Substrate supply for thermogenesis induced by the beta-adrenoceptor agonist BRL 26830A

The nature of the substrate that fuels the thermogenic response to the novel beta-adrenoceptor agonist BRL 26830A has been investigated. Respiratory quotient measurements indicated that the increase in metabolic rate produced by BRL 26830A in rats was fuelled wholly by lipid. BRL 26830A also produced a marked reduction in the lipid content of total dissectable brown adipose tissue. The energy content of this lipid lost during the 4-h period after dosing was equivalent to approximately 50% of the thermogenic effect of the compound over the same period, suggesting that lipid stored in brown adipose tissue is a major initial fuel for BRL 26830A induced thermogenesis. However, marked depletion of brown adipose tissue lipid prior to administration of BRL 26830A had no effect on the subsequent thermogenic response to the compound. Oral administration of glucose altered the pattern of fuel utilization for resting metabolism, but thermogenesis was still fuelled mainly by lipid. Administration of methyl palmoxirate, which inhibits oxidation of long-chain fatty acids, completely prevented the thermic effect of BRL 26830A, suggesting that lipid is a necessary fuel for this process. These results do not support suggestions that carbohydrate is quantitatively important as a fuel for nonshivering thermogenesis.     

An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor

The energy balance in vivo is maintained through inter-organ cross-talk involving several different tissues. As a first step towards recapitulating the metabolic circuitry, hepatocytes, endothelial cells and adipose tissue were connected in a multicompartmental modular bioreactor to reproduce salient aspects of glucose and lipid metabolism in vitro. We first examined how the two-way cellular interplay between adipose tissue and endothelial cells affects glucose and lipid metabolism. The hepatocyte cell line HepG2 was then added to the system, creating a three-way connected culture, to determine whether circulating metabolite concentrations were normalized, or whether metabolic shifts, which may arise when endothelial cells and adipose tissue are placed in connection, were corrected. The addition of hepatocytes to the system prevented the drop in the concentrations of glucose, L-alanine and lactate, and the rise in that of free fatty acids. There was no significant change in glycerol levels in either of the connected cultures. The results show that connected cultures recapitulate complex physiological systemic processes, such as glucose and lipid metabolism, and that the HepG2 hepatocytes normalize circulating metabolites in this in vitro environment in the presence of other cell types.     

Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.

Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA). From in vitro assays, HSL is known to hydrolyze TG, diglycerides (DG), cholesteryl esters, and retinyl esters. In the current study we have generated HSL knock-out mice and demonstrate three lines of evidence that HSL is instrumental in the catabolism of DG in vivo. First, HSL deficiency in mice causes the accumulation of DG in white adipose tissue, brown adipose tissue, skeletal muscle, cardiac muscle, and testis. Second, when tissue extracts were used in an in vitro lipase assay, a reduced FFA release and the accumulation of DG was observed in HSL knock-out mice which did not occur when tissue extracts from control mice were used. Third, in vitro lipolysis experiments with HSL-deficient fat pads demonstrated that the isoproterenol-stimulated release of FFA was decreased and DG accumulated intracellularly resulting in the essential absence of the isoproterenol-stimulated glycerol formation typically observed in control fat pads. Additionally, the absence of HSL in white adipose tissue caused a shift of the fatty acid composition of the TG moiety toward increased long chain fatty acids implying a substrate specificity of the enzyme in vivo. From these in vivo results we conclude that HSL is the rate-limiting enzyme for the cellular catabolism of DG in adipose tissue and muscle.     

