Eur. J. Biochem. 207, 377-382 (1992) 0FEBS 1992
Molecular and metabolic changes in white adipose tissue of the rat during development of ventromedial hypothalamic obesity Beatrice COUSIN ', Karen AGOU', Armelle LETURQUE', Pascal FERRE', Jean GIRARD' and Luc PENICAUD Laboratoire de Physiopathologie de la Nutrition, CNRS URA 307, Universite Paris VII, Paris, France Centre de Recherche sur I'Endocrinologie Moleculaire et le Dkveloppement, CNRS, Meudon, France (Received March 3/April 13, 1992) - EJB 92 0291
We have previously shown that rats made obese by lesion of ventromedial hypothalamus (VMH) nuclei, demonstrate an hyper-responsiveness to insulin with regard to whole-body glucose utilization one week after injury. This is mainly due to an increased glucose uptake in white adipose tissue. Six weeks after the lesion, glucose utilization in white adipose tissue returns to normal values. These modifications in insulin responsiveness could be mediated by altered activity and/or concentration of intracellular insulin effectors. In this study, we have measured the expression of the insulin-sensitive glucose transporter, Glut 4 and the activities and expression of key lipogenic enzymes (fatty-acid synthase and acetyl-CoA carboxylase) in white adipose tissue, one and six weeks after the lesion. All these parameters, as well as glucose transport and metabolism determined in white adipocytes, were markedly increased one week after the lesion. They returned to control values within six weeks in VMH-lesioned rats. These results indicate the existence of an increased expression of Glut 4 and lipogenic enzymes in white adipose tissue of VMH-lesioned rats which decreased with time and were parallel to glucose utilization determined in vivo.
In adipose tissue, insulin resistance, with regard to glucose uptake and utilization is a major feature of overt obesity (for reviews, see [I, 21). Nevertheless, it has been demonstrated that insulin resistance is preceded by a period of hypersensitivity and responsiveness to insulin in adipose tissue from obese human or rodents (for a review see [3]). In rats made obese by electrolytic lesion of the ventromedial hypothalamus (VMH), the onset of the syndrome is determined by the time of the lesion. Thus, this model allows the chronological study of the alterations associated with obesity. It has been shown in vivo that one week after the lesion, during the dynamic phase of obesity, insulin-induced whole-body glucose utilization is increased [4], and this could almost be accounted for by the differential increase observed in white adipose tissue glucose utilization [ 5 ] . Six weeks after VMH lesion, this state of increased insulin action disappears [4], and glucose utilization in white adipose tissue returns to near control values [5]. These changes in response to insulin of white adipose tissue, observed in vivo, are also present in young suckling (15 day old) or weaned (30 day old) genetically obese Zucker rats, although to a lesser extent [6-81, and in 13-year-old obese children [9]. These modifications could be mediated by changes in insulin receptor and/or intracellular insulin targets, for which glucose transporters and lipogenic enzymes are likely candidates. Correspondence to L. PCnicaud, Laboratoire de Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique URA 307, Universitk Paris VII, 2 place Jussieu, F-75251 Paris Cedex 05, France Fax: i33 1 44 27 18 36. Abbreviation. VMH, ventromedian hypothalamus. Enzymes. Acetyl-CoA carboxylase (EC 6.4.1.2); fatty-acid synthase (EC 2.3.1.85).
We have already shown that insulin-receptor kinase activity was increased in white adipose tissue of rats, one week after VMH lesion, and returned to control values six weeks after the lesion [lo]. Nevertheless, these modifications of insulinreceptor kinase activity could not totally explain the change in glucose utilization in white adipose tissue observed during the development of obesity. The aim of the present work was to determine whether other alterations located at post-receptor levels could mediate the variations of insulin responsiveness observed in vivo in white adipose tissue of VMH rats one and six weeks after the lesion. We have therefore studied the expression of the insulinsensitive glucose transporter (Glut 4) in white adipose tissue of control and obese rats. We have also studied activities and mRNA concentrations of two lipogenic enzymes: fatty-acid synthase and acetyl-CoA carboxylase. Finally, the consequences of these alterations on metabolic fluxes were determined in in vitro studies.
