Age-Dependent Regulation of Lipogenesis in Human and Rat

0021-972X/04/$15.00/0 Printed in U.S.A.

The Journal of Clinical Endocrinology & Metabolism 89(9):4601– 4606 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-030994

Age-Dependent Regulation of Lipogenesis in Human and Rat Adipocytes ¨ RNE, ASHRAF F. KAMEL, SVANTE NORGREN, KARIN STRIGÅRD, ANDERS THO HOSSEIN FAKHRAI-RAD, JOAKIM GALLI, AND CLAUDE MARCUS Department of Pediatrics (A.F.K., S.N., C.M.), Endocrine Research Unit, and Department of Surgery (K.S., A.T.), Huddinge University Hospital, S-141 86 Huddinge, Sweden; and Department of Molecular Medicine (H.F.-R., J.G.), Karolinska Hospital, L 602, S-171 76 Stockholm, Sweden The regulation of adipocyte metabolism is of importance for adipose tissue growth and therefore also for the development of obesity. This study was designed to investigate the regulation of basal and insulin-induced lipogenesis, glucose transport, and glucose transporter protein expression in human and rat adipocytes from different age groups. The study included 21 infants, 21 children, nine adults, and 80 male weaned and 20 male adult Fischer rats. The lipogenesis experiments were performed under conditions at which glucose transport is rate limiting. Basal lipogenesis was approximately three times higher in infants and children than in adults, whereas insulin-induced lipogenesis was two times higher in infants than in children and adults. In rats, basal

A

DIPOSE TISSUE FORMATION begins before birth (1), and (adipose) tissue mass expands rapidly during the first year of life due to increased adipocyte size and number (2– 4). Because more than 90% of the adipocyte volume consists of triglycerides (5), changes in adipocyte volume depend on the balance between synthesis (lipogenesis) and breakdown (lipolysis) of triglycerides. Therefore, studies of regulation of lipogenesis and lipolysis are of importance for the understanding of adipose tissue growth. There are distinct age-dependent variations in adiposity, which are reflected by body mass index (BMI) changes, thought to be of importance for adult obesity (6). The regulation of these BMI changes is unknown. The regulation of lipolysis during infancy and childhood has been investigated in some detail previously. In contrast to the situation in children and adults, TSH and not catecholamines is the main lipolytic hormone during the neonatal period (7–9). The lipolytic effect of catecholamines is poor in infants and increases gradually to reach the adult level by the age of 1–3 yr (7, 10, 11). The blunted lipolytic effect of catecholamines is probably due to enhanced ␣-2adrenoreceptor antilipolytic activity during the first months after birth (12, 13). A shift from higher ␤2-adrenoceptor activity during infancy to a combination of ␤1- and ␤2adrenoceptor in adults may also be of importance (14, 15). These factors act in concert to reduce the stress-induced adipocyte catabolism. Abbreviation: BMI, Body mass index. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

lipogenesis, insulin-induced lipogenesis, and insulin sensitivity were two times higher in weaned than in adult animals. Moreover, basal and insulin-induced glucose transport were two times higher in weaned than in adult rats. No differences were detected in GLUT1 or GLUT4 content between any of the age groups in human or in rat adipocytes. In conclusion, basal and insulin-stimulated lipogenesis are increased in adipocytes early in life. This may promote adipose tissue growth in early age. The data indicate that agedependent variation in basal and insulin-stimulated lipogenesis is differently regulated. (J Clin Endocrinol Metab 89: 4601– 4606, 2004)

The synthesis of triglycerides (lipogenesis) depends on the availability of fatty acids and glucose. Glucose transport into the cell is provided by a group of membrane carrier proteins called glucose transporters. GLUT1 and GLUT4 are the main glucose transporters in adipose tissue (16), in which GLUT4 is 10 times more abundant than GLUT1 (17). Under basal conditions, GLUT1 is concentrated in the plasma membrane and accounts for basal glucose transport (14), whereas GLUT4 is mainly concentrated in an intracellular compartment pool (18). Previous studies on age-dependent changes in glucose transport are scarce. One study (19) indicated that basal glucose transport is higher in adipocytes from children than adults. Insulin promotes adipocyte growth both via inhibition of lipolysis and stimulation of lipogenesis. The activation of lipogenesis is mediated by stimulation of glucose transport through recruitment of GLUT4 from intracellular compartments to the plasma membrane (18) and by intracellular events increasing the activity of acetyl-coenzyme A carboxylase, fatty acid synthetase, and glycerol-3-phosphate dehydrogenase (20, 21). We previously reported that neither the insulin-induced inhibition of lipolysis nor insulin receptor expression in isolated adipocytes differ among various age groups (22), but we are not aware of any studies on insulininduced lipogenesis during development. This study was designed to investigate the regulation of basal and insulin-induced lipogenesis and glucose transport as well as glucose transporter protein expression in both human adipocytes from different age groups (infants, children, and adults) and adipocytes from weaned and adult rats.

