OBJECTIVE: Lipids stored in adipose tissue can originate from dietary lipids or from de novo lipogenesis (DNL) from carbohydrates. Whether DNL is abnormal in adipose tissue of overweight individuals remains unknown. The present study was undertaken to assess the effect of carbohydrate overfeeding on glucose-induced whole body DNL and adipose tissue lipogenic gene expression in lean and overweight humans.
DESIGN: Prospective, cross-over study.
SUBJECTS AND METHODS: A total of 11 lean (five male, six female, mean BMI 21.0±0.5 kg/m2) and eight overweight (four males, four females, mean BMI 30.1±0.6 kg/m2) volunteers were studied on two occasions. On one occasion, they received an isoenergetic diet containing 50% carbohydrate for 4 days prior to testing; on the other, they received a hyperenergetic diet (175% energy requirements) containing 71% carbohydrates. After each period of 4 days of controlled diet, they were studied over 6 h after having received 3.25 g glucose/kg fat free mass. Whole body glucose oxidation and net DNL were monitored by means of indirect calorimetry. An adipose tissue biopsy was obtained at the end of this 6-h period and the levels of SREBP-1c, acetyl CoA carboxylase, and fatty acid synthase mRNA were measured by real-time PCR.
RESULTS: After isocaloric feeding, whole body net DNL amounted to 35±9 mg/kg fat free mass/5 h in lean subiects and to 49±3 mg/kg fat free mass/5 h in overweight subjects over the 5 h following glucose ingestion. These figures increased (P<0.001) to 156±21 mg/kg fat free mass/5 h in lean and 64±11 mg/kg fat free mass/5 h (P<0.05 vs lean) in overweight subjects after carbohydrate overfeeding. Whole body DNL after overfeeding was lower (P<0.001) and glycogen synthesis was higher (P<0.001) in overweight than in normal subjects. Adipose tissue SREBP-1c mRNA increased by 25% in overweight and by 43% in lean subjects (P<0.05) after carbohydrate overfeeding, whereas fatty acid synthase mRNA increased by 66 and 84% (P<0.05).
CONCLUSION: Whole body net DNL is not increased during carbohydrate overfeeding in overweight individuals. Stimulation of adipose lipogenic enzymes is also not higher in overweight subjects. Carbohydrate overfeeding does not stimulate whole body net DNL nor expression of lipogenic enzymes in adipose tissue to a larger extent in overweight than lean subjects.
Obesity is the result of lipid deposition in adipose tissue as a consequence of long-term positive energy and fat balance. The origin of the lipids stored in adipocytes can be from dietary lipids, de novo lipid synthesis from carbohydrates (de novo lipogenesis, DNL) or a mixture of both.
Under conditions of energy balance, DNL is active in hepatocytes, but remains quantitatively small.1 It can, however, be markedly stimulated when simple carbohydrates are overfed2 or by acute ethanol administration.3 In obese insulin-resistant patients, hepatic DNL is increased, and appears to be stimulated by hyperinsulinemia.4, 5 Interestingly, insulin has a dual signaling pathway in hepatocytes: it suppresses gluconeogenesis and glucose production but this effect is blunted in insulin-resistant individuals; in contrast, it stimulates hepatic glucose uptake and DNL through activation of SREBP-1c, a pathway that remains fully responsive in insulin-resistant subjects.6 This probably explains the impaired suppression of glucose production together with increased DNL observed in hyperinsulinemic insulin-resistant patients.4, 5, 7, 8
Adipose tissue has been shown to synthesize lipids de novo when studied in vitro. Furthermore, it was observed that DNL was more active in adipose tissue obtained from obese than lean subjects.9 The quantitative importance and physiological regulation of DNL in adipose tissue in vivo remains more elusive, due largely to the methodological limitations for measuring adipose tissue fat synthesis. Guo et al10 studied lean and obese individuals during uncontrolled energy intake, and reported that adipose tissue DNL, monitored by means of the incorporation of deuterated water into fatty acids, was very low. More recently, Strowford and collaborators used a novel approach to study adipose tissue kinetics in humans by monitoring deuterium incorporation into triglyceride and of adipocytes after a period of 5–9 weeks during which subjects drank deuterated water. They observed that DNL accounted for about 20% of newly deposited triglycerides after 9 weeks. The overall turnover of adipose tissue triglyceride was low, with a triglyceride half-life of 200–270 days.11 Owing to this slow turnover, the incorporation of label in adipose tissue triglyceride during short-term administration of labeled precursors is probably impractical to evaluate adipose tissue DNL.
