Adipose Tissue Of Cold-acclimated Rats Research Articles

Identification of serine phosphorylation in mitochondrial uncoupling protein 1

Native uncoupling protein 1 was purified from rat brown adipose tissue of cold-acclimated rats and rats kept at room temperature, in the presence of phosphatase inhibitors. The purified protein from cold-acclimated animals was digested with trypsin and immobilized metal affinity chromatography was used to select for phosphopeptides. Tandem mass spectroscopic analysis of the peptides derived from uncoupling protein 1, suggests phosphorylation of serine 3 or 4 and identified phosphorylation of serine 51. Furthermore, we were able to demonstrate that antibodies to phosphoserine detect full-length UCP 1 and that the proportion of phosphoserine on UCP1, purified from cold-acclimated rats, was significantly greater than that on UCP 1 from rats kept at room temperature (90±4% compared to 62±8%, p=0.013), respectively). We conclude that uncoupling protein 1 is a phosphoprotein and that cold-acclimation increases the proportion of UCP1 that is serine phosphorylated.

Leptin immunoexpression and innervation in rat interscapular brown adipose tissue of cold-acclimated rats: the effects of L-arginine and L-NAME.

The aim of the present study was to explore the effect of nitric oxide on leptin immunoexpression and innervation in interscapular brown adipose tissue (IBAT) of room- and cold- acclimated rats. Animals acclimated both to room-temperature (22 +/- 1 degrees C) and cold (4 +/- 1 degrees C) were treated with L-arginine, a substrate for nitric oxide synthases (NOSs), or N?-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, for 45 days. Leptin expression and localization in brown adipocytes was studied by immunohistochemistry, and innervation stained by the Bodian method. Strong leptin immunopositivity was observed in brown adipocytes cytoplasm of all room-acclimated groups, but nuclear leptin positivity was found only in L-NAME treated rats. In cold-acclimated control and L-NAME treated rats leptin immunopositivity was absent, while L-arginine treatment reversed the cold-induced suppression of leptin expression. Comparing to control, L-arginine, and even more L-NAME, at 22 +/- 1 degrees C induced greater innervation. In conclusion, L-arginine treatment changes leptin expression pattern on cold in rat IBAT.

Leptin immunoexpression and innervation in rat interscapular brown adipose tissue of cold-acclimated rats: the effects of L-arginine and L-NAME.

Leptin immunoexpression and innervation in rat interscapular brown adipose tissue of cold-acclimated rats: the effects of L-arginine and L-NAME.

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.

Modulation of fatty acid synthesis in vivo in brown adipose tissue, liver and white adipose tissue of cold-acclimated rats.

The present experiment was an appraisal of the relative importance of fatty acid synthesis in brown adipose tissue (BAT) in young 28 or 5 degrees C adapted rats (9 weeks old). With a low-fat diet in vivo incorporation of 3H2O into BAT fatty acids was 8 times lower during the day than during the night and was not modified by a 6-hour fast during the day (28 degrees C). Cold acclimation doubled (night) or increased 8 times (day) BAT lipogenesis. Fasting led to a halving of the diurnal rate. A high-fat diet led to large decrease in synthesis rate during the night but had a weak effect on diurnal synthesis. The specific activity of fatty acids was 3 times lower in phospholipids than in neutral lipids. A comparison between 9- and 15-week-old rats indicated that in older warm-adapted rats BAT lipogenesis decreased by half but that cold stimulation was unaltered. These results were compared with hepatic and epididymal white adipose tissue lipogenesis. In conclusion, we showed that BAT of 5 degrees C rats is an important but not the major site for the conversion of carbohydrate to fat and that the proportional involvement of each tissue is age-dependent.

Increased α1-adrenergic receptor density in brown adipose tissue of cold-acclimated rats and hamsters

Binding sites for [ 3H]prazosin were characterized in crude membrane fractions from rat brown adipose tissue. Based on agonist (norepinephrine ≅ phenylephrine ⪢ isoprenaline) and antagonist (prazosin ⪢ yohimbine > propranolol) potencies to compete with [ 3H]prazosin, the binding sites were identified as α 1-receptors, not previously described in rat brown adipose tissue. As the [ 3H]prazosin binding sites could be observed in isolated brown fat cell preparations, they were probably postsynaptic. The effect of cold acclimation was studied in crude membrane fractions from control and cold-acclimated (4°C) rats and hamsters. Cold acclimation did not change the affinity of the receptor for agonists and antagonists, but there was a significant increase in the number of α 1-receptors (per mg protein), both in rat (100% increase) and hamster (40% increase) brown fat. Based on these results and on earlier results on β-receptors from this and other laboratories, it is suggested that activation of brown adipose tissue is associated with an increase in the relative density of α 1-receptors (i.e. in the α 1/ β ratio) and an increased significance of α 1-adrenergic pathways for the function of the tissue.