4601

4602

J Clin Endocrinol Metab, September 2004, 89(9):4601– 4606

Kamel et al. • Age-Dependent Regulation of Lipogenesis

Subjects and Methods

at room temperature overnight before the radioactivity was measured by liquid scintillation counting.

Human studies The study included 21 infants aged between 0.5 and 2 months (mean 1.5 months), 21 children between 2 and 7 yr (mean 4 yr), and nine adults between 24 and 44 yr (mean 31 yr). All infants and children were undergoing inguinal hernia surgery and were otherwise healthy and of normal weight. Abdominal adipose tissue (100 –300 mg) was removed at the start of the operation. The adults were healthy volunteers or patients undergoing inguinal hernia surgery. Adipose tissue biopsies were obtained either at the start of surgery or by means of needle biopsies under local anesthesia. The amount of fat that was possible to obtain from infants and children was not enough to study both lipogenesis and GLUT4 protein expression in the same subject, and it was not enough to study glucose transport. Adipose tissue from 10 infants and 10 children was used to study lipogenesis, and adipose tissue from 11 infants and 11 children was used to study GLUT4 protein levels. In the adult group, adipose tissue from nine individuals was used to study lipogenesis, and from three of these, adipose tissue was also used to study GLUT4 protein levels. The adipose tissue was pooled from either three to four infants or three to four children to run Western blot analysis in each age group. The Karolinska Institute Ethics Committee approved the study, and informed consent was obtained from the guardians or participants.

Rat studies The study comprised 80 male weaned Fischer rats aged 4 wk and 20 male young adult Fischer rats aged 9 wk. Adipose tissue from five weaned and five adult rats was used to study lipogenesis. Adipose tissue from five weaned and five adult rats was used to study glucose transport, in which adipocytes were pooled from 10 weaned rats. Adipose tissue biopsies from 25 weaned rats and 10 adult rats were used to determine GLUT1 and GLUT4 protein expression levels. Adipose tissue was pooled from groups of five weaned and one to two adult rats for Western blot analysis. The rats were decapitated after carbon dioxide anesthesia, and an epididymal adipose tissue biopsy was removed.

Isolation of adipocytes The adipocytes were isolated as previously described (23). The adipose tissue was cut into fragments and isolated from stroma by incubation with collagenase. The adipose tissue was washed in Krebs Ringer phosphate buffer containing albumin, and aggregated material was removed by filtration through a silk cloth.

Determination of glucose incorporation into lipids The lipogenesis experiments were performed as described previously at low glucose concentrations (1 ␮mol/liter) in which glucose transport is rate limiting (24). The adipocytes were incubated at a final concentration of 2% vol/vol for 2 h at 37 C in Krebs Ringer phosphate buffer containing 40 mg/ml BSA, labeled glucose ([3-3H]glucose 5⫻ 106 cpm, 0.2 ␮mol/liter), unlabeled glucose (1 ␮mol/liter), and insulin (0 –10.000 ␮U/ml). Each incubation was performed in duplicates for 2 h at 37 C and stopped by rapidly chilling the vials to 4 C. The incubation volume was 0.5 ml. Incorporation of glucose into lipids was determined as described previously (25). Briefly, 45 ␮l of 6 mol/liter H2SO4 and 4 ml of toluene with 2.5 mg diphenyloxazole were added to each vial. The vials were left

Determination of glucose transport The glucose transport experiments were performed as described previously (26). The adipocytes were washed in Krebs-Ringer HEPES buffer [10 mmol/liter (pH 7.4)] containing 40 mg/ml BSA and diluted in the same buffer to a 40% (vol/vol) solution. Aliquots of 50 ␮l of the cell suspension were incubated in duplicates or triplicates for 30 min at 37 C in the presence of 10 ␮l insulin (4000 pmol/liter). Thereafter 50 ␮l of 3-O-methyl glucose was added at a final concentration of 20 mmol/liter. The reaction was stopped for 30 sec by adding 3 ml of 154 mmol/liter NaCl containing 0.3 mmol/liter of phloretin and 0.5% (vol/vol) of ethanol. Lastly, 0.8 ml silicone was layered on the top, and within 3 min the tubes were centrifuged for 1 min at 2500 ⫻ g. The supernatant cells were then removed for the determination of radioactivity.

