Relative importance of sympathetic outflow and insulin in the reactivation of brown adipose tissue lipogenesis in rats adapted to a high-protein diet.

The effect of denervation or acute insulin deficiency on brown adipose tissue lipogenesis was investigated in rats adapted to a high-protein diet before and after diet reversion to a balanced diet. Denervation of rats fed the balanced diet induced a 50% reduction in in vivo rates of brown adipose tissue fatty acid synthesis, with decreased activities of acetyl-CoA carboxylase and adenosine triphosphate (ATP)-citrate lyase. The markedly (80%) reduced fatty acid synthesis and enzyme activities in brown adipose tissue from rats adapted to the high-protein diet were not affected by denervation. Replacement of the high-protein diet by the balanced diet for 24 hours restored fatty acid synthesis to normal levels, but recovery of enzyme activities was only partial. Lipogenesis restoration and partial recovery of enzyme activities were impaired in denervated tissue from high-protein diet-fed rats. In all experimental conditions, the activities of acetyl-CoA carboxylase and ATP-citrate lyase showed a better correlation with brown adipose tissue lipogenesis than the generators of H(+), glucose-6-P dehydrogenase, and malic enzyme. Anti-insulin serum administration during the 12- to 24-hour period after diet reversion completely blocked lipogenesis recovery in innervated and denervated tissues and drastically reduced brown adipose tissue lipogenesis of concomitantly injected rats fed the balanced diet. The data suggest that efficient and rapid adjustments of brown adipose tissue lipogenesis require sympathetic activation, and that this tissue can maintain significant, albeit reduced, rates of lipogenesis in the absence of sympathetic activation, but not in the absence of insulin.

Glucose uptake and glycolytic flux in adipose tissue from rats adapted to a high-protein, carbohydrate-free diet.

Rates of glucose uptake by epididymal and retroperitoneal adipose tissue in vivo, as well as rates of hexose uptake and glycolytic flux in isolated adipocytes, were determined in rats adapted to a high-protein, carbohydrate-free (HP) diet and in control rats fed a balanced (N) diet. Adaptation to the HP diet induced a significant reduction in rates of glucose uptake, estimated with 2-deoxy-[1-(3)H]-glucose, both by adipose tissue (epididymal and retroperitoneal) in vivo and by isolated adipocytes. Twelve hours after replacement of the HP diet with the balanced diet, rates of adipose tissue uptake in vivo in HP-adapted rats returned to levels that did not differ significantly from those in N-fed rats. The rate of flux in the glycolytic pathway, estimated with (3)H[5]-glucose, was also significantly reduced in adipocytes from HP-fed rats. In agreement with the above findings, the activities of hexokinase (HK), phosphofructo-1-kinase (PFK-1), and pyruvate kinase (PK) were markedly reduced in adipose tissue from HP-adapted rats. The activity of pyruvate kinase was partially reverted by diet replacement for 12 hours. The low-plasma insulin and high-glucagon levels in HP-fed rats may have played an important role in the reduction of adipose tissue glucose utilization in these animals.

Effect of cold acclimation on brown adipose tissue fatty acid synthesis in rats adapted to a high-protein, carbohydrate-free diet.

The effect of cold acclimation on brown adipose tissue (BAT) fatty acid synthesis was investigated in rats adapted to a high-protein, carbohydrate-free diet. At an ambient temperature (25 degrees C), rates of fatty acid synthesis in BAT from rats adapted to the high-protein diet were reduced to 27% of rats fed the balanced diet and increased markedly after cold acclimation (10 days at 4 degrees C), although the increase was smaller than in control rats. BAT weight increase induced by cold acclimation was smaller in rats fed the high-protein diet (30%) than in controls (100%). When expressed per whole tissue, maximal activities of BAT glucose-6-phosphate dehydrogenase, malic enzyme, adenosine triphosphate (ATP)-citrate lyase, and acetyl-coenzyme A carboxylase were markedly reduced in high-protein diet-adapted rats at 25 degrees C and increased after cold acclimation in BAT from the 2 groups. However, when expressed per milligram protein, only acetyl-coenzyme A carboxylase showed an increase in both controls and in rats fed the high-protein diet. G6P-dehydrogenase, malic enzyme, and ATP-citrate lyase increased (per milligram protein) only in rats adapted to the high-protein diet and actually decreased in BAT from cold-acclimated control rats. Initial (before activation) pyruvate dehydrogenase (PDH) complex activity was lower in BAT from rats fed the high-protein diet at 25 degrees C and increased in cold-acclimated rats from the 2 groups. Circulating levels of insulin decreased in the 2 groups after cold acclimation. The data suggest that the cold acclimation-induced increase in BAT lipogenesis in rats adapted to the high-protein diet was due to a restoration of sympathetic activity, which induced both BAT hyperplasia and activation of adipocyte free fatty acid (FFA) synthesis, with an important participation of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase.

Glucose contribution to in vivo synthesis of glyceride-glycerol and fatty acids in rats adapted to a high-protein, carbohydrate-free diet.