Altered effect of insulin and catecholamines in brown adipose tissue of cold-acclimated rats

Data are presented indicating that in brown adipose tissue (BAT) of cold-acclimated (CA), but not cold-exposed (CE) rats, there was an alteration in the relative response to catecholamines and insulin as evidenced by increased binding of alprenolol and decreased binding of insulin to plasma membrane enriched fractions. In addition, the stimulatory effect of insulin on glucose incorporation into glycogen and its inhibitory action on adenylate cyclase activity were both blunted in the CA tissues. It is proposed that shifts in the capacity of BAT to respond to catecholamines and insulin may be involved in the mechanism of cold acclimation.

The effect of adrenergic agonists and cyclic AMP on the Na +/K + ATPase activity of brown adipose tissue

The Na +/K + ATPase activity of membrane fractions prepared from brown adipose tissue of cold-acclimated rats could be stimulated by addition of any one of the following: norepinephrine, isoproterenol, cyclic AMP or phenylephrine. These results are consistent with the proposal that in the intact brown adipocyte, the norepinephrine-induced stimulation of the Na +/K + membrane pump is associated with α- as well as β-adrenergic pathways and is, in part, mediated via cyclic AMP.

Decreased half-life of some mitochondrial proteins in skeletal muscle and brown adipose tissue of cold-acclimated rats.

Evidence is presented for a decreased half-life of certain mitochondrial proteins in skeletal muscle and brown adipose tissue of cold-acclimated rats living in the cold; nonshivering thermo-genesis is known to occur in both these tissues. No such decrease occurs in liver or kidney, tissues in which nonshivering thermogenesis does not occur. It is concluded that acclimation to cold causes a change in the rate of synthesis and/or in the amount of certain mitochondrial proteins and that this change may be associated with the development of the capacity for nonshivering thermogenesis.

Nonphosphorylating respiration of mitochondria from brown adipose tissue of rats.

Mitochondria from brown adipose tissue of cold-acclimated rats (6 degrees C) oxidize alpha-ketoglutarate at a rate twice that of controls (26 degrees C). In both groups, however, the phosphorus: oxygen ratio with alpha-ketoglutarate never exceeded unity, and it is essentially zero with either succinate or alpha-glycerophosphate. Adenosine triphosphatase activity of these mitochondria is very low and it is not stimulated by 2,4-dinitrophenol. In addition, both respiration and phosphorylation are unaffected by adenosine diphosphate, 2,4-dinitrophenol, bovine serum albumin, or glutathione. Endogenous respiration of tissue slices is not stimulated by 2-4-dinitrophenol. It is suggested that brown fat mitochondria are not capable of oxidative phosphorylation, but do phosphorylate at the substrate level. Since these findings provide an unusual example of electron transport by means of an energetically nonconservative pathway, their significance to thermogenesis by brown adipose tissue is particularly emphasized.

LIPID METABOLISM IN WARM-ACCLIMATED AND COLD-ACCLIMATED RATS EXPOSED TO COLD.

The turnover of triglycerides and phospholipids of various tissues was studied in warm-acclimated and cold-acclimated rats, exposed to either a cold or a warm environment, by injecting glucose-U-C14intraperitoneally and measuring its incorporation into glycerol, glycerophosphate, and fatty acid portions of triglycerides and phospholipids at various times after the injection. The incorporation of glucose carbon into brown (interscapular) adipose tissue lipids (principally into glyceride glycerol) was 10 to 50 times greater in cold-acclimated rats in the cold than in warm-acclimated rats in the warm. The results are interpreted to indicate a great acceleration of the triglyceride cycle (triglyceride hydrolysis and resynthesis with concomitant ADP formation, the latter occasioning an increase in the activity of the electron transport system) in brown adipose tissue of cold-acclimated rats in the cold. Other possible interpretations are discussed. This acceleration was not observed in cold-acclimated rats in the warm or in warm-acclimated rats in the cold. No evidence was obtained for acceleration of triglyceride cycle in liver, heart, skeletal muscle, or white (epididymal) adipose tissue. The hypothesis is advanced that acceleration of the triglyceride cycle of brown adipose tissue is a major mechanism of nonshivering thermogenesis in cold-acclimated rats; such a mechanism is known to be under the control of the sympathetic nervous system and is consistent with the requirement that non-shivering thermogenesis can be switched on and off in response to changes in the environmental temperature.

Effects of cold acclimation on lipid metabolism in adipose tissue.

Cold acclimation is known to alter hepatic lipid metabolism. Liver slices from cold-acclimated rats have a greatly depressed capacity to synthesize long-chain fatty acids from acctate-1-C14. Since adipose tissue is the major site of lipogenic activity in the intact animal, its fatty acid synthetic capacity was studied. In contrast to the liver, it was found that adipose tissue from the cold-acclimated rat synthesized three to six times as much long-chain fatty acids per milligram of tissue protein as the adipose tissue from the control rat living at 25°C. Evidence is presented indicating that adipose tissue from cold-acclimated and control rats esterify long-chain fatty acids at the same rate. The ability of adipose tissue to oxidize palmitic acid to CO2 was found to be unaltered by cold acclimation. The fate of the large amount of fatty acid synthesized in the adipose tissue of cold-acclimated rats is discussed.