Determination of glucose transporter content in adipose tissue GLUT1 and GLUT4 protein expression was measured as previously described (27). To prepare adipocyte membrane proteins, adipose tissue biopsy was homogenized for 5 sec in ice-cold buffer [25 mm HEPES, 250 mm sucrose, 4 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin, 1 U/ml aprotinin, and 0.1 mm sodium vanadate (pH 7.4)]. Homogenates were centrifuged at 5000 ⫻ g for 5 min at 4 C. To obtain the membrane fraction, the supernatant was centrifuged at 200,000 ⫻ g for 90 min at 4 C. The pellet was dissolved in ice-cold homogenization buffer. Protein concentration was determined by protein assay (Bio-Rad Laboratories, Hercules CA). Thirty micrograms of protein from rat adipose tissue or 80 ␮g from human adipose tissue per lane were separated on 10% polyacrylamide gels and electroblotted to polyvinyl difluoridenitrocellulose membranes (Amersham, Buckinghamshire, UK). All lanes from weaned and adult rats were run on one gel for analysis of each glucose transporter, and all human lanes were run on one separate gel. Blotted membranes were blocked in PBS with 0.01% Tween 20 supplemented with 10% dry milk overnight at 4 C and subsequently incubated with rabbit anti-GLUT1 or mouse anti-GLUT4 for 3 h at room temperature with mild agitation. The membranes were rinsed briefly with two changes of washing buffer (PBS with 0.01% Tween 20), washed twice for 15 min, and then rinsed again for 3⫻ 5 min with the washing buffer. The membranes were incubated with antirabbit or antimouse IgG (Amersham) for 1 h at room temperature followed by the same steps of washing as above. Equal loading was confirmed by Ponceau-red staining of the membranes. The signal was detected with chemiluminescence (ECL⫹plus, Amersham). The relative abundance of the glucose transporters was determined by scanning densitometry of exposed x-ray films (Hoefer, San Francisco, CA).

Expression of lipogenesis Lipogenesis was expressed per cell or per unit of cell surface area. The adipocyte diameter was measured during direct microscopy, and the mean adipocyte diameter was calculated from 100 cells in each subject. The mean adipocyte volume and cell surface area were calculated as previously described (28, 29). The insulin-induced lipogenesis (responsiveness) was calculated from each individual dose-response curve as

TABLE 1. Basal lipogenesis (i.e. in the absence of insulin), insulin-induced lipogenesis, and total lipogenesis (i.e. basal ⫹ insulin-induced lipogenesis) expressed per cell Human adipocytes

Basal lipogenesis Insulin induced-lipogensis Total lipogenesis

Rat adipocytes

Neonates

Children

Adults

Weaned

Adults

1.6 (0.9 –2.3) 0.8 (0.5–1.2) 2.4 (1.8 –3.5)

1.7 (1.2–2.2) 0.4 (0.4 – 0.7) 2.3 (1.4 –2.7)

1.2 (0.5–1.9) 0.8 (0.5–1.3) 1.8 (1.5–2.9)

0.8 (0.6 –1.2) 4.4 (2.9 – 6.6) 5.2 (3.5–7.7)

0.4 (0.3– 0.6)a 1.6 (1.2–2.4)a 2.0 (1.5–2.9)a

Lipogenesis was determined as glucose incorporation into lipids (␮mol/107 cells䡠2 h). Data are presented as median and upper and lower quartiles. Comparisons between data from different age groups were made by Wilcoxon signed ranks test. a P ⬍ 0.05, adults vs. weaned rats.


the difference between glucose incorporation at the maximum effective stimulatory concentration minus glucose incorporation in the absence of insulin. The EC50 of insulin (sensitivity) was calculated graphically from the individual dose-response curves.

Chemicals

J Clin Endocrinol Metab, September 2004, 89(9):4601– 4606 4603

insulin-induced lipogenesis in the total lipogenesis was greater in adults than children (P ⬍ 0.01; Table 2). We found no difference in lipogenesis between adipocytes from adults obtained during surgical operation or taken under local anesthesia (data not shown).