RNA biosynthesis in adipose tissue: effect of fasting

RNA metabolism has been examined in intact adipose tissue and isolated fat cells from rats. The lipocyte contains three species of RNA with sedimentation rates corresponding to those of ribosomal and transfer RNA. The de novo biosynthesis of RNA by adipose tissue cells in vitro was demonstrated. The base ratios of the RNA formed indicate that it was synthesized from a DNA template. Actinomycin D administered in vivo and in vitro decreased total RNA synthesis with the most marked effect on the synthesis of the heavy RNA components. Actinomycin D or puromycin added in vitro was not toxic: they did not inhibit total fatty acid biosynthesis or glucose utilization by the fat pad nor did they inhibit the immediate stimulation of fatty acid biosynthesis and glucose uptake by the addition of insulin in vitro. Starvation for 48-72 hr significantly depressed the synthesis of the heavy RNA components as measured by in vitro uridine incorporation into the individual RNA classes. Refeeding the fasted rat with glucose repaired the defect in RNA biosynthesis before the biosynthesis of monoenoic fatty acid was completely restored. Actinomycin D administered at the time of refeeding prevented the repair of monoenoic fatty acid synthesis. It is concluded that RNA metabolism is intimately involved in the control of biosynthetic reactions in adipose tissue.     

Effect of methoxyindole 2-carboxylic acid and 4-pentenoic acid on adipose tissue metabolism.

1. Administration of methoxyindole 2-carboxylic acid to rats caused an increase in circulating free fatty acids which was associated with rapid hypoglycemia in fasted rats and liver glycogenolysis without hypoglycemia in fed rats. 2. The incorporation of labeled glucose, pyruvate and acetate carbons into triacylglycerol-glycerol, triacylglycerol-fatty acids and CO2 was inhibited in epididymal fat pads from methoxyindole 2-carboxylic acid-treated rats and by the addition of methoxyindole 2-carboxylic acid in vitro. In contrast, palmitate esterification and oxidation were enhanced by methoxyindole 2-carboxylic acid. 3. The activity of enzymes associated with fatty acid synthesis was reduced to a varying degree in the presence of methoxyindole 2-carboxylic acid in the reaction mixture in concentrations lower than those used to inhibit glucose and pyruvate metabolism in the intact tissue in vitro. 4. 4-Pentenoic acid, a potent inhibitor of pyruvate and palmitate metabolism in the liver, was considerably less effective in adipose tissue. 5. The effect of the two hypoglycemic substances investigated on adipose tissue metabolism seems to be different.     

Effects of lactation and removal of pups on the rate of triacyglycerol/fatty acid substrate cycling in white adipose tissue of the rat.

The rate of the triacylglycerol/fatty acid substrate cycle was measured in vivo in adipose tissue of virgin and lactating rats with pups removed. The rate decreased by 70% in adipose tissue of lactating rats and increased 9-fold on removal of the pups. Similar differences in cycling rate were seen in adipose tissue incubated in vitro in the presence of isoprenaline.     

Brown adipose tissue cell respiration in hypo- and hyperthyroidism after stimulation with selective and non selective beta-adrenergic agonists.

Brown adipocyte respiration was measured in isolated cells from hypothyroid, hyperthyroid and euthyroid Sprague-Dawley male rats. Hypothyroidism was induced by providing drinking water containing methimazole and hyperthyroidism was induced by addition of thyroid powder to the diet. Brown adipose tissue (BAT) cells were isolated by collagenase digestion and oxygen consumption (VO2) was measured by Clark type oxygen electrodes. BAT cell respiration was stimulated by selective and nonselective beta-adrenergic agonists: BRL 35135A (BRL) and Isoprenaline (ISO). Basal BAT cells respiration did not differ according to thyroid status. Maximal VO2 responses of BAT adipocytes from hypothyroid rats were significantly lower than in euthyroidism after ISO and BRL. The reduced response was more marked for ISO than for BRL. The thermogenic sensitivity was significantly greater in euthyroid than is hypothyroid cells for ISO, but not for BRL. The euthyroid-hyperthyroid differences were not significantly different. These results suggest: basal respiration of BAT cells in hypo- and hyperthyroidism does not reflect the overall changes in whole body metabolism; the decreased thermogenic response in hypothyroidism might be due to decreased beta-adrenoceptor numbers and/or decreased intracellular thyroxine-triiodothyronine conversion; changes in sensitivity to ISO and BRL in vitro reflect the changes seen in VO2 in vivo.     

