Biomedical subjects

L J Bukowiecki

Publications and source records attributed to L J Bukowiecki.

At least 19 recordsLinked to original sources

PubMed2001

Lowering plasma free fatty acids with Acipimox mimics the antidiabetic effects of the beta 3-adrenergic agonist CL-316243 in obese Zucker diabetic fatty rats.

We previously reported that long-term treatment of Zucker diabetic fatty (ZDF) rats with the selective beta(3) agonist CL-316243 normalizes glycemia, decreases plasma free fatty acids (FFA) concentration, improves insulin responsiveness, and increases glucose uptake, not only in brown and white adipose tissues, but also in skeletal muscles. Because muscles do not express typical beta(3) adrenoceptors, we postulated that the muscle effect was indirect and that it was possibly mediated by an activation of the glucose-fatty acid cycle. To test this hypothesis, we investigated the effects of Acipimox, a potent inhibitor of lipolysis in adipose tissue. Similar to CL-316243, Acipimox (150 mg/kg orally) markedly decreased plasma FFA, glucose, and insulin concentrations and improved glucose tolerance while reducing the insulin response in obese (350 to 400 g) ZDF rats. Plasma FFA concentrations were significantly correlated with plasma glucose and insulin concentrations (r =.72 and.83, respectively; P <.01), indicating strong metabolic relationships between these parameters. Euglycemic-hyperinsulinemic clamps combined with the 2-[(3)H]deoxyglucose method revealed that Acipimox markedly improved insulin responsiveness and significantly increased glucose uptake (Rg') in the diaphragm, the heart, and various skeletal muscles. Unlike CL-316243, Acipimox did not increase glucose use in brown or white adipose tissues. This selectivity shows that it is possible to improve diabetes in obese ZDF rats without necessarily stimulating thermogenesis in adipose tissues. Thus, decreasing plasma FFA with 2 drugs (Acipimox or CL-316243) that act via different mechanisms (acute inhibition of lipolysis or chronic stimulation of FFA oxidation) is associated with increased glucose uptake in muscles and enhanced insulin responsiveness. These observations support the hypothesis that CL-316243 may indirectly stimulate glucose uptake in muscles of type II diabetic rats by first stimulating brown adipose tissue (increasing uncoupling protein content and fatty acid oxidation) and progressively decreasing the levels of circulating FFA, resulting in activation of the glucose-fatty acid cycle or other mechanisms regulating insulin responsiveness in skeletal muscles.

Adrenergic beta-3 Receptor Agonists↗

PubMed1998

Effects of chronic treatment with noradrenaline or a specific beta3-adrenergic agonist, CL 316 243, on energy expenditure and epididymal adipocyte lipolytic activity in rat.

1. The effects of 7 days exposure to a specific beta3-adrenergic agonist, CL 316 243 (1 mg/kg x 24 hr), or to the physiological hormone, noradrenaline (5 mg/kg x 24 hr), were tested on energy expenditure and on in vitro lipolysis in male Sprague-Dawley rats. 2. At the second day of treatment, the total energy expenditure and the resting metabolic rate were increased by 20 and 30%, respectively, in the CL-treated group. Under the same conditions, a dose five times higher of NA increased the resting metabolic rate by 11% without any significant change in the total daily energy expenditure. 3. The CL-treated group showed a lower weight gain, correlated with a significant reduction in retroperitoneal adipose tissue weight. Both treatments resulted in a marked desensitization (increased EC50 values) of the NA stimulated lipolysis of epididymal adipocytes. The effects of both treatments on maximal lipolysis were opposite. Indeed, chronic NA-treatment decreased the responsiveness of lipolysis while chronic treatment with CL increased the maximal stimulation of lipolysis to NA. Furthermore, dose-response curve for CL on lipolysis showed a marked functional desensitization of beta3-adrenergic response. 4. Our results demonstrate the high selectivity of beta3-adrenergic agonists to stimulate whole body energy expenditure and lipid mobilization in rodents. The present results point out for the first time an adrenergic desensitization of the lipolytic response after chronic administration of a beta3-agonist.

Adipocytes↗

PubMed1998

Mechanisms of the antidiabetic effects of the beta 3-adrenergic agonist CL-316243 in obese Zucker-ZDF rats.