BSA (fraction V), A-6918 for human and A-7030 for rats, and collagenase prepared from Clostridium histolyticum (type 1, C-1030) were purchased from Sigma (St. Louis, MO). Radioactive glucose ([3-3H]glucose) was from Amersham. Within the same species, the same batches of collagenase and albumin were used in all experiments. Phenylmethylsulfonyl fluoride was from Roche Molecular Biochemicals (Biochemica, Mannheim, Germany). Leupeptin (A-2023), aprotinin (A-1153), and sodium vanadate (S-6383) were from Sigma. Polyvinyl difluoridenitrocellulose membrane (RPN 2020 F), ECL⫹plus Western blotting detection system (RPN 2132), and hyperfilm ECL (RPN 3103 H) were from Amersham. Monoclonal mouse anti-GLUT4 (1262– 00) was from Genzyme Diagnostics (Novakemi AB, Stockholm, Sweden), and rabbit anti-GLUT1 (4670 –1607) was from Biogenesis (Novakemi AB). Antimouse Ig horseradish peroxidase (NA 931) and antirabbit Ig horseradish peroxidase (NA 934) were from Amersham.

Statistics Data were analyzed employing the Statistical Package for Social Sciences (version 5.0; SPSS Inc., Chicago, IL) and expressed as median (interquartile range) in the text and tables. A Mann-Whitney U test was used for comparisons between two time points and linear regression analysis for correlation studies. Significance was defined as P ⬍ 0.05.

Results Adipocyte volume

Adipocyte volume was 149 pl (86 –186) in the infant group, 226 pl (164 –276) in children, and 486 pl (341– 879) in adults. There was a positive correlation between the age of the subjects and adipocyte volume (r ⫽ 0.730, P ⬍ 0.001). These data confirm our previous results (30). Adipocyte volume was 83 pl (6 –96) in weaned rats and 103 pl (91–116) in adult rats. No significant difference between the two groups was found. Lipogenesis

Human adipocytes. There were no significant differences in basal, insulin-induced, or total lipogenesis (i.e. basal ⫹ insulin-induced lipogenesis) in adipocytes from the different age groups when data were expressed per cell (Table 1). Expressed per cell surface area, basal lipogenesis was approximately 3 times higher in infants and children than adults (P ⬍ 0.01; Fig. 1A), whereas no difference was found between infants and children. The basal lipogenesis showed a negative correlation with age (r ⫽ 0.524, P ⬍ 0.01). The insulin-induced lipogenesis was higher in infants than both children and adults (P ⬍ 0.01 and P ⬍ 0.05, respectively; Fig. 1A). No difference in insulin-induced lipogenesis was found between the children and adults. Consequently, insulininduced lipogenesis expressed as a percentage of basal lipogenesis was significantly higher in adults than children. The total lipogenesis was higher in infants than both children and adults (P ⬍ 0.05 and P ⬍ 0.001, respectively; Fig. 1A) and higher in children than adults (P ⬍ 0.01). The insulin sensitivity, expressed as EC50, did not vary between the groups (Fig. 1B). Basal lipogenesis was the major contributor to total lipogenesis in infants and children. Thus, the contribution of

FIG. 1. A, Age-dependent variation in basal, insulin-induced, and total lipogenesis in human adipocytes. Adipocytes from 10 infants (open bars), nine children (hatched bars), and nine adults (gray bars) were incubated in the absence and presence of increasing concentrations of insulin. Glucose incorporation into lipids in the absence (basal lipogenesis), presence of maximum effective concentration of insulin with basal lipogenesis subtracted (insulin-induced lipogenesis), and total lipogenesis (basal ⫹ insulin-induced lipogenesis) are shown. The horizontal lines in the box denote the 25th, 50th, and 75th percentile values. The error bars denote the fifth and 95th percentile values. The stars denote the first and 99th percentile values. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. B, Concentration of insulin resulting in EC50 of lipogenesis in adipocytes from 10 infants, 10 children, and nine adults. Horizontal bars denote median levels. No significant difference was found between the insulin sensitivity of the different age groups.

