Neural and vascular provisions of rat interscapular brown adipose tissue

The innervation of rat interscapular brown adipose tissue has been studied by light and fluorescence microscopy and electron microscopy after treatment with "false" adrenergic neurotransmitters 5- and 6-hydroxydopamine. The vascular markers neoprene latex and thioflavin S were used to define the blood vascular arrangements within the around the tissue. Catecholaminergic innervation was revealed by fluorescence microscopy at both parenchymal and vasomotor sites. In animals injected with 6-hydroxydopamine, this catecholaminergic fluorescence was extinguished in the parenchymal nerve distribution and markedly reduced in the vasomotor plexus. Identification of an extensive network of noradrenergic vasomotor and parenchymal nerve terminals was established by electron microscopy after 5- and 6-hydroxydopamine administration, but unmarked terminals were also observed in both distributions. These unmarked terminals might represent an additional nonnoradrenergic nerve supply to interscapular brown adipose tissue. The thoracodorsal veins draining the fat pads are directly tributary to a large median perforating vein, which joins the azygos vein, and are also continuous with the axillary vein. In addition to the recognized vascular distribution pattern of lobular arteries supplying an abundant capillary plexus drained by lobular veins, direct arteriovenous anastomoses were observed within the interscapular brown fat pad. It is postulated that these additional vascular arrangements are determinant in the phenomenal increase in blood flow through brown adipose tissue during metabolic stimulation.     

In vitro alanine utilization by rat interscapular brown adipose tissue

The in vitro oxidation to CO2 and tissue incorporation of alanine label by pieces of rat interscapular brown adipose tissue (IBAT) has been investigated. Insulin increased both uptake and oxidation of alanine, as well as the incorporation of alanine label into tissue. This effect only was observed in the presence of glucose in the incubation medium. Noradrenaline hampered alanine incorporation, not affecting its rate of oxidation. IBAT from 4-h cold-exposed rats showed a higher alanine utilization than that of controls; however, IBAT pieces from both 36-h starved and 30-day cold-exposed rats presented lower rates of alanine utilization. The main fate of alanine taken up by the IBAT pieces was its oxidation to CO2. Part of the label was also incorporated into the fatty acid fraction of lipids. The results obtained in this study agree with a possible role of alanine as alternative energetic substrate for IBAT.     

Development of fatty acid-synthetic capacity in interscapular brown adipose tissue during suckling in genetically obese Zucker rats.

The development of the lipogenic capacity in brown adipose tissue was studied in suckling lean (Fa/fa) and obese (fa/fa) Zucker pups aged from 7 to 22 days. In both lean and obese pups, activities of the two key lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and of citrate cleavage enzyme rose from the early to the late suckling period. Compared with lean pups, 7-day-old fa/fa pups showed a 35% increase in fat accumulation in interscapular brown adipose tissue and a 25% increase in fatty acid synthetase activity. By 10 days of age, fat deposition, lipogenesis in vivo (assessed by the incorporation of 3H from 3H2O into fatty acids) and fatty acid synthetase activity were 1.5-2-fold higher in pre-obese than in lean pups. Compared with lean pups, the increased lipogenesis in vivo observed in brown adipose tissue of 10-day-old pre-obese pups could not entirely account for the difference in fat deposition observed in this tissue, suggesting that additional mechanisms are at play to explain the increased fat content of this tissue.     

The utilization of ketone bodies by the interscapular brown adipose tissue of the rat

The activities of 3-oxo acid-CoA transferase (EC 2.8.3.5, 13-15 micromol/min per g) and acetoacetyl-CoA thiolase (EC 2.3.1.9, 18-21 micromol/min per g) in interscapular brown adipose tissue of the rat are comparable to the activities reported for heart and kidney. The incorporation of D-3-hydroxy[3-14C]butyrate into lipid in vivo was about 30-fold higher in interscapular brown adipose tissue than in white adipose tissue of virgin rats. In lactating rats, the mammary gland was the major site of ketone body incorporation into lipid and incorporation of D-3-hydroxy-[3-14C]butyrate into lipid in brown adipose tissue was lower than in virgin rats. After an oral load of medium chain triacylglycerol, which inhibits lipogenesis in lactating mammary gland, the incorporation of ketone bodies into lipid was decreased in mammary gland but increased in brown adipose tissue. The rate of oxidation of D-3-hydroxy[3-14C]butyrate by brown adipose tissue slices in vitro was higher than the rate of incorporation into lipid.     

Evidence for a high fatty acid synthesis activity in interscapular brown adipose tissue of genetically obese Zucker rats.

Obese (fa/fa) rats (30 days old) exhibited a 50% increase in the weight of interscapular brown adipose tissue compared with their lean (Fa/fa) littermates. The tissue weight increase was accounted for by an increased fat content. Lipogenesis in vivo, as assessed by the incorporation of 3H from 3H2O into lipid, was increased 5-fold in brown adipose tissue of obese as compared with lean rats. Accordingly, acetyl-CoA carboxylase, fatty acid synthetase, citrate-cleavage enzyme and malic enzyme in this tissue were 4-8 times more active in obese than in lean rats.     

Lipogenesis in interscapular brown adipose tissue of virgin, pregnant and lactating rats. The effects of intragastric feeding.