Previous studies have demonstrated that chronic cold exposure activates the sympathetic nervous system, increases energy expenditure, improves glucose uptake in peripheral tissues [brown and white adipose tissues (BAT and WAT) and muscles] of normal rats. The goal of the present studies was to test whether the selective beta 3-adrenergic agonist CL-316243 (CL) would mimic the beneficial beneficial effects of cold exposure in lean and obese ZDF/Gmi-fa male (ZDF) rats, a new model of type II diabetes. In obese ZDF rats, chronic infusion of CL (1 mg.kg-1.day-1 for 14 days) significantly decreased body weight gain, food intake, and WAT weight. It also increased total tissue cytochrome oxidase activity, not only in BAT (15 times), but also in WAT (2-4) times, suggesting that it progressively enhanced mitochondriogenesis in adipose tissues. CL treatment normalized hyperglycemia and reduced hyperinsulinemia and circulating free fatty acid (FFA) levels. It also improved glucose tolerance and reduced insulin response during an intravenous glucose tolerance test. In general, the beneficial effects of CL were more pronounced in obese than in lean rats. Hyperinsulinemic-euglycemic glucose clamps combined with the [2-3H]deoxyglucose method revealed that CL markedly improved insulin responsiveness in obese rats (3-4 times) and increased glucose uptake in BAT (21 times), WAT (3 times), skeletal muscles (2-3 times), and in the diaphragm (2.8 times), but not in the heart. It is concluded that chronic CL treatment improves glucose tolerance and insulin responsiveness in obese ZDF rats by a mechanism similar to that induced by chronic cold exposure, i.e., by stimulating facultative thermaogenesis, mitochondriogenesis, and glucose utilization in BAT and WAT. In addition to this mechanism, the reduction in plasma FFA levels induced by chronic CL treatment may further contribute to enhance glucose uptake in skeletal muscles (a tissue that does not express typical beta 3-adrenoceptors) via the "glucose-fatty acid" cycle. The antiobesity and antidiabetic properties of CL suggest that selective beta 3-adrenergic agonists may represent useful agents for the treatment of type II diabetes.

Adipose Tissue, Brown↗

PubMed1997

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women.

Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2-3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = -0.79 to -0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated.

Adipocytes↗

PubMed1997

Role of beta1- and beta3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes.

To evaluate the physiological functions of beta1-, beta2-, and beta3-adrenoceptors (ARs) in brown adipose tissue, the lipolytic and respiratory effects of various adrenergic agonists and antagonists were studied in rat brown adipocytes. The beta-agonists stimulated both lipolysis and respiration (8-10 times above basal levels), with the following order of potency (concentration eliciting 50% of maximum response): CL-316243 (beta3) > BRL-37344 (beta3) > isoproterenol (mainly beta1/beta2) > norepinephrine (NE; mainly beta1/beta2) > epinephrine (mainly beta1/beta2) >> dobutamine (beta1) >> procaterol (beta2). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (beta1 antagonist) revealed that more than one type of receptor mediates NE action. It is concluded from our results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through beta1-ARs, 2) NE, at higher levels, stimulates lipolysis and respiration via both beta1- and beta3-ARs, 3) beta2-ARs play only a minor role, and 4) beta3-ARs may represent the physiological receptors for the high NE concentrations in the synaptic cleft, where the high-affinity beta1-ARs are presumably desensitized. It is also suggested that lipolysis represents the flux-generating step regulating mitochondrial respiration.

Adipocytes↗

PubMed1997

In situ lipolysis measured by in vivo microdialysis during acute cold exposure.

The lipolytic responsiveness of interscapular white adipose tissue was measured, in male Sprague-Dawley rats (355 +/- 12 g, n = 7), by microdialysis before, during, and after an acute cold exposure (1 h at 4 degrees C). Microdialysis probes were perfused with standard Krebs-Ringer buffer to determine basal and stimulated rates of lipolysis. The concentration of glycerol in the dialysate was measured and considered as the lipolytic index. During the experiment, energy expenditure was measured by indirect calorimetry. Cold exposure at 4 degrees C doubled energy expenditure. At the same time, it resulted in a 2.7-fold increase in glycerol release. The present study shows that microdialysis is a perfectly adapted tool to investigate in vivo regulation of adipose tissue on awake, unrestrained rats.