4604



TABLE 2. Insulin-induced lipogenesis as percentage of basal lipogenesis Human adipocytes

Percentage of basal lipogenesis

Rat adipocytes

Infants

Children

Adults

Weaned

Adults

30.6 (20.9.0 –52.7)

22.4 (14.6 –28.5)

51.9 (24.9 – 63.2)a

83.3 (79.7– 85.8)b

80.6 (77.1– 81.3)b

Data are presented as median and upper and lower quartiles. Comparisons between data from different age groups were made by Wilcoxon signed ranks test. a P ⬍ 0.05 adults vs. children; b P ⬍ 0.001 rat adipocytes vs. human adipocytes.

adipocytes (P ⬍ 0.001), but there was no difference between weaned and adult rats (Table 2). Glucose transport

Basal glucose transport was higher in weaned rats: 129.00 pmol (101.00 – 430.50) per 30 sec per gram triglycerides than in adult rats [61.00 pmol (23.00 –113.50; P ⬍ 0.05)]. Glucose transport at insulin concentration 4000 pmol/liter was higher in weaned rats [331.00 pmol (221.00 – 656.50) per 30 sec per gram triglycerides] than in adult rats [163.00 pmol (83.00 –172.50)] (P ⬍ 0.01). GLUT1 and GLUT4 protein expression

The difference in basal and insulin-induced lipogenesis may have been explained by changes in the expression of glucose transporters. GLUT1 and GLUT4 protein contents were measured by Western blot analysis. The amount of protein was not enough to detect GLUT1 in human adipocytes. No difference was detected in GLUT1 or GLUT4 contents between any of the age groups in humans (Fig. 3) or rats (Fig. 4). Discussion

FIG. 2. A, Age-dependent variation in basal, insulin-induced, and total lipogenesis in rat adipocytes. Adipocytes from five weaned (open bars) and five adult rats (gray bars) were incubated in the absence and presence of increasing concentrations of insulin. Glucose incorporation into lipids in the absence (basal lipogenesis), presence of maximum effective concentration of insulin (insulin-induced lipogenesis), and total lipogenesis (basal ⫹ insulin-induced lipogenesis) are shown. The horizontal lines in the box denote the 25th, 50th, and 75th percentile values. The error bars denote the fifth and 95th percentile values. The stars denote the first and 99th percentile values. *, P ⬍ 0.05. B, Concentration of insulin resulting in EC50 of lipogenesis (in adipocytes from five weaned and five adult rats). Horizontal bars denote median levels. The insulin sensitivity was significantly higher in weaned rats, compared with adult rats (P ⬍ 0.05).

Rat adipocytes. Expressed both per cell and per cell surface area, basal lipogenesis was higher in weaned rats than in adult rats (P ⬍ 0.05; Table 1 and Fig. 2A). Also, maximum insulin-induced lipogenesis and insulin sensitivity was increased in weaned rats than adult rats (P ⬍ 0.05; Table 1 and Fig. 2, A and B). The contribution of insulin-induced lipogenesis to total lipogenesis was greater in rats than human

Human adipocytes exhibited marked age-dependent differences in lipogenesis. When expressed per cell surface area, basal lipogenesis was higher in adipocytes from infants and children than adipocytes from adults. Under the conditions used in this study, lipogenesis primarily reflects variation in glucose transport (24), and our results are in accordance with one previous report showing that basal glucose transport is higher in adipocytes from children than adults (19). In contrast to basal lipogenesis, insulin-induced lipogenesis declined to adult levels already during childhood. Thus, data indicated that the age-dependent variations in basal and insulin-induced lipogenesis are regulated by distinct mechanisms. Insulin-induced lipogenesis, expressed as a percentage of basal lipogenesis, was also higher in adults than children, which also supports this hypothesis. The adipocyte volume was much smaller in infants and children, and it is a methodological problem how to compare the metabolic activity in small and large fat cells (see Ref. 10 for a detailed discussion). It is well established that large adipocytes have higher metabolic activity than small cells when the metabolic activity is expressed per cell (10, 31). Despite that, there were no significant variations in lipogenesis in adipocytes from different human age groups when expressed per cell. This further emphasizes the high metabolic activity in adipocytes from infants and children. The observed age-dependent shift from predominantly basal to


FIG. 3. Western blot analysis of GLUT4 expression in adipocytes from infants, children, and adults. Adipocyte membrane protein preparation, electrophoresis, and immunoblotting were performed as described in Subjects and Methods. Adipose tissue was pooled from a group of three to four infants and three to four children per lane.