Alterations of mitochondrial protein metabolism in liver, brown adipose tissue and skeletal muscle. During cold-acclimation.

Incorporation of L-[U-14C] leucine into liver, brown adipose tissue and skeletal muscle mitochondrial proteins was determined in vivo and in vitro during cold-acclimation. Major alterations in mitochondrial protein metabolism were observed in brown adipose tissue and skeletal muscle but not in liver. Immediate cold-exposure is accompanied by an inhibition of the in vivo incorporation of L-[U-14C] leucine into mitochondrial proteins of all tissues. However, during cold-acclimation the incorporation of leucine increases markedly in brown adipose tissue, continues to decrease in skeletal muscle, nut does not change appreciably in the liver. Because increased incorporation of L-[U-14C]-leucine into brown adipose tissue mitochondrial proteins was observed both in vivo and in vitro, it can be concluded that the mitochondrial protein-synthesizing system of this tissue is directly affected by the acclimation process. The observed changes in mitochondrial protein metabolism of brown adipose tissue and skeletal muscle might be responsible for the development of several morphological and biochemical alterations that characterize the establishment in these tissues of the cold-acclimated state.     

The mobilization of free fatty acids in relation to adipose tissue triglyceride fatty acids in the rat

Three diets, consisting respectively of formulations high in oleic and stearic acid, linolenic acid, and lauric acid, were fed to rats until the adipose tissue TGFA largely reflected the dietary pattern of fatty acids. The composition of the serum FFA under basal conditions and following noradrenaline-stimulated lipolysis, were examined in relation to the respective adipose tissue TGFA. It was found in both in vivo and in vitro studies that lauric acid appeared to be less easily mobilized than longer chain acids. The in vitro studies indicated that this could not be explained either by positional preference of the shorter chain acids for the alpha-position of esterification or by increased reesterification of the shorter chain acids. The possibility remains that the difference is due to some specificity of tissue lipases for certain ester linkages.     

Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes

Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans.     

Selective mobilization of fatty acids from adipose tissue triacylglycerols

Adipose tissue triacylglycerols represent the main storage of a wide spectrum of fatty acids differing by molecular structure. The release of individual fatty acids from adipose tissue is selective according to carbon chain length and unsaturation degree in vitro and in vivo in animal studies and also in humans. The mechanism of selective fatty acid mobilization from white fat cells is not known. Lipolysis is widely reported to work at a lipid-water interface where only small amounts of substrate are available. A preferential hydrolysis of a small triacylglycerol fraction enriched in certain triacylglycerol molecular species at the lipid-water interface and enzymological properties of hormone-sensitive lipase could explain the selective mobilization of fatty acids from fat cells. This selectivity could affect the individual fatty acid supply to tissues.     

Inhibition by potential metabolic inhibitors of in vitro adipose tissue lipogenesis

To study the pathway of lactate utilization as a carbon source for fatty acid synthesis, the effect of (-)-hydroxycitrate, agaric acid, sodium oxamate, 2-n-butyl malonate and alpha-cyano-4-hydroxycinnamate on the rate of in vitro conversion of lactate, acetate and glucose to fatty acids was measured in bovine and rat adipose tissues. Sodium oxamate and hydroxycitrate caused less fatty acid to be synthesized from lactate in bovine adipose tissue. Hydroxycitrate depressed fatty acid synthesis from glucose in rat adipose tissue. alpha-Cyano-4-hydroxycinnamate was an effective inhibitor of lipogenesis from all substrates and may act as a specific inhibitor in adipose tissue. Although the inhibitors were absorbed poorly into adipocytes, the results indicate that conversion of lactate to fatty acids probably occurs by way of the citrate cleavage pathway.     