Adipose Tissue↗

PubMed1996

Beta 3-adrenoceptor in guinea pig brown and white adipocytes: low expression and lack of function.

In the guinea pig, cold acclimation induced a conversion of unilocular to multilocular adipocytes in interscapular (IS) and retroperitoneal (RP) fat depots but not in the epididymal (EP) fat pad. The conversion was associated with an increase in mitochondriogenesis and the appearance of the uncoupling protein. The maximal lipolytic responses to norepinephrine and dibutyryl adenosine 3',5'-cyclic monophosphate were decreased in IS cells, unchanged in RP cells, and increased in EP cells, suggesting a site-specific regulation of lipolysis at the postreceptor level. beta 3-Adrenergic agonists were not lipolytic regardless of the depot and the thermal environment of the animal. These agents did not inhibit glucose transport and lipogenesis, as was previously reported for rodents. Cloning and sequencing of the guinea pig beta 3-adrenoceptor gene revealed a slightly higher amino acid sequence similarity with the human than with the rodent beta 3-adrenoceptors. beta 3-Adrenoceptor transcripts were present at a very low level in guinea pig adipocytes, and mRNA levels did not increase to a significant extent after cold acclimation. The guinea pig thus differs from rodents by an absence of beta 3-adrenergic effects and by low beta 3-adrenoceptor expression in brown and white adipose tissues.

Acclimatization↗

PubMed1995

Characterization of beta 1- and beta 3-adrenoceptors in intact brown adipocytes of the rat.

1. The binding properties of beta 1-, beta 2- and beta 3-adrenoceptors were determined in isolated brown adipocytes of the rat rather than in membrane preparations from tissue homogenates, because typical brown adipocytes represent only about 40% of the various cells present in brown adipose tissue. Binding characteristics were assessed with the hydrophilic beta-adrenoceptor radioligand, (-)-[3H]-CGP 12177. The potent beta-antagonist, bupranolol (100 microM) was used to determine nonspecific binding. Characterization was essentially performed by saturation and competition studies. 2. The saturation curve of (-)-[3H]-CGP 12177 was clearly biphasic (Hill coefficient, nH = 0.57 +/- 0.11, P < 0.01) indicating the presence of two different beta-adrenoceptor populations of high (KD = 0.24 +/- 0.04 nM) and low (KD = 80 +/- 7 nM) affinity. The low affinity sites were more numerous (Bmax = 121,000 +/- 30,000 sites/cell) than the high affinity sites (Bmax = 12,000 +/- 1,000 sites/cell). 3. (-)-[3H]-CGP 12177 (25 nM) was displaced by adrenaline (Ad), noradrenaline (NA), isoprenaline (Iso), phenylephrine (Phe) and by the new beta 3 agonist, CL 316,243 (CL) in a biphasic pattern. The order of potency for (-)-[3H]-CGP 12177 displacement from the small population of high affinity sites (Iso >> NA > Ad >> CL >> Phe was in agreement with a beta 1/beta 2-classification. In contrast, the potencies of the same agonists for displacing the radioligand from the low affinity binding sites (CL >> Iso > NA > Ad >> Phe) revealed the presence of a distinct population of adrenoceptors obeying a beta 3-classification. 5-HT did not displace (-)-[3H]-CGP 12177 (25 nM) when used at concentrations as high as 0.1 mM.4. The beta-adrenoceptor antagonist, (-)-bupranolol, was more effective than (-)-propranolol for displacing(- )-[3H]-CGP 12177 (25 nM) from the high (Ki= 0.029 =/- 0.011 and 0.19 +/- 0.07 nM, respectively and low (Ki= 0.27 +/- 0.04 microM and 1.6 +/- 0.2 lM, respectively) affinity binding sites. The selective beta 1 antagonist CGP 20712A efficiently displaced the radioligand from a small population (Ki = 65 +/- 19 pM)of binding sites, confirming the presence of beta 1-adrenoceptors.5. To evaluate whether beta 2-adrenoceptors could be identified in the population of high affinity binding sites, displacement studies were performed at a low concentration of (- )-[3H]-CGP 12177 (4 nM) that mainly labelled beta 1/beta 2-adrenoceptors. ICI 118 551 ( a selective beta 2-antagonist) and procaterol (a selective beta 2-agonist) displaced (- )-[3H]-CGP 12177 from its binding sites with very low affinity (Ki = 0.17 +/- 0.02 micro M and Ki = 11 +/- 2 micro M respectively).6. From these observations, we conclude that: (1) two kinds of binding sites with low and high affinities for (-)-[H]-CGP 12177 can be detected in intact brown adipocytes, (2) there are 10 times more low than high affinity beta-adrenoceptors, as determined by saturation or competition curve analysis, (3) the high affinity binding sites mainly correspond to beta1-adrenoceptors, whereas the low affinity sites represent beta 3-adrenoceptors, and (4) beta 2-adrenoceptors are undetectable.7. It is suggested that the low affinity beta 3-adrenoceptors represent the physiological receptors for noradrenaline secreted from sympathetic nerve endings when the concentration of the neurohormone in the synaptic cleft is very high and/or when the high affinity beta 1-adrenoceptors are desensitized by prolonged sympathetic stimulation such as chronic cold exposure.