FIG. 4. Western blot analysis of GLUT1 and GLUT4 expression in adipocytes from weaned and adult male Fisher rats. Adipocyte membrane protein preparation, electrophoresis, and immunoblotting were performed as described in Subjects and Methods. Adipose tissue was pooled from a group of five weaned and one to two adult rats per lane.

insulin-induced lipogenesis in adults is independent of whether results are expressed per cell or cell surface area. The insulin-stimulated lipogenesis, expressed as a percentage of basal lipogenesis in adults, was similar to previous data (32–34). The sensitivity of insulin-induced lipogenesis was similar in all human age groups. This indicates that the observed difference in insulin-induced lipogenesis probably is a postbinding effect because an increased number of insulin receptors or increased insulin receptor binding should result in greater sensitivity. In accordance with these results, we previously demonstrated that the insulin receptor RNA expression as well as the sensitivity to the antilipolytic effect of insulin was similar in adipocytes from different age groups (22). In rats, both basal and insulin-induced lipogenesis and glucose transport in adipocytes were higher from weaned than adult rats. However, in contrast to human adipocytes, the insulin sensitivity was higher in weaned rats, indicating age-dependent differences also in insulin signaling. The contribution of insulin-induced lipogenesis to total lipogenesis was greater in rat than human adipocytes. In contrast to humans, the age-dependent differences were significant when expressed both per cell and per cell surface area, most probably because the increase in fat cell volume in young adult rats was only 25%, compared with the 3-fold increase in humans. It has previously been shown that GLUT1 and GLUT4 are under developmental control in human skeletal muscles (35, 36). Furthermore, the glucose transporters GLUT1 and GLUT4 expression have previously been shown to correlate to changes in glucose transport in man (37, 38) and could therefore be of importance for the differences in basal and insulin-induced lipogenesis observed in this study. Only GLUT4 protein levels were studied in humans due to the limited amounts of adipose tissue obtained from infants and children. We were not able to detect any difference in GLUT1

J Clin Endocrinol Metab, September 2004, 89(9):4601– 4606 4605

or GLUT4 protein expression, in neither human nor rat adipose tissue from the different age groups. This may be due to the limited sample size and the marked variation within each age group. Furthermore, whole adipose tissue was studied and the contribution of other cell types than adipocytes may also affect the results. However, it is also possible that factors involved in insulin-induced translocation of GLUT4 such as protein kinase B/Akt (39) are expressed differently during development. In neonates, adipose tissue forms approximately 11% of total body weight. By 4 months of age, 26% of the body weight is fat (40). Thus, under this time period, the weight of the adipose tissue increases 4-fold. This is due an increase in both adipocyte size and number (3). Between 1 and 8 yr of age, the total number of adipocytes progressively increases (2), and the mean adipocyte size decreases (30), probably due to the increased proportion of small, newly differentiated adipocytes from the preadipocyte pool. In young rats, compared with adult animals, there is also an increased recruitment of adipocytes from preadipocytes in the epidydimal fat pads (41– 43). In both humans and rats, a higher lipogenesis was found in adipocytes obtained during periods coinciding with an increase in adipocyte number. Thus, newly differentiated adipocytes may have an increased insulin-independent lipogenesis, which, in combination with a reduced catecholamine-induced lipolysis (12), favors adipocyte expansion. However, whether this reflects intrinsic properties of newly differentiated adipocytes or endocrine regulation is unknown. BMI decreases after 2 yr of life, and around the age of 6 yr, BMI usually rises again, the so-called adiposity rebound. An early adiposity rebound is thought to predispose adult obesity (6), and it has recently been shown that type 2 diabetes is associated with early adiposity rebound (44). It is possible that changes in lipolysis and lipogenesis are of importance for the age-dependent variations of BMI during the first years of life. In conclusion, we found higher unstimulated and insulininduced lipogenesis in both human and rat adipocytes obtained early in life. The underlying mechanism for this remains unclear because no difference in GLUT1 or GLUT4 protein expression was observed. Variations in lipogenesis may be of importance for age-dependent changes of adipose tissue growth and the development of childhood obesity. Acknowledgments Received June 9, 2003. Accepted May 20, 2004. Address all correspondence and requests for reprints to: Professor Dr. Claude Marcus, Department of Pediatrics, National Childhood Obesity Centre, Children’s Hospital, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden. E-mail: [email protected]. This work was supported by grants from the Swedish Medical Research Council (9941 and 11332), Karolinska Institute, the Frimurare Barnhuset Foundation, and the Sven Jerring Foundation.