MATERIALS AND METHODS Animals Female Wistar rats were obtained from a local supplier (Iffa-Credo, L'Arbresle, France) at 10 - 11 weeks of age, and were housed at 24"C, in an animal quarter, with a constant light/dark cycle (light from 7 a.m. to 7 p.m.). Rats were anesthetized with 95 mg ketaminelkg intraperitoneally (Imalgene, Bio Mkrieux, France), and VMH lesions or sham operations (control rats) were carried out as described previously [Ill. Rats were then housed in individual cages. A lesion was considered successful when daily body mass gain was greater than 5 g compared to 2 g in control rats. VMH-
378 lesioned rats and their controls had free access to water and chow pellets [65% (by mass) carbohydrate, 11% fat, 24% protein; UAR, Villemoisson, France]. Experiments were then performed one or six weeks after the operation, at 3 p. m., and after 6 h of food deprivation to reduce possible differences due to hyperphagia in obese rats [12]. Lesioned and control rats were killed by cervical dislocation. Periovarian white adipose tissue was rapidly removed, and either used for adipocyte preparation or frozen in liquid nitrogen and stored at - 80°C for subsequent analysis. Blood samples were collected for determination of blood glucose and plasma insulin concentrations. Preparation of isolated white adipocytes
White adipocytes were isolated as described previously by Rodbell [13]. The two periovarian fat pads were removed and washed in Krebs/Ringer/phosphate/Hepes buffer, pH 7.4 [14], containing 0.5 mM calcium. The tissue was cut into small pieces and digested for 45 min in the same buffer containing 1 mg/ml collagenase (Sigma Chemical, St Louis, MO, USA), 2% bovine serum albumin (fraction V, fat free; Sigma Chemical, St Louis, MO, USA), and 2 mM pyruvate, in a shaking water bath at 37°C. The digested tissue was then filtered through a 200-pm nylon mesh, the isolated cells were washed three times in the buffer devoid of collagenase and collected in the incubation medium. Glucose transport in isolated adipocytes
Glucose-transport studies were performed by measuring radiolabelled glucose in cells after a rapid incubation of white adipocytes with 50 pM glucose [15, 161. Isolated adipocytes, at a final concentration of 2 x lo5 cells/ml, were preincubated for 1 h, with or without insulin (0.1 - 1 mU/ml), in Krebs/ Ringer/phosphate buffer, pH 7.4, containing 2 mM pyruvate. The assays were initiated by adding 50 pM [6-'4C]glucose (45 Ci/mol; CEA, Gif-sur-Yvette, France). The radioactivity in the cell layer was corrected for ['4C]glucose trapped in the extracellular water using [3H]inulin (0.4 Ci/g; NEN, Du Pont de Nemours, Paris, France). The assays were ended after 20 s by a rapid centrifugation of 200 p1 cell suspension with dinonylphthalate oil (Merck, Darmstadt, FRG). The radioactivity in the cell layer was measured. Glucose metabolism in isolated adipocytes
Isolated white adipocytes, at a concentration of lo5 cells/ ml, were incubated for 2 h in a shaking water bath at 37°C. The incubations were performed in 2.5 ml Krebs/Ringer/ phosphate/Hepes buffer, pH 7.4, containing 1 mM calcium, 2% fatty-acid-free albumin, and 5 mM [U-'4C]glucose (0.5 pCi/ml; CEA, Gif-sur-Yvette, France), with or without insulin (0.1 - 1 mU/ml). The flasks were oxygenated and tightly closed, and assays were stopped after 2 h by adding 0.5 ml 40% (by vol.) perchloric acid. To determine the amount of I4C-labelled C 0 2 formed from glucose, 0.3 ml hyamine was added to a center well. 14C-labelled COz was collected during an additional 45-min incubation. Two aliquots were taken from the content of the incubation flask. The first was used to measure glucose incorporation into lactate; [14C]lactate accumulating in the incubation medium was separated from ['4C]glucose using ionexchange resin columns [17]. With the second aliquot, total
lipids were extracted according to Dole [18] and were used to measure I4C incorporation into lipids. Northern-blot analysis