Adipocytes↗

PubMed1994

Comparison of the lipolytic effects of norepinephrine and BRL 37344 in rat brown and white adipocytes.

The lipolytic effects of norepinephrine (a non-selective beta-agonist) and BRL 37344 (a selective beta3-agonist) were compared in isolated rat brown and white adipocytes. Norepinephrine and BRL 37344 maximally stimulated lipolysis in brown and white adipocytes, approximately 10 times above basal values. However, adipocyte sensitivity for BRL 37344 was greater than that for norepinephrine, particularly in brown adipocytes [the EC50 values (nM) for BRL 37344 and norepinephrine were 5 +/- 1 and 103 +/- 31 in brown adipocytes (P < 0.01) versus 56 +/- 9 and 124 +/- 17 in white adipocytes (P < 0.05), respectively]. On the other hand, the lipolytic effects of norepinephrine were totally blocked by 20-40 times superior concentrations of propranolol or bupranolol in brown as well as in white adipocytes. In contrast, the lipolytic effects of BRL 37344 were fully inhibited by concentrations of propranolol or bupranolol that were 200-1000 superior to the beta3 agonist concentration. The results demonstrate that: (1) the beta3-agonist BRL 37344 is as effective as norepinephrine for maximally stimulating lipolysis in rat brown and white adipocytes, (2) both adipocyte types are more sensitive to the lipolytic effects of BRL 37344 than to those of norepinephrine, (3) although bupranolol is a better antagonist than propranolol on BRL 37344-stimulated lipolysis, it cannot be considered as a specific beta3-antagonist, (4) brown adipocytes are 10 times more sensitive than white adipocytes to the lipolytic effects of BRL 37344, suggesting an important role of beta3-receptors in brown adipose tissue.

Adipocytes↗

PubMed1994

Chronic norepinephrine infusion stimulates glucose uptake in white and brown adipose tissues.

Cold exposure activates the sympathetic nervous system and markedly stimulates glucose uptake in rat peripheral tissues [A. L. Vallerand, F. Pérusse, and L. J. Bukowiecki. Am. J. Physiol 259 (Regulatory Integrative Comp. Physiol. 28): R1043-R1049, 1990]. To test whether norepinephrine (NE) mimics the effects of cold exposure, we estimated the effects of chronic NE treatment on tissue glucose uptake by determining the glucose metabolic index using the 2-[1,2-3H(N)]deoxy-D-glucose method. NE was administered in conscious rats at various doses (ranging from 1.9 to 25.1 nmol.kg-1.min-1) during 4 days via minipumps implanted subcutaneously. At doses > 10 nmol.kg-1.min-1, NE maximally stimulated glucose uptake in interscapular brown adipose tissue (approximately 50 times above controls) and epididymal white adipose tissue (approximately 3 times above controls). NE infusion (18.8 nmol.kg-1.min-1) increased the circulating levels of NE from 1.1 +/- 0.1 to 19.2 +/- 0.4 nM (P < 0.001), which is in the range of concentrations for the stimulatory effects of NE on glucose uptake in isolated brown adipocytes. At all concentrations tested, NE infusion did not stimulate glucose uptake in the heart and skeletal muscles. NE treatment did not significantly alter plasma insulin or glucose levels but increased the concentration of circulating free fatty acids. The capacity of brown adipose tissue for NE stimulation of glucose uptake (expressed per g of tissue) was much higher than that of white adipose tissue (100 times), various types of white or red skeletal muscles (10-80 times), or the heart (3-4 times).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1993

Tissue-specific alterations in lipoprotein lipase activity in the rat after chronic infusion of isoproterenol.