Lipogenesis in brown adipose tissue of virgin rats increased 8--10-fold after intragastric feeding with glucose or medium-chain triacylglycerol, and this increase was prevented by short-term insulin deficiency. Brown adipose tissue increased in weight during pregnancy, regressed during lactation and hypertrophied again on weaning; the rate of lipogenesis paralleled these changes. Glucose did not increase brown-adipose-tissue lipogenesis at mid-lactation.     

Relationship between hypothalamic noradrenergic activity and the sympathetic activity in interscapular brown adipose tissue after cold-swim stress in rats.

Noradrenaline (NA) activities in both hypothalamus and interscapular brown adipose tissue (IBAT) were simultaneously assessed before and after cold-swim stress in rats. The technique of gas chromatography-mass spectrometry was employed for the analysis of NA and its primary neuronal metabolite, 3,4-dehydroxy-phenylethylenglycol (DHPG), and the ratio of DHPG to NA was used as an index of NA activity. The ratios of DHPG/NA in both hypothalamus and IBAT were significantly elevated 5 and 20 min after cold-swim stress. Moreover, we found that there is a highly significant positive relationship between the hypothalamic DHPG/NA ratio and the ratio of DHPG/NA in IBAT (r = 0.872, p less than 0.0001). This observation strongly supports the concept in which hypothalamic NA neurons play an important role in modulating the sympathetic outflow.     

Responses to cafeteria feeding in mice after the removal of interscapular brown adipose tissue

Feeding acafeteria diet to mice resulted in an increased energy intake of approximately 30% and this led to increases in the wet weight, total protein content , and total cytochrome oxidase activity of interscapular and dorso-cervical brown adipose tissue. Surgical removal of interscapular brown adipose tissue, followed by cafeteria feeding, gave rise to an elevation in dorso-cervical brown adipose tissue wet weight, total protein content, and total cytochrome oxidase activity, compared to intact cafeteria-fed mice. Cafeteria feeding with or without the removal of interscapular brown adipose tissue did not lead to significant increases in body weight compared to stock-fed control mice, but both cafeteria-fed groups of mice showed significant elevations in body fat content indicating that the induced hyperphagia led to a relative obesity in the cafeteria-fed groups. The results presented are consistent with an increased thermogenic activity in the brown adipose tissue of cafeteria-fed mice, and the effect of the removal of interscapular brown adipose tissue further indicates the quantitative importance of the tissue in the control of body weight.     

Norepinephrine and dopamine content in the brown adipose tissue of developing eu- and hypothyroid rats.

The norepinephrine and dopamine content of interscapular brown adipose tissue of developing rats was studied at intervals from birth to 50 days. Throughout this period, neonatal hypothyroidism is associated with a decreased norepinephrine content and with an increased dopamine content.     

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.     

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.     

Sympatho-adrenal activity and metabolic responses in fasted rats--the role of interscapular brown adipose tissue

1. Sympatho-adrenal (SA) and metabolic responses to fasting were studied in sham-operated (SHAM) rats and in those with interscapular brown adipose tissue (IBAT) removed. 2. Fasting significantly increased adrenaline (A) excretion and serum free fatty acids (FFA), but decreased noradrenaline (NA) excretion and blood glucose level in SHAM rats. 3. IBAT removal did not change metabolic responses while it markedly altered the SA activity. Fasting in animals void of IBAT potentiated the activity of adrenal medulla, inhibited the FFA rise and prevented glucose reduction, which is normally observed in SHAM-fasted rats. 4. Results suggest the significance of IBAT in the regulation of the blood level of energy substrates in fasted rats and in maintaining the basal level of NA excretion.     

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.     

The influence of vitamin C supplementation on the oxidative status of rat interscapular brown adipose tissue

The interscapular brown adipose tissue (IBAT) thermogenesis is accompanied with oxidative stress. In spite of the ability of rats to synthesize vitamin C, we tested the possibility that its additional intake may improve the tissue antioxidative protection. Thus, we studied the IBAT oxidative status in rats supplemented by two doses of ascorbic acid over a 4-week period of time.

Our results confirmed that the additional intake of ascorbate improves the tissue antioxidative defense. Probably acting through enhanced insulin release, vitamin C also exerted some metabolic effects, which emphasize its role in the regulation of IBAT functions under normal physiological conditions.     

Implications of interscapular brown adipose tissue removal and sucrose overfeeding on the sympatho-adrenal activity and metabolic responses

1. The influence of sucrose overfeeding on the sympatho-adrenal (SA) and metabolic responses was studied in sham-operated (SHAM) rats and in those with interscapular brown adipose tissue (IBAT) removed. 2. Sucrose feeding significantly increased the SA activity, mobilized the free fatty acids (FFA), but did not change glucose homeostasis in sham-operated rats. 3. IBAT removal in control rats fed a stock diet modified the SA activity whereas the levels of both blood glucose and serum FFA were unchanged. 4. However, sucrose in rats void of IBAT potentiated the activity of sympathetic nervous system only and prevented the FFA rise, which is seen in sham-operated sucrose fed rats indicating that the enhanced level of serum FFA in these animals principally originated from the IBAT.