Total RNA was extracted from 1 - 3 g white adipose tissue using guanidine thiocyanate [19]. The concentration of RNA was determined by absorbance at 260 nm, and the RNA solutions were stored in H20/diethyl pyrocarbonate (0.020/) at - 80 "C until yse. All samples had an A260/A280 ratio of about 2. For Northern-blot analysis, 20 pg RNA was denatured, sized-fractionated by electrophoresis in 1% agarose gel and transferred to a nylon membrane (Hybond N; Amersham, Bucks, UK). Blots were hybridized with several probes. The cDNA probes were kindly provided by Dr D. James for Glut 4 [20], Dr A. G. Goodridge for fatty-acid synthase [21] and Dr K. H. Kim for acetyl-CoA carboxylase [22]. The probes were labelled with 32P using a Multiprime labelling system kit (Amersham, Bucks, UK). Hybridations with Glut 4 were realized as described by James et al. [20], and with fatty-acid synthase and acetyl-CoA carboxylase according to Coup6 et al. [23]. To verify the amount of total RNA in each lane, blots were hybridized with a synthetic oligonucleotide specific for ribosomal 18s RNA, in solutions containing 0.1YODenhardt's solution, 5 x NaCI/Cit (NaCl/Cit: 0.15 M NaC1, 15 mM trisodium citrate), 0.05 M sodium phosphate, pH 6.5, 0.1% SDS and salmon sperm DNA (250 pg/ml) denaturated at 42 "C overnight. The membranes were washed twice for 20 min each, with 2 x NaCl/Cit, at 42°C. The blots were exposed for 4 - 48 h at - 80 "C with intensifying screens. Quantification was performed by scanning densitometry. Values were normalized for the corresponding amount of 18s mRNA. Western-blot analysis
200 - 300 mg white adipose tissue was homogenized in ice-cold 1 mM EDTA, 20 mM Tris/HCl and 0.25 M sucrose (pH 7.4). Equal amounts of proteins (50 pg) were solubilized in Laemmli buffer and submitted to SDSjPAGE (12% acrylamide gels). The proteins were electrophoretically transferred to nitrocellulose membrane (BA 85; Schleicher and Schuell, Dassel, FRG). Protein markers (Rainbow; Amersham, Bucks, UK) were used as molecular mass standards and also to assess the efficiency of the transfer. The blots were blocked with non-fat dried milk (Carnation, Los Angeles, CA, USA) in buffer A (10 mM Tris, 0.9O/0 NaC1, 0.01% NaN3, pH 7.5), and incubated either with an antibody directed against Glut 4 (East-Acres Biological, MA, USA) or with an antibody directed against Glut 1 (East-Acres Biological, MA, USA). The immune complex was detected using 2 pCi/ml '251-labelled protein A (1 Ci/mol, Amersham, Bucks, UK). After several washes in buffer A with 0.1% Tween 20 (Sigma, St Louis, MO, USA), the blots were autoradiographed (Hyperfilm MP, Amersham, Bucks, UK) and quantified by scanning densitometry (Hoeffer GS 300, San Francisco, CA, USA). Analytical techniques
Blood samples were centrifuged and plasma glucose concentrations were determined using a glucose analyser (Beckman 2, CA, USA). Plasma insulin concentrations were measured by a radioimmunoassay kit (SB-INSI-5, ORIS, Gifsur-Yvette, France) using rat insulin as standard. DNA was
379 Table 1. Plasma glucose and insulin concentrations, and concentration of proteins, DNA and RNA in white adipose tissue of control and obese rats, one and six weeks after VMH lesion. Values are means f SEM of 10 rats. Sample
Plasma glucose
Plasma insulin
Protein
RNA
DNA
PUW
mg/g wet tissue
control VMH
+
31 + 4 32+4
mg/mg D N A 7.5 f 0.6 11.1 f 0.7b
vg/mg DNA
1
mM 6.1 f 0.2 5.2 0.2”
1.5 0.3 1.5 f 0.2
52+ 8 110+ 4b
6
control VMH
5.7 f 0.2 5.6 f 0.3
42f4 129 9b
10.9 k 0.9 10.1 1.2“
1.3 0.3 0.6 f 0.1
Time after lesion week
a
+
+
+
54+ I 59 I l b
+
P < 0.05. P < 0.002 P < 0.01.
measured in tissue homogenates by a spectrofluorometric assay [24]. Proteins were quantified using the Bio-Rad protein assay (Bio-Rad, Munich, FRG). The maximal activity of fatty-acid synthase was determined using the spectrophotometric assay of Linn [25].The maximal activity of acetylCoA carboxylase was determined in the presence of 10 mM citrate, according to Maeda et al. [26] with minor modifications.
Glut 4
4
28kb
FAS
4 4
9.4kb
ACC
4-
9.5kb
18 s
4
2okb
a5lrb
Statistical analysis Results were expressed as mean k SEM. Statistical analysis was performed by Student’s t test for unpaired data.