This study describes the consequences of sustained exposure to the nonspecific beta adrenergic agonist isoproterenol on the activity of lipoprotein lipase (LPL) in tissues that are sites for a large portion of the intravascular hydrolysis of triacylglycerols. Chronic exposure to isoproterenol was accomplished by means of osmotic pump infusion during four consecutive days, and assessment of the activity of LPL was performed at the end of the treatment. Four days of ISO infusion resulted in a large decrease in LPL activity of parametrial white adipose tissue, which declined to half of that found in the untreated group. In contrast, brown adipose tissue LPL activity was greatly increased (approximately threefold) by ISO treatment. The beta agonist raised LPL activity in red vastus lateralis muscle by a factor of four over control values, whereas heart enzyme activity was not significantly altered by the drug. These results demonstrate that catecholamines are part of the factors which determine the tissue specificity of LPL regulation. The tissue-specific changes in enzyme activity in response to isoproterenol, which are in very good agreement with the known metabolic actions of catecholamines in adipose and skeletal muscle tissues, attest to the key role of the enzyme in the partitioning of lipid substrates in situations of increased sympathetic activity.

Adipose Tissue↗

PubMed1993

Specific decrease of mitochondrial thermogenic capacity in brown adipose tissue of obese SHR/N-cp rats.

The metabolic properties of brown adipose tissue (BAT), liver, and skeletal muscles were compared in lean and obese diabetic SHR/N-cp rats (a new model of type II diabetes) to test whether the severe insulin resistance of obese animals is specifically associated with a thermogenic defect in BAT. The respiratory response of brown adipocytes to norepinephrine and to agents bypassing the adenylate cyclase complex (dibutyryl cyclic AMP and palmitate) was decreased by two-thirds in obese rats, thereby indicating the presence of a major postreceptor defect. Significantly, total BAT cytochrome oxidase activity, uncoupling protein content, and mitochondrial guanosine 5'-diphosphate binding (3 indexes of BAT thermogenic capacity) were also decreased by two-thirds. The specific activities of these parameters expressed per total BAT mitochondrial protein were not altered either. This indicates that the total number of mitochondria per cell is decreased in BAT of obese rats. In contrast, total tissue cytochrome oxidase activity, protein content, and DNA content all increased by two to three times in the liver of obese SHR/N-cp rats, but these parameters remained unchanged in skeletal muscles (vastus lateralis and soleus). Such a remarkable liver hypertrophy may have occurred as a consequence of the persistent hyperphagia-hyperinsulinemia of obese rats that induced a hyperplasia and/or a hepatocyte polyploidization. This observation together with the fact that daily energy expenditure associated with food intake was markedly increased in obese rats (representing as much as 25% of the total energy expenditure) strongly suggests that the liver plays a major role in energy balance in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue, Brown↗

PubMed1993

Norepinephrine- and insulin-resistant glucose transport in brown adipocytes from diabetic SHR/N-cp rats.

The effects of norepinephrine and insulin on glucose transport were investigated in brown adipocytes isolated from obese nondiabetic Lister and Albany (LA/N-cp strain) rats (O-LA), obese diabetic spontaneously hypertensive (SHR/N-cp strain) rats (O-SHR), and from their lean (L) controls to test whether the decreased calorigenic response to norepinephrine of O-SHR adipocytes was specifically associated with alterations in glucose metabolism. Norepinephrine and insulin independently stimulated glucose transport in L-LA, O-LA, and L-SHR brown adipocytes, but their stimulatory effects were markedly reduced in O-SHR cells. Both insulin responsiveness and the total number of insulin receptors were significantly decreased in O-SHR adipocytes but not in O-LA cells. The number of high-affinity beta 1/beta 2-adrenoceptors was significantly increased (+70%) in O-LA adipocytes but was similar in L-SHR and O-SHR cells. These results indicate that 1) major metabolic defects are present in brown adipose tissue (BAT) of O-SHR but not of O-LA, although these two strains are homozygous for the cp allele, 2) postreceptor defects are predominantly involved in O-SHR adipocyte refractoriness to norepinephrine, and 3) a reduced mitochondrial content may represent the principal metabolic alteration explaining the decreased effects of norepinephrine on both thermogenesis and glucose transport. It is postulated that the marked insulin resistance of O-SHR leads to a decreased mitochondriogenesis in BAT, resulting in a diminished tissue thermogenic capacity and reduced glucose metabolism, thereby contributing to obesity and diabetes.