Triacylglycerol (TAG) synthesis from all carbon sources and from glucose carbon was evaluated in rats fed a high-protein, carbohydrate-free (HP) diet or control diet by determining simultaneously in the same animal the rate of incorporation of 3H2O and of 14C-glucose into the two TAG moieties in the carcass, liver, and retroperitoneal and epididymal adipose tissue. Incorporation rates of 3H2O into TAG-fatty acids (FAs) in the two adipose tissues and in liver were reduced in HP rats to about 20% and 50%, respectively, of the rates in control rats. In the two experimental groups, glucose was a poor precursor for FA synthesis, contributing only 22.8% of whole-body (carcass plus liver) total FA synthesis in control rats and even less (14%) in HP rats. In contrast to the reduction in FA synthesis, incorporation of 3H2O into TAG-glycerol in HP rats did not differ significantly or was even higher (in epididymal tissue) versus the control level. In all tissues of both HP and control rats, the rate of 14C-glucose incorporation into TAG-glycerol was much higher than the rate of incorporation into FA. Glyceroneogenesis, estimated by subtracting TAG-glycerol synthesis from glucose from the rate obtained with 3H2O, was significantly increased in adipose tissue from HP rats, with almost all of the glycerol formed by this route being used to esterify preformed FAs. It is suggested that the increased adipose tissue glyceroneogenesis is important for esterification of diet-derived FA and preservation of body fat stores in rats adapted to the HP diet.

Glucose uptake, glucose transporter GLUT4, and glycolytic enzymes in brown adipose tissue from rats adapted to a high-protein diet.

In vivo rates of glucose uptake, insulin-responsive glucose transporter (GLUT4) content, and activities of glycolytic enzymes were determined in brown adipose tissue (BAT) from rats adapted to a high-protein, carbohydrate-free (HP) diet. Adaptation to the HP diet resulted in marked decreases in BAT glucose uptake and in GLUT4 content. Replacement of the HP diet by a balanced control diet for 24 hours restored BAT glucose uptake to levels above those in rats fed the control diet, with no changes in GLUT4 levels in 4 of 5 animals examined. BAT denervation of rats fed the control diet induced a 50% reduction in glucose uptake, but did not significantly affect the already markedly reduced BAT hexose uptake in HP diet-fed rats. It is suggested that the pronounced decrease in BAT glucose uptake in these animals is due to the combined effects of the HP diet-induced reductions in plasma insulin levels and in BAT sympathetic activity. Adaptation to the HP diet was accompanied by decreased activities of hexokinase, phosphofructo-1-kinase, and pyruvate kinase (PK). The activity of BAT PK in HP diet-fed rats was reduced to about 50% of controls, and approached normal levels 24 hours after diet reversion. BAT denervation induced a small (15%) decrease in BAT PK activity in control rats, but did not affect the activity of the enzyme in HP diet-adapted rats. Also, denervation did not interfere with the restoration of PK activity induced by diet substitution. Treatment with anti-insulin serum resulted in an almost 50% reduction in PK activity in both innervated and denervated BAT from rats fed the control diet, but caused a much smaller ( thick approximate 20%) decrease in BAT from HP diet-fed rats. Furthermore, anti-insulin serum administration completely suppressed the restoration of BAT PK activity induced by diet reversion. These data suggest that, differently from glucose uptake, BAT PK activity is predominantly controlled by hormonal/metabolic factors.

Intra-ventromedial hypothalamic injection of cholinergic agents induces rapid hyperglycemia, hyperlactatemia and gluconeogenesis activation in fed, conscious rats.

Microinjections of carbachol (50 nmol) or neostigmine (25 nmol) in 0.5 ml saline into the ventromedial nucleus of the hypothalamus (VMH) (but not into the lateral hypothalamic area) of fed, conscious rats produced marked increases in plasma glucose and lactate, which were suppressed or markedly reduced by previous adrenodemedullation. The rate of incorporation of 14C from infused bicarbonate (0.60 microliter, 0.20 microCi/min), an index of gluconeogenic activity, increased significantly after VMH administration of neostigmine. The data suggest that cholinergic synapses in the VMH participate of a central glucoregulatory system that increases hepatic glucose production mainly through a stimulation of adrenal medulla epinephrine secretion.

Centrally injected atropine reduces hyperglycemia caused by 2-DG or immobilization stress in awake rats.

The physiological significance of central cholinergic neurons was investigated by verifying the effect of previous intracerebroventricular administration of atropine on the hyperglycemia induced by 2-deoxyglucose (2-DG) or by immobilization stress in unrestrained, nonanesthetized rats. Intravenous 2-DG induced a marked increase in plasma glucose that was not affected by atropine injected intracerebroventricularly 30 min before. However, the hyperglycemia induced by intracerebroventricular 2-DG was significantly reduced by previous intracerebroventricular injection of atropine. Immobilization induced a rapid increase of plasma glucose levels that was reduced by about 50% by intracerebroventricular injection of atropine. The increase in plasma lactate induced by intravenous 2-DG, or immobilization, was not significantly affected by previous intracerebroventricular injection of atropine. The data suggest that central cholinergic neurons participate in the complex neural events responsible for the hyperglycemic response to neurocytoglucopenia and to stressful situations.