Results Characteristics of animals Body mass was significantly higher in VMH-lesioned rats than in controls, at both one (293 k 7 g in obese versus 240 f 3 g in control rats, P < 0.001) and six weeks after the lesion (402 k 20 g in obese versus 263 k 5 g in control rats, P < 0.001). One week after the lesion, plasma glucose concentration was lower in VMH-lesioned rats, whereas plasma insulin concentration was identical to that of controls (Table 1). Six weeks after the lesion, VMH-lesioned rats were normoglycemic, but their plasma insulin concentration was significantly higher compared to age-matched controls (Table 1). One week after the VMH lesion, DNA concentration was similar in control and obese rats, but protein and RNA concentrations were higher after six weeks. This suggests that overall transcription is increased one week after the lesion. Six weeks after VMH lesion, DNA, protein and RNA concentrations were less important in obese than in control rats, although the difference was not significant for DNA (Table 1). This is in agreement with an increased cell size in obese rats. Glut 4 mRNA concentrations Fig. 1 shows a representative Northern blot hybridized with a cDNA specific for Glut 4, and with the probe which revealed rRNA 18s in both control and obese rats, one and six weeks after VMH lesion. A quantitative analysis of eight animals in each group revealed that the concentration of Glut 4 mRNA was similar in the two control groups (Table 2). One week after VMH lesion, the concentration of Glut 4 mRNA,
1
2 3 1 week
4
5
6 7 6 weeks
8
Fig. 1. Northern-blot analysis of Glut 4, fatty-acid synthase (FAS), acetyl-CoA carboxylase (ACC) mRNA, and 18s rRNA of periovarian white adipose tissue from control (lanes 1,2,5,6) and obese rats (lanes 3, 4, 7, S), one (lanes 1 - 4) and six weeks (lanes 5 and 6) after VMH lesion.
expressed as DNA, was markedly increased in obese over control animals, whereas six weeks after the lesion, the increase in obese animals was always significant but less important than one week after the lesion. Glucose-transporter concentration It has been demonstrated that adipose tissue expresses a distinct glucose-transporter protein that is recognized specifically by an antiserum. We used this antiserum to compare the relative abundance of this protein in white adipose tissue of control and obese rats one and six weeks after VMH lesion. An autoradiogram of a representative Western blot is presented in Fig. 2 and reveals a protein which migrated as a diffuse band near 43 kDa. Equal amounts of proteins (50 pg) were loaded in each lane. Quantification of Glut 4 protein by densitometry (Table 3) shows that one week after the lesion, white adipose tissue of obese rats possessed more Glut 4 protein than control rats. Six weeks after VMH lesion, there was no significant difference in Glut 4 amount between obese and control rats. Western blots have also been incubated with an antibody against Glut 1 (Fig. 2); and quantification showed that Glut
380 Table 2. mRNA concentrations of Glut 4, fatty-acid synthase (FAS) ayd acetyl-CoA carboxylase (ACC) in white adipose tissue of lean and obese rats, one and six weeks after the lesion. Values are means k SEM of 4 - 8 scanning determinations measured as DNA. Time after lesion
Sample
mRNA concentration Glut 4
FAS
ACC
1.0k0.2 16 + 1 "
l.OIfr0.2 22 + 3 "
week 1
control VMH
1.0k0.2 13 k 0 . 5 "
6
control VMH
1.2k0.3 5 fI b
a
1.OkO.1 5.0 k0.4"
1.0+0.1 3.0 k0.2"
P < 0.001 P < 0.05.
with cDNA encoding for fatty-acid synthase and for acetylCoA carboxylase is shown in Fig. 1. The quantitative analysis of eight rats in each group revealed that one week after VMH lesion, fatty-acid synthase and acetyl-CoA carboxylase mRNA concentrations were markedly increased over control values (Table 2). Six weeks after the lesion, the degree of stimulation over control values observed for these two enzymes was decreased when compared to one week lesioned rats. When we measured fatty-acid synthase and acetyl-CoA carboxylase activities on a cellular basis, we observed that these activities were markedly increased in white adipose tissue of rats one week after VMH lesion (Table 3). Later on, these activities remained more important in obese than in control rats, but lower than those from one-week lesioned rats. Glucose transport and utilization in isolated adipocytes
Glut 4
4-
Glut I
1
2 1
3
4
4
5
6
7
42kDa
8
6-
Fig. 2. Western-blot analysis of Glut 4 and Glut 1, in periovarian white adipose tissue of control (lanes 1, 2, 5, 6) and obese (lanes 3, 4, 7, 8) rats, one (lanes 1 - 4) and six weeks (lanes 5 - 8) after VMH lesion. Table 3. Protein concentrations of Glut 4 and Glut 1, and maximal activities of fatty-acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in control and obese rats, one and six weeks after VMH lesion. Values are means k SEM of eight animals. Time after lesion