Adipose Tissue, Brown↗

PubMed1993

Immunohistochemical detection of human brown adipose tissue uncoupling protein in an autopsy series.

We used an immunohistochemical method for the inner mitochondrial membrane uncoupling protein (UCP) to examine whether human brown adipose tissue UCP could be detected by an anti-rat UCP antibody. Samples of human brown adipose tissue were obtained at medicolegal autopsies. Fat tissue was excised from around the common carotid arteries and in the subscapular region and from around the thoracic aorta. The subjects were either known alcohol consumers, in which thermogenically active brown adipose tissue (BAT) is often found, or victims of sudden infant death syndrome (SID). UCP was detected in all the cases examined, even when the post-mortem time from death to autopsy reached several days, but the intensity of the immunostaining was variable. Intense staining was observed in three cases with a post-mortem time under 24 hr, but in the SID cases a strong positive staining was seen even with a post-mortem delay of 4 days. These results show that an anti-rat UCP antibody can be used for immunohistochemical detection of UCP in human brown adipose tissue and that it provides a useful method for distinguishing between white and brown fat in paraffin-embedded samples. It can be used to detect UCP in the BAT of obese and diabetic individuals and probably also in the histopathological diagnosis of brown adipose tissue lipoma, known as hibernoma.

Adipose Tissue, Brown↗

PubMed1993

Differential regulation of GLUT1 and GLUT4 glucose transporters in skeletal muscle of a new model of type II diabetes. The obese SHR/N-cp rat.

The obese diabetic SHR/N-cp rat is a newly developed strain that inherits obesity as an autosomal recessive trait. These rats display early-onset hyperinsulinemia and hyperglycemia, which are hallmarks of type II diabetes. This study was undertaken to determine the expression and the subcellular distribution of the GLUT1 and GLUT4 glucose transporters in skeletal muscle of obese diabetic SHR rats. D-glucose-protectable cytochalasin-B binding to subcellular membrane fractions of hindlimb muscles was used to determine glucose transporter number. GLUT1 and GLUT4 glucose transporter isotypes were detected using antibodies to the COOH-terminal region of the GLUT1 and GLUT4 proteins. Glucose transporter number was significantly lower (-40%) in crude unfractionated membranes of obese diabetic SHR than of lean SHR muscles. When crude membranes were fractionated to separate plasma membranes and the intracellular membranes containing glucose transporters, the number of cytochalasin-B binding sites was found to be markedly lower (-50%) in intracellular membranes and slightly but not significantly reduced (-20%) in plasma membranes of muscle from obese diabetic SHR compared with lean SHR rats. Western blot analysis revealed that a lower GLUT4 protein abundance (-40%) accounts for the reduced glucose transporter number in intracellular membranes of obese diabetic SHR compared with lean SHR muscles. GLUT4 protein content was also reduced by 50% in plasma membranes from obese SHR muscles relative to lean rat muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Morphometric-stereologic analysis of brown adipocyte differentiation in adult mice.