Comparison of glycerolipid biosynthesis in homogenates from human, ovine, bovine and rat adipose tissue in vitro.

1. Assay conditions were compared for glycerolipid biosynthesis in homogenates prepared from human abdominal, ovine and bovine subcutaneous, and rat epididymal adipose tissues. 2. In contrast to other species, longer incubation time and greater homogenate concentration resulted in decreased glycerolipid biosynthesis with rat adipose tissue homogenates. 3. Species differences were observed in concentration-dependency for ATP and fatty acids (palmitate, oleate and palmitoleate). 4. Results indicated that glycerolipid biosynthesis transpired at different rates in the four species, and that ovine and human adipose tissue homogenates had similar properties.     

Metabolic characteristics of human subcutaneous abdominal adipose tissue after overnight fast

Subcutaneous abdominal adipose tissue is one of the largest fat depots and contributes the major proportion of circulating nonesterified fatty acids (NEFA). Little is known about aspects of human adipose tissue metabolism in vivo other than lipolysis. Here we collated data from 331 experiments in 255 healthy volunteers over a 23-year period, in which subcutaneous abdominal adipose tissue metabolism was studied by measurements of arterio-venous differences after an overnight fast. NEFA and glycerol were released in a ratio of 2.7:1, different (P < 0.001) from the value of 3.0 that would indicate no fatty acid re-esterification. Fatty acid re-esterification was 10.2 ± 1.4%. Extraction of triacylglycerol (TG) (fractional extraction 5.7 ± 0.4%) indicated intravascular lipolysis by lipoprotein lipase, and this contributed 21 ± 3% of the glycerol released. Glucose uptake (fractional extraction 2.6 ± 0.3%) was partitioned around 20-25% for provision of glycerol 3-phosphate and 30% into lactate production. There was release of lactate and pyruvate, with extraction of the ketone bodies 3-hydroxybutyrate and acetoacetate, although these were small numerically compared with TG and glucose uptake. NEFA release (expressed per 100 g tissue) correlated inversely with measures of fat mass (e.g., with BMI, r(s) = -0.24, P < 0.001). We examined within-person variability. Systemic NEFA concentrations, NEFA release, fatty acid re-esterification, and adipose tissue blood flow were all more consistent within than between individuals. This picture of human adipose tissue metabolism in the fasted state should contribute to a greater understanding of adipose tissue physiology and pathophysiology.     

In vitro glucose and 2-aminoisobutyric acid uptake by rat interscapular brown adipose tissue

The dependence upon substrate and insulin concentrations, as well as on sodium and potassium concentrations in the medium of the uptake of glucose and 2-aminoisobutyric acid, was determined for fragments of brown and white adipose tissues incubated in vitro. Brown adipose tissue showed a high capacity for glucose uptake at high glucose concentrations, this uptake being dependent on both glucose and insulin concentration. White adipose tissue showed much more limited uptake capabilities. The presence of Na+ and K+ had little effect on the uptake. The uptake of 2-aminoisobutyric acid was similar in both adipose tissues, being enhanced by physiological levels of insulin and depressed by ouabain. This amino acid transport was dependent on Na+ and K+ concentrations, and the overall transporting capability was two to three orders of magnitude lower than that for glucose. It was concluded that amino acids could not play a significant role as bulk thermogenic substrates for brown adipose tissue, as their transporters lack the plasticity of response to high substrate and insulin concentrations which characterize brown adipose tissue uptake of glucose.     

Prostacyclin as a potent effector of adipose-cell differentiation.