It has long been documented, using indirect calorimetry, that carbohydrate overfeeding stimulates whole body net DNL.12, 13 More recently, Aarsland et al14 simultaneously monitored whole body net DNL with indirect calorimetry and hepatic DNL by incorporation of 13C-labeled acetate into VLDL-triglyceride. These authors reported that whole body net DNL far exceeded hepatic lipogenesis, suggesting that adipose tissue could be a major site of DNL under such conditions. We have recently reported that whole body DNL and the expression of lipogenic enzymes in adipose tissue were stimulated by carbohydrate overfeeding in lean male and female subjects, corroborating the lipogenic role of adipose tissue.15 However, Diraison et al16 reported that healthy subjects fed an isoenergetic or hyperenergetic diet had very little adipose DNL, evaluated from incorporation of either 13C-acetate or deuterated water into adipose tissue fatty acids. This suggests that monitoring tracer incorporation into adipose tissue lipids might be impractical due to the huge dilution of tracer in the large adipose triglyceride pool. The regulation of whole body and adipose tissue DNL in overweight human subjects has not been extensively investigated. The aim of this study was therefore to evaluate whether short-term carbohydrate overfeeding affects whole body DNL and the expression of lipogenic enzymes differently in adipose tissue of lean and overweight healthy humans.
Subjects and methods
A total of 11 lean subjects (five male, six female : mean age 27±1 y, mean BMI 21.0±0.5 kg/m2) and eight overweight subjects (four males, four females: mean age 29±1 y, mean BMI 30.1±0.6 kg/m2) were studied twice, after 4 days of isocaloric feeding and after 4 days of carbohydrate overfeeding. All subjects were in apparent good health and had no family history of obesity and/or diabetes mellitus. Anthropometric measurements (fat mass, fat-free mass (FFM)) were assessed by skinfold thickness measurement17 after each dietary intervention (Table 1). Female subjects were studied during the follicular phase of their menstrual cycle. The experimental data regarding four lean male and four lean female subjects have been reported previously.15 The expression of adipose tissue lipogenic enzymes of these subjects has been reassessed in the present study to ensure comparable methodology in both lean and overweight subjects. The study protocol was reviewed and approved by the Lausanne University School of Medicine Ethical Committee and written informed consent was obtained from all participants.
At inclusion in the study, resting metabolic rate (RMR) was measured in the morning after an overnight fast, using an open circuit indirect calorimeter (Deltatrac II, Datex, Finland). Daily energy requirements were calculated as RMR* 1.6, to account for usual physical activity. Each subject was instructed to follow a prescribed diet for two periods of 4 days, with an interval of at least 2 weeks for men and 4 weeks for women between dietary treatments. During one period, subjects received an isocaloric diet providing 50% carbohydrates (of which 58% complex carbohydrate and 42% mono- and disaccharides), 35% lipid and 15% protein as energy and during the second period of carbohydrate overfeeding, the subjects received the same basic diet to which an extra amount of carbohydrates, corresponding to 75% (of which 32% complex carbohydrates and 68% mono- and disaccharides) of their calculated energy requirements was added. The amount of protein and lipid consumed remained identical in the two conditions (Table 2).
All subjects consumed prepacked food items during the first 4 days and were instructed not to consume any other food or beverages except for water. The two meals preceding the metabolic studies were taken at the Department of Physiology to ensure compliance. The prepacked foods consisted of pasta, rice, beans, sweetcorn, milk, cheese, yoghurt and canned fruits.
