Effect of 36-hour starvation on in vivo amino acid and glucose uptake by rat brown adipose tissue

The effect of 36-hour starvation on the net uptake/release of amino acids and glucose by interscapular brown adipose tissue (IBAT) of the rat has been studied by means of the determination of the arterio-venous differences in their blood concentrations. Starvation induced a net release of non-essential amino acids by the tissue, mainly alanine, glutamine, glycine and citrulline. In food deprived animals there was not a net glucose uptake by the IBAT. The results obtained in this study are in accordance with a typical peripheral tissue metabolic pattern of IBAT under food deprivation situations.     

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.     

Uptake of glucose and release of fatty acids and glycerol by rat brown adipose tissue in vivo

The net in vivo uptake or release of free fatty acids glycerol, glucose, lactate, and pyruvate by the interscapular brown adipose tissue (IBAT) of barbital-anesthetized, cold-acclimated rats was determined from measurements of plasma arteriovenous concentration differences across IBAT and tissue blood flow. Measurements were made without stimulation of the tissue and also during submaximal and maximal stimulation by infused noradrenaline (NA), the physiological activator of BAT thermogenesis. There was no appreciable uptake of glucose or release of fatty acids and glycerol by the nonstimulated tissue. At both levels of stimulation there was significant uptake of glucose (1.7 and 2.0 mumol/min) and release of glycerol (0.9 and 1.2 mumol/min), but only at maximal stimulation was there significant release of fatty acids (1.9 mumol/min). Release of lactate and pyruvate accounted for 33% of the glucose taken up at submaximal stimulation and 88% at maximal stimulation. By calculation, the remainder of the glucose taken up was sufficient to have fueled about 12% of the thermogenesis at submaximal stimulation, but only about 2% at maximal stimulation. As estimated from the rate of glycerol release, the rate of triglyceride hydrolysis was sufficient at submaximal stimulation to fuel IBAT thermogenesis entirely with the resulting fatty acids, but it was not sufficient to do so at maximal stimulation when some of the fatty acid was exported. It is suggested that at maximal NA-induced thermogenesis a portion of lipolysis proceeded only to the level of mono- and di-glycerides with the result that glycerol release did not fully reflect the rate of fatty acid formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid and glucose uptake by rat brown adipose tissue. Effect of cold-exposure and acclimation.

The net uptake/release of glucose, lactate and amino acids from the bloodstream by the interscapular brown adipose tissue of control, cold-exposed and cold-acclimated rats was estimated by measurement of arteriovenous differences in their concentrations. In the control animals amino acids contributed little to the overall energetic needs of the tissue; glucose uptake was more than compensated by lactate efflux. Cold-exposure resulted in an enhancement of amino acid utilization and of glucose uptake, with high lactate efflux. There was a net glycine and proline efflux that partly compensated the positive nitrogen balance of the tissue; amino acids accounted for about one-third of the energy supplied by glucose to the tissue. Cold-acclimation resulted in a very high increase in glucose uptake, with a parallel decrease in lactate efflux and amino acid consumption. Branched-chain amino acids, however, were more actively utilized. This was related with a much higher alanine efflux, in addition to that of glycine and proline. It is suggested that most of the glucose used during cold-exposure is returned to the bloodstream as lactate under conditions of active lipid utilization, amino acids contributing their skeletons largely in anaplerotic pathways. On the other hand, cold-acclimation resulted in an important enhancement of glucose utilization, with lowered amino acid oxidation. Amino acids are thus used as metabolic substrates by the brown adipose tissue of rats under conditions of relatively scarce substrate availability, but mainly as anaplerotic substrates, in parallel to glucose. Cold-acclimation results in a shift of the main substrates used in thermogenesis from lipid to glucose, with a much lower need for amino acids.     