Brown adipocyte differentiation from interstitial stem cells was analyzed by morphometric-stereologic methods in adult mice. Confirming previous studies, four different stages of development were identified: 1) interstitial cells----2) protoadipocytes (interstitial cells with tiny lipid droplets)----3) preadipocytes----4) mature brown adipocytes. Brown adipocyte precursor cells (interstitial cells and protoadipocytes) occupied only a small fraction of total brown adipose tissue (BAT) volume (1.7 and 1.8%, respectively). Most of the BAT volume was occupied by fully differentiated multilocular cells (65% vol/vol), preadipocytes (12%), and blood capillaries (10%). The differentiation of protoadipocytes into preadipocytes was characterized by a doubling in the cellular volume (from 800 to 1,500 microns 3) that was associated with a fivefold increase in the number of mitochondria (221 to 1,464), an eightfold augmentation in mitochondrial size (from 0.042 to 0.37 microns 3), a fourfold increase in the surface density of the inner mitochondrial membrane (from 8 to 35 microns 2/microns 3), resulting in a 14-fold enlargement in the relative volume of the mitochondrial compartment (from 2 to 29%). This remarkable mitochondrial proliferation was accompanied by an increase in the number and volume of cytosolic lipid droplets. In contrast, the differentiation of preadipocytes into brown adipocytes was mainly characterized by a doubling in the size of the lipid compartment; the mean volume of single droplets increased 35 times but their number decreased 6-7 times. The mitochondrial modifications were minor; there was only a slight increase in the surface density of the inner membrane. In conclusion, the major step of brown adipocyte differentiation consists in the transformation of protoadipocytes into preadipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue, Brown↗

PubMed1992

Role of sympathetic innervation in brown adipocyte proliferation.

The role of sympathetic nerves in the control of brown adipose tissue (BAT) growth was studied by quantitative photonic autoradiography using [3H]-thymidine to follow mitotic activity. The effects of cold exposure (4 days at 5 degrees C) and chronic norepinephrine treatment (0.375 mumol/h for 4 days at 25 degrees C) were compared in intact and denervated interscapular BAT pads of adult rats. The denervated pads were paler than the contralateral innervated sides and contained numerous adipocytes filled with large lipid droplets. In the innervated BAT pad, cold exposure markedly enhanced the labeling frequency in brown adipocyte precursor cells (interstitial cells and preadipocytes) and in endothelial cells forming the numerous blood capillaries (P < 0.01). However, in the denervated BAT pads, the effects of cold exposure on cell proliferation were markedly inhibited. Remarkably, norepinephrine infusion at 25 degrees C mimicked the effects of cold exposure on the coordinated proliferation of the different cellular types, both in the innervated and denervated pads. These results demonstrate that intact sympathetic nerves are required for the stimulation of mitotic activity in BAT by cold exposure but not by norepinephrine infusion. It appears therefore that norepinephrine release from sympathetic nerves represents the initial stimulus in a chain of metabolic events leading to BAT hyperplasia. However, sympathetic or sensory nerves may still contain factors modulating cell proliferation and differentiation in BAT.

Adipose Tissue, Brown↗

PubMed1991

Noradrenaline stimulates glucose transport in rat brown adipocytes by activating thermogenesis. Evidence that fatty acid activation of mitochondrial respiration enhances glucose transport.

The mechanisms by which noradrenaline, lipolytic agents and long-chain fatty acids stimulate glucose transport were investigated in rat brown adipocytes. Glucose transport was evaluated with tracer D-[U-14C]glucose and cell respiration was measured polarographically. Noradrenaline increased basal oxygen consumption (8-10-fold) and glucose transport (4-5-fold) in a dose-dependent manner, with a maximal stimulation at 100 nM. The stimulatory effects of noradrenaline on respiration and glucose transport were selectively mimicked by dibutyryl cyclic AMP (DBcAMP), 3-isobutyl-1-methylxanthine, cholera toxin and physiological concentrations of palmitic acid. Cytochalasin B completely blocked the effects of these agents on glucose transport. The beta-adrenergic antagonist propranolol inhibited noradrenaline-induced glucose transport, but did not affect the action of DBcAMP, palmitic acid or cholera toxin on this process. The specific inhibitor of mitochondrial carnitine palmitoyltransferase, 2-tetradecylglycidic acid (McN 3802) (50 microM), inhibited the stimulatory effects of noradrenaline (100 nM) and palmitic acid (0.5 mM) on both glucose transport and mitochondrial respiration. Significantly, McN 3802 failed to affect insulin (1 nM) action under identical experimental conditions. These results demonstrate that (a) the stimulatory effects of noradrenaline on brown-adipocyte respiration and glucose transport can be dissociated from those induced by insulin, and (b) noradrenaline increases glucose transport indirectly, by activating adenylate cyclase via beta-adrenergic pathways and by stimulating mitochondrial oxidation of fatty acids.

1-Methyl-3-isobutylxanthine↗