References 1. Poissonnet CM, Burdi AR, Bookstein FL 1983 Growth and development of adipose tissue during early gestation. Early Hum Dev 8:1–11 2. Ha¨ger A, Sjo¨stro¨m L, Arvidsson B, Bjo¨rntorp P, Smith U 1977 Body fat and adipose tissue cellularity in infants: a longitudinal study. Metab Clin Exp 26:607– 614

4606


3. Knittle JL, Timmers K, Ginsberg-Fellner F 1979 The growth of adipose tissue in children and adolescents: cross-sectional and longitudinal studies of adipose cell number and size. J Clin Invest 63:239 –246 4. Poissonnet CM, Lavelle M, Burdi AR 1988 Growth and development of adipose tissue. J Pediatr 113:1–9 ¨ stman J 1971 Human adipose tissue. Dynamics and regulation. 5. Bjo¨ rntorp P, O Adv Metab Disord 5:277–327 6. Guo SS, Huang C, Maynard LM, Demerath E, Towne B, Chumlea WC, Siervogel RM 2000 Body mass index during childhood, adolescence and young adulthood in relation to adult overweight and adiposity: the Fels Longitudinal Study. Int J Obes Relat Metab Disord 24:1628 –1635 7. Marcus C, Ehren H, Bolme P, Arner P 1988 Regulation of lipolysis during the neonatal period: importance of thyroprotein. J Clin Invest 82:1793–1797 8. Janson A, Karlsson FA, Micha-Johansson G, Bolme P, Bro¨ nnegård M, Marcus C 1995 Effects of stimulatory and inhibitory thyrotropin receptor antibodies on lipolysis in infant adipocytes. J Clin Endocrinol Metab 80:1712–1716 9. Janson A, Rawet H, Perbeck L, Marcus C 1998 Presence of thyrotropin receptor in infant adipocytes. Pediatr Res 43:555–558 10. Marcus C, Karpe B, Bolme P, Sonnenfeld T, Arner P 1987 Changes in catecholamine-induced lipolysis in isolated human fat cells during the first year of life. J Clin Invest 79:1812–1818 11. Marcus C, Sellde´ n H, Rickardsson E, Lo¨ nnqvist PA, Bro¨ nnegård M, Arner P 1993 Lack of lipolytic response in infants after endotracheal intubation. Arch Dis Child 68:402– 404 12. Marcus C, Sonnenfeld T, Karpe B, Bolme P, Arner P 1989 Inhibition of lipolysis by agents acting via adenylate cyclase in fat cells from infants and adults. Pediatr Res 26:255–259 13. Rosenbaum M, Presta E, Hirsch J, Leibel R 1991 Regional differences in adrenoceptor status of adipose tissue in adults and prepubertal children. J Clin Endocrinol Metab 73:341–347 14. Marcus C, Bolme, P, Karpe B, Bro¨ nnegård M, Sellde´ n H, Arner P 1993 Expression of B1- and B2-receptor genes and correlation to lipolysis in human adipose tissue during childhood. J Clin Endocrinol Metab 76:879 – 884 15. Janson A, Marcus C 1997 The lipolytic effects of thyrotropin and isoprenaline are not affected by growth hormone in infant adipocytes. Horm Metab Res 29:164 –167 16. Pilch PF, Wilkinson W, Garvey WT, Ciaraldi, TP, Hueckstaedt TP, Olefsky JM 1993 Insulin-responsive human adipocytes express two glucose transporter isoforms and target them to different vesicles. J Clin Endocrinol Metab 77: 286 –289 17. Zorzano A, Wilkinson W, Kotliar N, Thoidis G, Wadzinski BE, Ruoho AE, Pilch PE 1989 Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J Biol Chem 264:12358 –12363 18. Kozka IJ, Clark AE, Reckless JPD, Cushman SW, Gould GW, Holman GD 1995 The effects of insulin on the level and activity of the GLUT4 present in human adipose cells. Diabetologia 38:661– 666 19. Nyberg G, Ha¨ ger A, Smith U 1977 Effect of age on human adipose tissue metabolism and hormonal responsiveness. Acta Paediatr 66:495–500 20. Moustaid N, Jones BH, Taylor JW 1996 Insulin increases lipogenic enzyme activity in human adipocytes in primary culture. J Clin Nutr 126:865– 870 21. Assimacopoulos-Jeannet F, Brichard S, Rencurel F, Cusin L, Jeanrenaud B 1995 In vivo effects of hyperinsulinemia on lipogenic enzymes and glucose transporter expression in rat liver and adipose tissue. Metabolism 44:228 –233 22. Kamel A, Norgren S, Ehren H, Hildingsson U, Marcus C 1997 Antilipolytic effect of insulin and insulin receptor messenger RNA expression in adipocytes of infants, children, and adults. Pediatr Res 41:563–567 23. Rodbell M 1964 Metabolism of isolated fat cells. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–380