Sample
Glut 4 protein
Glut 1 protein
Activity of FAS
week
mg DNA-'
ACC
U/g DNA
1
control VMH
1.OkO.1 3.4f0.3"
1.0+0.03 1.5f0.1b
15k 2 253 k 6 6 "
13 i 2 6 3 k 13b
6
control VMH
1.OkO.2 1.6k0.5
1.0+0.1 0.8 k0.2
37f 2 253 k 6 4 "
6k 2 11 k 2
a
P
Molecular and metabolic changes in white adipose tissue of the rat during development of ventromedial hypothalamic obesity Beatrice COUSIN ', Karen AGOU', Armelle LETURQUE', Pascal FERRE', Jean GIRARD' and Luc PENICAUD Laboratoire de Physiopathologie de la Nutrition, CNRS URA 307, Universite Paris VII, Paris, France Centre de Recherche sur I'Endocrinologie Moleculaire et le Dkveloppement, CNRS, Meudon, France (Received March 3/April 13, 1992) - EJB 92 0291
We have previously shown that rats made obese by lesion of ventromedial hypothalamus (VMH) nuclei, demonstrate an hyper-responsiveness to insulin with regard to whole-body glucose utilization one week after injury. This is mainly due to an increased glucose uptake in white adipose tissue. Six weeks after the lesion, glucose utilization in white adipose tissue returns to normal values. These modifications in insulin responsiveness could be mediated by altered activity and/or concentration of intracellular insulin effectors. In this study, we have measured the expression of the insulin-sensitive glucose transporter, Glut 4 and the activities and expression of key lipogenic enzymes (fatty-acid synthase and acetyl-CoA carboxylase) in white adipose tissue, one and six weeks after the lesion. All these parameters, as well as glucose transport and metabolism determined in white adipocytes, were markedly increased one week after the lesion. They returned to control values within six weeks in VMH-lesioned rats. These results indicate the existence of an increased expression of Glut 4 and lipogenic enzymes in white adipose tissue of VMH-lesioned rats which decreased with time and were parallel to glucose utilization determined in vivo.
In adipose tissue, insulin resistance, with regard to glucose uptake and utilization is a major feature of overt obesity (for reviews, see [I, 21). Nevertheless, it has been demonstrated that insulin resistance is preceded by a period of hypersensitivity and responsiveness to insulin in adipose tissue from obese human or rodents (for a review see [3]). In rats made obese by electrolytic lesion of the ventromedial hypothalamus (VMH), the onset of the syndrome is determined by the time of the lesion. Thus, this model allows the chronological study of the alterations associated with obesity. It has been shown in vivo that one week after the lesion, during the dynamic phase of obesity, insulin-induced whole-body glucose utilization is increased [4], and this could almost be accounted for by the differential increase observed in white adipose tissue glucose utilization [ 5 ] . Six weeks after VMH lesion, this state of increased insulin action disappears [4], and glucose utilization in white adipose tissue returns to near control values [5]. These changes in response to insulin of white adipose tissue, observed in vivo, are also present in young suckling (15 day old) or weaned (30 day old) genetically obese Zucker rats, although to a lesser extent [6-81, and in 13-year-old obese children [9]. These modifications could be mediated by changes in insulin receptor and/or intracellular insulin targets, for which glucose transporters and lipogenic enzymes are likely candidates. Correspondence to L. PCnicaud, Laboratoire de Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique URA 307, Universitk Paris VII, 2 place Jussieu, F-75251 Paris Cedex 05, France Fax: i33 1 44 27 18 36. Abbreviation. VMH, ventromedian hypothalamus. Enzymes. Acetyl-CoA carboxylase (EC 6.4.1.2); fatty-acid synthase (EC 2.3.1.85).
We have already shown that insulin-receptor kinase activity was increased in white adipose tissue of rats, one week after VMH lesion, and returned to control values six weeks after the lesion [lo]. Nevertheless, these modifications of insulinreceptor kinase activity could not totally explain the change in glucose utilization in white adipose tissue observed during the development of obesity. The aim of the present work was to determine whether other alterations located at post-receptor levels could mediate the variations of insulin responsiveness observed in vivo in white adipose tissue of VMH rats one and six weeks after the lesion. We have therefore studied the expression of the insulinsensitive glucose transporter (Glut 4) in white adipose tissue of control and obese rats. We have also studied activities and mRNA concentrations of two lipogenic enzymes: fatty-acid synthase and acetyl-CoA carboxylase. Finally, the consequences of these alterations on metabolic fluxes were determined in in vitro studies.
