 and that foetal liver may contain a mixture of adult liver- and muscle-type enzymes. In rats made hypothyroid by administration of propylthiouracil and an iodine-deficient diet, hepatic CPT1 activity was decreased by 83%. However, CPT1 activity in extrahepatic tissues showed no adaptive decrease in hypothyroidism.
Acyl Coenzyme A↗