Assessment of brown adipose tissue activity in rats by 99mTc-sestamibi uptake

Brown adipose tissue (BAT) physiology and imaging have recently attracted considerable attention. BAT is characterized both by enhanced perfusion and increased mitochondrial activity. (99m)Tc-sestamibi is a lipophilic cationic tracer that concentrates in mitochondria. Data on the accumulation of (99m)Tc-sestamibi in BAT are currently lacking. This study investigates the in vivo (99m)Tc-sestamibi uptake in rat BAT. (99m)Tc-sestamibi was administered in male Wistar rats of various age and body size. (99m)Tc-sestamibi uptake was measured in vitro in BAT and white fat (WF) together with cytochrome c oxidase activity. Both (99m)Tc-sestamibi uptake and cytochrome c oxidase activity were higher in BAT than in WF (P<0.05). (99m)Tc-Sestamibi uptake in both BAT and WF was negatively related to body weight (r = -0.96 and -0.89, respectively) as was the BAT/WF uptake ratio (r = -0.85). These data show a higher (99m)Tc-sestamibi uptake in BAT compared to WF, in agreement with the high mitochondrial content and respiratory activity of the former. The strong negative correlation between (99m)Tc-sestamibi uptake in BAT and body weight (negative allometry), is in accordance to increased needs of thermogenesis in smaller animals. Implications of increased (99m)Tc-sestamibi uptake in BAT in radionuclide imaging are also discussed.     

Characteristics of Ca uptake in mitochondria of brown adipose tissue

The rate of calcium uptake in brown adipose tissue mitochondria is here shown to be a sensitive parameter of energisation in this tissue, as demonstrated by high susceptibility to purine nucleotides and albumin. Complete uptake of low amounts of calcium generally requires added phosphate. Bicarbonate can at least partially substitute for phosphate, whereas acetate cannot. Calcium transport in brown fat mitochondria is of interest due to recent indications of an important role of this organelle in regulation of cytosolic calcium levels.     

Modulation of glucose uptake in adipose tissue by nitric oxide-generating compounds

There is increasing evidence that endogenous nitric oxide (NO) influences adipogenesis, lipolysis and insulin-stimulated glucose uptake. We investigated the effect of NO released from S-nitrosoglutathione (GSNO) and S-nitroso N-acetylpenicillamine (SNAP) on basal and insulin-stimulated glucose uptake in adipocytes of normoglycaemic and streptozotocin (STZ)-induced diabetic rats. GSNO and SNAP at 0.2, 0.5, and 1 mM brought about a concentration-dependent increase in basal and insulin-stimulated 2-deoxyglucose uptake in adipocytes of normoglycaemic and STZ-induced diabetic rats. SNAP at 1.0 mM significantly elevated basal 2-deoxyglucose uptake (115.8 ± 10.4%) compared with GSNO at the same concentration (116.1 ± 9.4%;P 0.05) in STZ-induced diabetic rats. Conversely, SNAP at concentrations of 10 mM and 20 mM significantly decreased basal 2-deoxyglucose uptake by 50.0 ± 4.5% and 61.5 ± 7.2% respectively in adipocytes of STZ-induced diabetic rats (P 0.05). GSNO at concentrations of 10 mM and 20 mM also significantly decreased basal 2-deoxyglucose uptake by 50.8 ± 6.4% and 55.2 ± 7.8% respectively in adipocytes of STZ-induced diabetic rats (P 0.05). These observations indicate that NO released from GSNO and SNAP at 1 mM or less stimulates basal and insulin-stimulated glucose uptake, and at concentrations of 10 mM and 20 mM inhibits basal glucose uptake. The additive effect of GSNO or SNAP, and insulin observed in this study could be due to different mechanisms and warrants further investigation.     

Plasma glucose kinetics and tissue uptake in brown trout in vivo: effect of an intravascular glucose load