24. Arner P, Engfeldt P 1987 Fasting-mediated alteration studies in insulin action on lipolysis and lipogenesis in obese women. Am J Physiol 253:E193–E201 25. Moody AJ, Stan MA, Stan M, Gliemann J 1974 A simple free fat cell bioassay for insulin. Horm Metab Res 6:12–16 26. Whitesell RR, Gliemann J 1979 Kinetic parameters of transport of 3-O-methylglucose and glucose in adipocytes. J Biol Chem 254:5276 –5283 27. Castello A, Rodriguez-Manzaneque JC, Camps M, Pe´ rez-Castillo A, Testar X, Palacin M, Santos A, Zorzano A 1994 Perinatal hypothyroidism impairs the normal transition of GLUT4 and GLUT1 glucose transporters from fetal to neonatal levels in heart and brown adipose tissue. Evidence for tissue-specific regulation of GLUT4 expression by thyroid hormone. J Biol Chem 269:5905– 5912 28. Hirsch J, Gallian E 1968 Method for the determination of adipose cell size and cell number in man and animals. J Lipid Res 12:91–95 29. Zinder Z, Shapiro B 1971 Effect of cell size on epinehrine and ACTH-induced fatty acid release from isolated fat cells. J Lipid Res 12:521–530 30. Marcus C 1988 Regulation of lipolysis in human adipocytes, a developmental study. Thesis, Karolinska Institutet, Stockholm 31. Holm G, Jacobsson H, Bjo¨ rntorp P, Smith U 1975 Effects of age and cell size on rat adipose tissue metabolism. J Lipid Res 16:461– 464 32. Hissin P, Foley JE, Wardzala LJ, Karnieli E 1982 Mechanism of insulinresistant glucose transport activity in the enlarged adipose cell of the aged, obese rat. Relative depletion of intracellular glucose transport systems. J Clin Invest 70:780 –790 33. Yki-Jarvinen H, Kiviluoto T, Nikkila EA 1986 Insulin binding and action in adipocytes in vitro in relation to insulin action in vivo in young and middle-aged subjects. Acta Endocrinol 113:88 –92 34. Di-Girolamo M, Rudman D 1967 Variations in glucose metabolism and sensitivity to insulin of the rat’s adipose tissue, in relation to age and body weight. Endocrinology 82:1133–1141 35. Gaster M, Handberg A, Beck-Nielsen H, Schro¨ der HD 2000 Glucose transporter expression in human skeletal muscle fibers. Am J Physiol Endocrinol Metab 279:E529 –E538 36. Sarabia V, Lam L, Burdett E, Leiter LA, Klip A 1992 Glucose transport in human skeletal muscle cells in culture. Stimulation by insulin and metformin. J Clin Invest 90:1386 –1395 37. Garvey TW, Huecksteadt TP, Matthaei S, Olefsky JK 1988 Role of glucose transporters in the cellular insulin resistance of type II non-insulin-dependent diabetes mellitus. J Clin Invest 81:1528 –1536 38. Garvey TW, Maianu L, Huecksteadt TP, Birnbaum MJ, Molina JM, Ciaraldi TP 1991 Pretransitional suppression of a glucose transporter protein causes insulin resistance in adipocytes from patients with NIDDM and obesity. J Clin Invest 87:1072–1081 39. Downward J 1998 Mechanisms and consequences of activation of protein kinase B/Akt. Curr Opin Cell Biol 10:262–267 40. Baker GL 1969 Human adipose tissue composition and age. Am J Clin Nutr 22:829 – 835 41. Bjo¨ rntorp P, Karlsson M, Pettersson P, Sypniewska G 1980 Differentiation and function of rat adipocyte precursor cells in primary culture. J Lipid Res 21:714 –723 42. Bjo¨ rntorp P, Karlsson M, Pettersson P 1982 Expansion of adipose tissue storage capacity at different ages in rats. Metabolism 31:366 –373 43. Djian P, Roncari DAK, Hollenberg CH 1983 Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J Clin Invest 72:1200 –1208 44. Eriksson JG, Forsen T, Tuomilehto J, Osmond C, Barker DJ 2003 Early adiposity rebound in childhood and risk of type 2 diabetes in adult life. Diabetologia 46:190 –194

JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.