MATERIALS AND METHODS Animals Female Wistar rats were obtained from a local supplier (Iffa-Credo, L'Arbresle, France) at 10 - 11 weeks of age, and were housed at 24"C, in an animal quarter, with a constant light/dark cycle (light from 7 a.m. to 7 p.m.). Rats were anesthetized with 95 mg ketaminelkg intraperitoneally (Imalgene, Bio Mkrieux, France), and VMH lesions or sham operations (control rats) were carried out as described previously [Ill. Rats were then housed in individual cages. A lesion was considered successful when daily body mass gain was greater than 5 g compared to 2 g in control rats. VMH-
378 lesioned rats and their controls had free access to water and chow pellets [65% (by mass) carbohydrate, 11% fat, 24% protein; UAR, Villemoisson, France]. Experiments were then performed one or six weeks after the operation, at 3 p. m., and after 6 h of food deprivation to reduce possible differences due to hyperphagia in obese rats [12]. Lesioned and control rats were killed by cervical dislocation. Periovarian white adipose tissue was rapidly removed, and either used for adipocyte preparation or frozen in liquid nitrogen and stored at - 80°C for subsequent analysis. Blood samples were collected for determination of blood glucose and plasma insulin concentrations. Preparation of isolated white adipocytes
White adipocytes were isolated as described previously by Rodbell [13]. The two periovarian fat pads were removed and washed in Krebs/Ringer/phosphate/Hepes buffer, pH 7.4 [14], containing 0.5 mM calcium. The tissue was cut into small pieces and digested for 45 min in the same buffer containing 1 mg/ml collagenase (Sigma Chemical, St Louis, MO, USA), 2% bovine serum albumin (fraction V, fat free; Sigma Chemical, St Louis, MO, USA), and 2 mM pyruvate, in a shaking water bath at 37°C. The digested tissue was then filtered through a 200-pm nylon mesh, the isolated cells were washed three times in the buffer devoid of collagenase and collected in the incubation medium. Glucose transport in isolated adipocytes
Glucose-transport studies were performed by measuring radiolabelled glucose in cells after a rapid incubation of white adipocytes with 50 pM glucose [15, 161. Isolated adipocytes, at a final concentration of 2 x lo5 cells/ml, were preincubated for 1 h, with or without insulin (0.1 - 1 mU/ml), in Krebs/ Ringer/phosphate buffer, pH 7.4, containing 2 mM pyruvate. The assays were initiated by adding 50 pM [6-'4C]glucose (45 Ci/mol; CEA, Gif-sur-Yvette, France). The radioactivity in the cell layer was corrected for ['4C]glucose trapped in the extracellular water using [3H]inulin (0.4 Ci/g; NEN, Du Pont de Nemours, Paris, France). The assays were ended after 20 s by a rapid centrifugation of 200 p1 cell suspension with dinonylphthalate oil (Merck, Darmstadt, FRG). The radioactivity in the cell layer was measured. Glucose metabolism in isolated adipocytes
Isolated white adipocytes, at a concentration of lo5 cells/ ml, were incubated for 2 h in a shaking water bath at 37°C. The incubations were performed in 2.5 ml Krebs/Ringer/ phosphate/Hepes buffer, pH 7.4, containing 1 mM calcium, 2% fatty-acid-free albumin, and 5 mM [U-'4C]glucose (0.5 pCi/ml; CEA, Gif-sur-Yvette, France), with or without insulin (0.1 - 1 mU/ml). The flasks were oxygenated and tightly closed, and assays were stopped after 2 h by adding 0.5 ml 40% (by vol.) perchloric acid. To determine the amount of I4C-labelled C 0 2 formed from glucose, 0.3 ml hyamine was added to a center well. 14C-labelled COz was collected during an additional 45-min incubation. Two aliquots were taken from the content of the incubation flask. The first was used to measure glucose incorporation into lactate; [14C]lactate accumulating in the incubation medium was separated from ['4C]glucose using ionexchange resin columns [17]. With the second aliquot, total
lipids were extracted according to Dole [18] and were used to measure I4C incorporation into lipids. Northern-blot analysis