The object of the present study was to elucidate whether a glucose load modifies glucose uptake by tissues in brown trout in vivo. By the use of 2-[1,2-³H]-deoxyglucose, plasma glucose disappearance rate and tissue glucose uptake were measured after an intraaortic glucose load of 500 mg·kg^-1 (glucose load group) and under normoglycemic conditions (control). We also attempted to determine whether fasting modifies the glucose load disposal (fasted glucose load group). The procedure used to calculate 2-deoxyglucose uptake by tissues was evaluated, and the levels of 2-deoxyglucose uptake were compared with those of 2-deoxyglucose phosphorylation. Uptake and phosphorylation rates were similar in all tissues, except in brain and heart. In all the groups glucose uptake rates were highest in spleen, kidney, brain and gills, and lowest in red muscle, heart and white muscle. However, white muscle was the main site of glucose uptake on a whole tissue basis. The glucose load led to strong, long-lasting hyperglycemia, in spite of the increases observed in plasma insulin levels and in glucose uptake rate by the whole body (control: 4.9 mol·min^-1·kg^-1; glucose load group: 6.5 mol·min^-1·kg^-1). This higher rate was due to the higher glucose uptake only in white and red muscles (four- and threefold, respectively). Fasting halved the uptake of glucose by both red and white muscles in the load condition. In consequence the use of exogenous glucose decreased with fasting (fasted glucose load group: 5.1 mol·min^-1·kg^-1), causing still longer hyperglycemia.Abbreviations bw body weight - 2DG 2-[1,2-³H]-deoxyglucose - 2DG-P 2-[1,2-³H]-deoxyglucose phosphate - dpm disintegrations per min - FGL fasted glucose load group - GL glucose load group - G-6-Pase glucose-6-phosphatase - LG L-[1-¹⁴C]-glucose - MS-222 3-aminobenzoic acid ethyl ester methanesulphonate salt     

In vivo quantitative lipidic map of brown adipose tissue by chemical shift imaging at 4.7 Tesla.

In the present paper, chemical shift imaging techniques are applied to quantitative in vivo evaluation of fat and water content in interscapular brown adipose tissue (BAT). The experiments have been carried out on five female Sprague-Dawley rats after calibration and testing with suitable phantoms containing known amounts of water and oil. We found that, in the interscapular BAT, the fat is about 50% at the surface (mainly unilocular) region, but its percentage drops to 20;-30% in the deepest (mainly multilocular) portion. The perirenal deposits of white adipose tissue (WAT) contained significantly higher amount of fat with large areas ranging from 70 to 90%. Later the rats were killed and the same procedure was repeated with dead animals. Experiments performed in dead rats show a modification of the hydro-lipidic ratio more evident in the multilocular portions of the deposit. The present work demonstrates that MRI-based methods allow a non-invasive, in vivo quantitative mapping of the lipid content which can be applied to investigation of brown adipose tissue deposits in small experiment animals.     

Effect of exercise training on in vivo insulin-stimulated glucose uptake in intra-abdominal adipose tissue in rats

Intra-abdominal obesity may be crucial in the pathogenesis of the insulin-resistance syndrome, and training may alleviate this condition. We compared insulin-mediated glucose uptake in vivo in three intra-abdominal adipose tissues (ATs; retroperitoneal, parametrial, and mesenteric) and in subcutaneous AT and also studied the effect of training. Rats were either swim trained (15 wk, n = 9) or sedentary (n = 16). While the rats were under anesthesia, a hyperinsulinemic ( approximately 900 pM), euglycemic clamp was carried out and local glucose uptake was measured by both the 2-deoxy-D-[(3)H]glucose and microdialysis techniques. Blood flow was measured by microspheres. Upon insulin stimulation, blood flow generally decreased in AT. Flow was higher in mesenteric tissue than in other ATs, whereas insulin-mediated glucose uptake did not differ between ATs. Training doubled the glucose infusion rate during hyperinsulinemia, in part, reflecting an effect in muscle. During hyperinsulinemia, interstitial glucose concentrations were lower, glucose uptake per 100 g of tissue was higher in AT in trained compared with sedentary rats, and training influenced glucose uptake identically in all ATs. In conclusion, differences between ATs in insulin sensitivity with respect to glucose uptake do not explain that insulin resistance is associated with intra-abdominal rather than subcutaneous obesity. Furthermore, training may be beneficial by enhancing insulin sensitivity in intra-abdominal fat depots.     