Thermogenic capacity of brown adipose tissue is reduced in rats fed a high protein, carbohydrate-free diet.

The functional state of interscapular brown adipose tissue (IBAT) was examined in rats fed for 20-30 d a high protein, carbohydrate-free diet [70% (wt/wt) protein, 8% fat] or a balanced diet (66% carbohydrate, 17% protein, 8% fat). In rats fed the high protein diet, body weight did not differ from that of control rats, but relative IBAT weight (grams per 100 g body wt) and lipid concentration (per gram of tissue) were 37% and 14% lower, respectively. In vivo rates of lipogenesis in IBAT, epididymal and retroperitoneal adipose tissue of rats fed the high protein diet were 20, 30 and 40%, respectively, of control values. Mitochondrial protein and cytochrome oxidase activity per total IBAT were significantly lower in rats fed the high protein diet than in controls; GDP binding was lower even when expressed per total tissue or per milligram of mitochondrial protein. The increase of IBAT temperature following norepinephrine infusion was significantly smaller than in controls. It is suggested that the decrease in IBAT capacity in the rats fed the high protein diet was due, at least in part, to a sustained reduction of sympathetic activity.

Sympathetic activity in brown adipose tissue from rats adapted to a high protein, carbohydrate-free diet.

Previous studies have shown that adaptation of rats to a high protein, carbohydrate-free (HP) diet results in a marked reduction in brown adipose tissue (BAT) thermogenic capacity. The present experiments were carried out to assess BAT sympathetic activity in HP diet-adapted rats. It was found that interscapular brown adipose tissue (IBAT) norepinephrine (NE) content, fractional turnover rate and calculated rate of turnover were markedly reduced in HP-fed rats. Replacement of the HP diet by a control, balanced diet for 24 h did not affect BAT NE content significantly, but restored fractional turnover rate and turnover rate values to those of control animals. Exposure to cold (4 degrees) for a short period (8 h) induced a marked increase in IBAT NE fractional turnover rate and calculated turnover rate in both HP and control rats. The cold-induced rise of turnover rate over values at ambient temperature (25 degrees C) was higher in HP rats than in controls. Rectal temperature after 8 h of cold exposure did not differ in HP and control rats. The data suggest that the decreased thermogenic capacity of rats adapted to a high protein, carbohydrate-free diet is due to a reduced sympathetic outflow to BAT, which can be rapidly reactivated by cold stimulation.

Brown adipose tissue triacylglycerol synthesis in rats adapted to a high-protein, carbohydrate-free diet.

Adaptation of rats to a high-protein, carbohydrate-free (HP) diet induced a marked reduction of brown adipose tissue (BAT) fatty acid (FA) synthesis from both 3H2O and [14C]glucose in vivo, with pronounced decreases in the activities of four enzymes associated with lipogenesis: glucose-6-phosphate dehydrogenase, malic enzyme, citrate lyase, and acetyl-CoA carboxylase. In both HP-adapted and control rats, in vivo incorporation of 3H2O and [14C]glucose into BAT glyceride-glycerol was much higher than into FA. It could be estimated that most of the glycerol synthetized was used to esterify preformed FA. Glycerol synthesis from nonglucose sources (glyceroneogenesis) was increased in BAT from HP rats, as evidenced by an increased capacity of tissue fragments to incorporate [1-14C]pyruvate into glycerol and by a fourfold increase in the activity of phosphoenolpyruvate carboxykinase activity, a key glyceroneogenic enzyme. The data suggest that high rates of glyceroneogenesis and of esterification of preformed FA in BAT from HP-adapted rats are essential for preservation of tissue lipid stores, necessary for heat generation when BAT is recruited in nonshivering thermogenesis.

Glycerokinase activity in brown adipose tissue: a sympathetic regulation?

The effect of brown adipose tissue (BAT) sympathetic hemidenervation on the activity of glycerokinase (GyK) was investigated in different physiological conditions. In rats fed a balanced diet, the activity of the enzyme was approximately 50% lower in BAT-denervated pads than in intact, innervated pads. In rats adapted to a high-protein, carbohydrate-free diet, norepinephrine turnover rates and BAT GyK activity were already reduced, and BAT denervation resulted in a further decrease in the activity of the enzyme. Cold acclimation of normally fed rats at 4 degrees C for 10 days markedly increased the activity of the enzyme. Cold exposure (4 degrees C) for 6 h was insufficient to stimulate BAT GyK, but the activity of the enzyme was already increased after 12 h of cold exposure. The cold-induced BAT GyK stimulation was completely blocked in BAT-denervated pads. The data indicate that an adequate sympathetic flow to BAT is required for the maintenance of normal levels of GyK activity and for the enzyme response to situations, such as cold exposure, which markedly increase BAT sympathetic flow.