Total RNA was extracted from 1 - 3 g white adipose tissue using guanidine thiocyanate [19]. The concentration of RNA was determined by absorbance at 260 nm, and the RNA solutions were stored in H20/diethyl pyrocarbonate (0.020/) at - 80 "C until yse. All samples had an A260/A280 ratio of about 2. For Northern-blot analysis, 20 pg RNA was denatured, sized-fractionated by electrophoresis in 1% agarose gel and transferred to a nylon membrane (Hybond N; Amersham, Bucks, UK). Blots were hybridized with several probes. The cDNA probes were kindly provided by Dr D. James for Glut 4 [20], Dr A. G. Goodridge for fatty-acid synthase [21] and Dr K. H. Kim for acetyl-CoA carboxylase [22]. The probes were labelled with 32P using a Multiprime labelling system kit (Amersham, Bucks, UK). Hybridations with Glut 4 were realized as described by James et al. [20], and with fatty-acid synthase and acetyl-CoA carboxylase according to Coup6 et al. [23]. To verify the amount of total RNA in each lane, blots were hybridized with a synthetic oligonucleotide specific for ribosomal 18s RNA, in solutions containing 0.1YODenhardt's solution, 5 x NaCI/Cit (NaCl/Cit: 0.15 M NaC1, 15 mM trisodium citrate), 0.05 M sodium phosphate, pH 6.5, 0.1% SDS and salmon sperm DNA (250 pg/ml) denaturated at 42 "C overnight. The membranes were washed twice for 20 min each, with 2 x NaCl/Cit, at 42°C. The blots were exposed for 4 - 48 h at - 80 "C with intensifying screens. Quantification was performed by scanning densitometry. Values were normalized for the corresponding amount of 18s mRNA. Western-blot analysis
200 - 300 mg white adipose tissue was homogenized in ice-cold 1 mM EDTA, 20 mM Tris/HCl and 0.25 M sucrose (pH 7.4). Equal amounts of proteins (50 pg) were solubilized in Laemmli buffer and submitted to SDSjPAGE (12% acrylamide gels). The proteins were electrophoretically transferred to nitrocellulose membrane (BA 85; Schleicher and Schuell, Dassel, FRG). Protein markers (Rainbow; Amersham, Bucks, UK) were used as molecular mass standards and also to assess the efficiency of the transfer. The blots were blocked with non-fat dried milk (Carnation, Los Angeles, CA, USA) in buffer A (10 mM Tris, 0.9O/0 NaC1, 0.01% NaN3, pH 7.5), and incubated either with an antibody directed against Glut 4 (East-Acres Biological, MA, USA) or with an antibody directed against Glut 1 (East-Acres Biological, MA, USA). The immune complex was detected using 2 pCi/ml '251-labelled protein A (1 Ci/mol, Amersham, Bucks, UK). After several washes in buffer A with 0.1% Tween 20 (Sigma, St Louis, MO, USA), the blots were autoradiographed (Hyperfilm MP, Amersham, Bucks, UK) and quantified by scanning densitometry (Hoeffer GS 300, San Francisco, CA, USA). Analytical techniques
Blood samples were centrifuged and plasma glucose concentrations were determined using a glucose analyser (Beckman 2, CA, USA). Plasma insulin concentrations were measured by a radioimmunoassay kit (SB-INSI-5, ORIS, Gifsur-Yvette, France) using rat insulin as standard. DNA was
379 Table 1. Plasma glucose and insulin concentrations, and concentration of proteins, DNA and RNA in white adipose tissue of control and obese rats, one and six weeks after VMH lesion. Values are means f SEM of 10 rats. Sample
Plasma glucose
Plasma insulin
Protein
RNA
DNA
PUW
mg/g wet tissue
control VMH
+
31 + 4 32+4
mg/mg D N A 7.5 f 0.6 11.1 f 0.7b
vg/mg DNA
1
mM 6.1 f 0.2 5.2 0.2”
1.5 0.3 1.5 f 0.2
52+ 8 110+ 4b
6
control VMH
5.7 f 0.2 5.6 f 0.3
42f4 129 9b
10.9 k 0.9 10.1 1.2“
1.3 0.3 0.6 f 0.1
Time after lesion week
a
+
+
+
54+ I 59 I l b
+
P < 0.05. P < 0.002 P < 0.01.
measured in tissue homogenates by a spectrofluorometric assay [24]. Proteins were quantified using the Bio-Rad protein assay (Bio-Rad, Munich, FRG). The maximal activity of fatty-acid synthase was determined using the spectrophotometric assay of Linn [25].The maximal activity of acetylCoA carboxylase was determined in the presence of 10 mM citrate, according to Maeda et al. [26] with minor modifications.
Glut 4
4
28kb
FAS
4 4
9.4kb
ACC
4-
9.5kb
18 s
4
2okb
a5lrb
Statistical analysis Results were expressed as mean k SEM. Statistical analysis was performed by Student’s t test for unpaired data.