Anatomical and functional assessment of brown adipose tissue by magnetic resonance imaging

Brown adipose tissue (BAT) is the primary tissue responsible for nonshivering thermogenesis in mammals. The amount of BAT and its level of activation help regulate the utilization of excessive calories for thermogenesis as opposed to storage in white adipose tissue (WAT) which would lead to weight gain. Over the past several years, BAT activity in vivo has been primarily assessed by positron emission tomography-computed tomography (PET-CT) scan using 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) to measure glucose utilization associated with BAT mitochondrial respiration. In this study, we demonstrate the feasibility of mapping and estimating BAT volume and metabolic function in vivo in rats at a 9.4T magnetic resonance imaging (MRI) scanner using sequences available from clinical MR scanners. Based on the morphological characteristics of BAT, we measured the volume distribution of BAT with MRI sequences that have strong fat-water contrast. We also investigated BAT volume by utilizing spin-echo MRI sequences. The in vivo MRI-estimated BAT volumes were correlated with direct measurement of BAT mass from dissected samples. Using MRI, we also were able to map hemodynamic responses to changes in BAT metabolism induced pharmacologically by β3-adrenergic receptor agonist, CL-316,243 and compare this to BAT activity in response to CL-316,243 assessed by PET 18F-FDG. In conclusion, we demonstrate the feasibility of measuring BAT volume and function in vivo using routine MRI sequences. The MRI measurement of BAT volume is consistent with quantitative measurement of the tissue ex vivo.     

Effect of alanine on in vitro glucose utilization by rat interscapular brown adipose tissue

The rates of either glucose or alanine incorporation into tissue and oxidation to CO2 were studied in rat interscapular brown adipose tissue in order to evaluate the mutual influence of both substrates on their uptake and utilization. Tissue fragments were incubated in vitro in the presence of 1-10 mM glucose and 0.3-1.5 mM alanine. The highest glucose oxidation rate was obtained with the lowest alanine concentrations tested. This suggests that alanine inhibits glucose utilization by this tissue at concentrations that are within the physiological plasmatic range. Glucose levels had little effect upon alanine oxidation, but glucose had a permissive effect on the utilization of alanine. On the basis of these results, it is postulated that this glucose conservation effect of alanine on brown adipose tissue can help to prevent glucose wastage in postprandrial situations.     

Cold exposure increases glucose utilization and glucose transporter expression in brown adipose tissue.

When rats were exposed to a cold environment (4 degrees C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for non-shivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the cold-induced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4.     

Sympathetic activation of glucose utilization in brown adipose tissue in rats.

The effects of norepinephrine (NE) infusion and surgical denervation or electrical stimulation of the sympathetic nerves on 2-deoxyglucose (2-DG) uptake in interscapular brown adipose tissue (BAT) were investigated in vivo in rats to obtain direct evidence for sympathetic control of glucose utilization in this tissue. 2-DG uptake was rather low in fasted rats, but after refeeding it increased in the BAT as well as the heart, skeletal muscle, and white adipose tissue, in parallel with an increase in plasma insulin level. Cold exposure also enhanced 2-DG uptake in the BAT without the increase in plasma insulin level, while it had no appreciable effect on 2-DG uptake in other tissues. Sympathetic denervation greatly attenuated the stimulatory effect of cold exposure on 2-DG uptake in BAT, but it did not affect the increased 2-DG uptake after refeeding. Electrical stimulation of the sympathetic nerves entering BAT or NE infusion produced a marked increase in 2-DG uptake in BAT without noticeable effects in other tissues. beta-Adrenergic blockade, but not alpha-blockade, abolished the increased 2-DG uptake in BAT. It was concluded that glucose utilization in BAT is activated directly, independently of the action of insulin, by sympathetic nerves via the beta-adrenergic pathway.     

Adrenergic regulation of AMP-activated protein kinase in brown adipose tissue in vivo

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In β(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or β-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in β(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.     

The effects of meal-feeding and the diurnal cycle on lipogenesis in brown adipose tissue of rats

A significant diurnal variation in the rates of lipogenesisin vivo in brown adipose tissue occurred in both virgin and lactating rats. On a meal-feeding regime of either a chow, high-sucrose, or high-lipid diet, there was a very large increase in BAT lipogenesis following the meal. The rates observed after the sucrose meal are the highest so far reported. There was no significant difference in BAT lipogenesis between lactating and virgin rats, contrary to previous reports by others. The pattern of stimulation of BAT lipogenesis by these feeding regimes was different from that for white adipose tissue and liver and was not correlated with plasma insulin levels.