The terminal differentiation of Ob1771 pre-adipose cells induced by arachidonic acid in serum-free hormone-supplemented medium containing insulin, transferrin, growth hormone, tri-iodothyronine and fetuin (5F medium) was strongly diminished in the presence of inhibitors of prostaglandin synthesis, namely aspirin or indomethacin. Carbaprostacyclin, a stable analogue of prostacyclin (prostaglandin I2) known to be synthesized by pre-adipocytes and adipocytes, behaved as an efficient activator of cyclic AMP production and was able, when added to 5F medium, to mimic the adipogenic effect of arachidonic acid. Prostaglandins E2, F2 alpha and D2, unable to affect the cyclic AMP production, failed to substitute for carbaprostacyclin. However, prostaglandin F2 alpha, which is another metabolite of arachidonic acid in pre-adipose and adipose cells, able to promote inositol phospholipid breakdown and protein kinase C activation, potentiated the adipogenic effect of carbaprostacyclin. In addition, carbaprostacyclin enhanced both a limited proliferation and terminal differentiation of adipose precursor cells isolated from rodent and human adipose tissues maintained in primary culture. These results demonstrate the critical role of prostacyclin and prostaglandin F2 alpha on adipose conversion in vitro and suggest a paracrine/autocrine role of both prostanoids in the development of adipose tissue in vivo.     

Changes in medium radioactivity and composition accompany high-affinity uptake of glutamate and aspartate by mouse brain slices

In measurements of high affinity transport in tissue slices, the incubation medium is often treated as an infinitely large pool. External substrate concentrations, even at the micromolar level, are assumed to be constant and metabolic interactions between tissue and medium are neglected. In the present report we describe experiments in which glutamic and aspartic acid uptake by mouse brain slices were studied using techniques that could test these assumptions. Cerebral hemispheres were cut into 0.1 mm sections and about 90 mg of tissue incubated in 10 ml of oxygenated medium. After 45 minutes of equilibration, radioactive substrates were added and the concentrations and specific activities of the amino acids and their metabolites in the medium were determined. During the first 10 min following substrate addition, rapid decreases in glutamic and aspartic acid concentrations in the medium were accompanied by large decreases in specific activity caused by the continuous release of these amino acids from the tissue. In addition, extensive conversion of both substrates to glutamine and the preferential accumulation of this metabolite, in the medium, was found. These results demonstrate that metabolism and release occur simultaneously with uptake during transport experiments in vitro and that these processes can take place in specific tissue compartments. It is therefore necessary to measure the tissue and medium concentration levels of amino acids along with their radioactivity in such experiments, since all three processes (transport, metabolism, and compartmentation) are interrelated in the clearance of amino acids from the incubation medium and probably from the extracellular spaces in vivo as well.     

Clenbuterol stimulated increase of plasma free fatty acids in reserpinized pigs

1. Previous studies indicate the beta-adrenergic agonist, clenbuterol, does not stimulate porcine adipose tissue lipolysis or cAMP concentration in vitro but increases plasma free fatty acid concentrations when infused, implying an indirect mechanism in vivo. 2. One indirect mechanism is the release of endogenous catecholamines to increase adipose tissue lipolysis and raise plasma free fatty acids. 3. In pigs treated with reserpine to deplete endogenous catecholamines, clenbuterol infusion increased plasma free fatty acids concentration suggesting that this increase in vivo did not result from release of endogenous catecholamines.     

Reduced maximum capacity of glycolysis in brown adipose tissue of genetically obese, diabetic (db/db) mice and its restoration following treatment with a thermogenic beta-adrenoceptor agonist

The maximal activities of the key glycolytic enzymes hexokinase and 6-phosphofructokinase, were reduced in brown adipose tissue in db/db mice compared to their lean littermates. Treatment of db/db mice with the thermogenic beta-adrenoceptor agonist, BRL 26830, restored normoglycaemia. The only significant increase in activity of hexokinase and 6-phosphofructokinase in the BRL 26830-treated db/db mice occurred in brown adipose tissue where the total tissue activity increased 10- and 11-fold respectively. These changes together with increased 2-deoxyglucose uptake in vivo suggest that brown adipose tissue can play a quantitatively important role in the removal of glucose from the blood.