Results Characteristics of animals Body mass was significantly higher in VMH-lesioned rats than in controls, at both one (293 k 7 g in obese versus 240 f 3 g in control rats, P < 0.001) and six weeks after the lesion (402 k 20 g in obese versus 263 k 5 g in control rats, P < 0.001). One week after the lesion, plasma glucose concentration was lower in VMH-lesioned rats, whereas plasma insulin concentration was identical to that of controls (Table 1). Six weeks after the lesion, VMH-lesioned rats were normoglycemic, but their plasma insulin concentration was significantly higher compared to age-matched controls (Table 1). One week after the VMH lesion, DNA concentration was similar in control and obese rats, but protein and RNA concentrations were higher after six weeks. This suggests that overall transcription is increased one week after the lesion. Six weeks after VMH lesion, DNA, protein and RNA concentrations were less important in obese than in control rats, although the difference was not significant for DNA (Table 1). This is in agreement with an increased cell size in obese rats. Glut 4 mRNA concentrations Fig. 1 shows a representative Northern blot hybridized with a cDNA specific for Glut 4, and with the probe which revealed rRNA 18s in both control and obese rats, one and six weeks after VMH lesion. A quantitative analysis of eight animals in each group revealed that the concentration of Glut 4 mRNA was similar in the two control groups (Table 2). One week after VMH lesion, the concentration of Glut 4 mRNA,
1
2 3 1 week
4
5
6 7 6 weeks
8
Fig. 1. Northern-blot analysis of Glut 4, fatty-acid synthase (FAS), acetyl-CoA carboxylase (ACC) mRNA, and 18s rRNA of periovarian white adipose tissue from control (lanes 1,2,5,6) and obese rats (lanes 3, 4, 7, S), one (lanes 1 - 4) and six weeks (lanes 5 and 6) after VMH lesion.
expressed as DNA, was markedly increased in obese over control animals, whereas six weeks after the lesion, the increase in obese animals was always significant but less important than one week after the lesion. Glucose-transporter concentration It has been demonstrated that adipose tissue expresses a distinct glucose-transporter protein that is recognized specifically by an antiserum. We used this antiserum to compare the relative abundance of this protein in white adipose tissue of control and obese rats one and six weeks after VMH lesion. An autoradiogram of a representative Western blot is presented in Fig. 2 and reveals a protein which migrated as a diffuse band near 43 kDa. Equal amounts of proteins (50 pg) were loaded in each lane. Quantification of Glut 4 protein by densitometry (Table 3) shows that one week after the lesion, white adipose tissue of obese rats possessed more Glut 4 protein than control rats. Six weeks after VMH lesion, there was no significant difference in Glut 4 amount between obese and control rats. Western blots have also been incubated with an antibody against Glut 1 (Fig. 2); and quantification showed that Glut
380 Table 2. mRNA concentrations of Glut 4, fatty-acid synthase (FAS) ayd acetyl-CoA carboxylase (ACC) in white adipose tissue of lean and obese rats, one and six weeks after the lesion. Values are means k SEM of 4 - 8 scanning determinations measured as DNA. Time after lesion
Sample
mRNA concentration Glut 4
FAS
ACC
1.0k0.2 16 + 1 "
l.OIfr0.2 22 + 3 "
week 1
control VMH
1.0k0.2 13 k 0 . 5 "
6
control VMH
1.2k0.3 5 fI b
a
1.OkO.1 5.0 k0.4"
1.0+0.1 3.0 k0.2"
P < 0.001 P < 0.05.
with cDNA encoding for fatty-acid synthase and for acetylCoA carboxylase is shown in Fig. 1. The quantitative analysis of eight rats in each group revealed that one week after VMH lesion, fatty-acid synthase and acetyl-CoA carboxylase mRNA concentrations were markedly increased over control values (Table 2). Six weeks after the lesion, the degree of stimulation over control values observed for these two enzymes was decreased when compared to one week lesioned rats. When we measured fatty-acid synthase and acetyl-CoA carboxylase activities on a cellular basis, we observed that these activities were markedly increased in white adipose tissue of rats one week after VMH lesion (Table 3). Later on, these activities remained more important in obese than in control rats, but lower than those from one-week lesioned rats. Glucose transport and utilization in isolated adipocytes
Glut 4
4-
Glut I
1
2 1
3
4
4
5
6
7
42kDa
8
6-
Fig. 2. Western-blot analysis of Glut 4 and Glut 1, in periovarian white adipose tissue of control (lanes 1, 2, 5, 6) and obese (lanes 3, 4, 7, 8) rats, one (lanes 1 - 4) and six weeks (lanes 5 - 8) after VMH lesion. Table 3. Protein concentrations of Glut 4 and Glut 1, and maximal activities of fatty-acid synthase (FAS) and acetyl-CoA carboxylase (ACC) in control and obese rats, one and six weeks after VMH lesion. Values are means k SEM of eight animals. Time after lesion
Sample
Glut 4 protein
Glut 1 protein
Activity of FAS
week
mg DNA-'
ACC
U/g DNA
1
control VMH
1.OkO.1 3.4f0.3"
1.0+0.03 1.5f0.1b
15k 2 253 k 6 6 "
13 i 2 6 3 k 13b
6
control VMH
1.OkO.2 1.6k0.5
1.0+0.1 0.8 k0.2
37f 2 253 k 6 4 "
6k 2 11 k 2
a
P