On the morning of the fifth day, following each dietary intervention, the subject came to the laboratory at 0800 hours after an overnight fast. Body weight and skinfolds were measured. The subject lay in bed in a semirecumbent position until the end of the test. A venous catheter (Venflon) was inserted into an antecubital vein for collection of hourly blood samples. Before each blood sample, the subject's hand was heated in a warming box to achieve partial arterialization of venous blood. The first blood collection was performed at 1000, that is, after a 2-h rest and indirect calorimetry was started at the same time. Respiratory exchange monitored continuously throughout the experiment by means of a Deltatrac II (Datex Instruments, Helsinki, Finland), using a ventilated hood to collect expiratory gases. Energy expenditure, respiratory quotient (RQ), substrate oxidation and whole body net DNL were calculated using the equations of Livesey and Elia.18 Oral glucose was administered at mid-day (1.625 g/kg FFM), at 1400 hours (0.8125 g/kg FFM), and again at 1500 hours (0.8125 g/kg FFM). This fractional mode of administration was chosen to avoid gastrointestinal side effects. Indirect calorimetry was continued until 1700 hours. At the end of the test, a subcutaneous adipose tissue sample was obtained by needle biopsy, as previously described.19 Tissues were immediately frozen in liquid nitrogen and stored at −80°C until mRNA extraction. Urine was collected before, during, when necessary, and at the end of each metabolic study and was analyzed for total urinary urea excretion.
Plasma glucose concentrations were measured with a glucose-lactate analyzer YSI 2300 STAT plus (Yellow Springs, OH, USA). Plasma insulin (kit from Biochem Immunosystems GmbH, Freiburg, Germany) and leptin (kit from Linco, St Charles, MO, USA) were determined by radioimmunoassay. Plasma free fatty acids (FFA) and triglyceride (TG) concentrations were analyzed by a colorimetric method using kits from Wako (Freiburg, Germany) for FFA and from Biomérieux (Biomérieux Vitek Inc., Switzerland) for TG.
Analysis of messenger RNA in adipose biopsy
Total RNA was extracted from adipose tissue biopsies using the RNeasy total RNA minikit (Qiagen, Courtaboeuf, France). First-strand cDNAs were first synthesized from 250 ng total RNA in the presence of 100 U of Superscript II (Invitrogen, Eragny, France) using both random hexamers and oligo (dT) primers (Promega, Charbonnières, France). Real-time PCR was performed using a LightCycler (Roche Diagnostics, Meylan, France) in a final volume of 20 μl containing 5 μl of a 100-fold dilution of the RT reaction and 15 μl of reaction buffer from the FastStart DNA Master SYBR Green kit (Roche Diagnostics, Meylan, France) with 3 mM of MgCl2 and the specific forward primers. After amplification, a melting curve analysis was performed to verify the specificity of the reactions. For quantification, a standard curve was systematically generated with six different amounts (150–30 000 molecules/tube) of cDNA of the human target mRNA cloned in the pGEM plasmid (Promega). The expression level of a reference gene (cyclophilin) was measured in all samples using the specific forward (5′-IndexTermGCCATGGAGCGCTTTGG-3′) and reverse (5′-IndexTermCCACAGTCACGAATGGTGATC-3′) cyclophilin primers. Values were expressed as the ratio of target mRNA to cyclophilin mRNA. The analysis was performed using the LightCycler software (Roche Diagnostics, Basel, Switzerland).
Calculation of substrate oxidation and whole body DNL
Substrate oxidation and energy expenditure were calculated from respiratory gas exchange and urinary nitrogen excretion, as described in detail elsewhere.20 Whole body nonprotein RER>1.0 indicates net DNL. Under such conditions, fat oxidation rates becomes negative and corresponds to net DNL, while net carbohydrate oxidation calculated using standard equations reflects the sum of glucose oxidized and glucose converted into fat. Under such conditions, carbohydrate oxidation can be calculated as:21
where VCO2 is carbon dioxide production in ml/min and N is urinary nitrogen excretion in mg/min. For all these calculations, urinary nitrogen excretion was assumed to be equal to (urinary urea nitrogen excretion)/0.85.
Glycogen synthesis was calculated as glucose intake −glucose oxidation −glucose converted into fat.
All data are presented as mean ±s.e.m. A two-way analysis of variance (ANOVA) was performed for statistical analysis. Post hoc analysis was performed using Fisher's PLSD test for blood parameters, whole body DNL, and substrate oxidation. Comparison of gene expression between the two groups of subjects was performed by the Mann–Whitney U-test. The effect of dietary interventions on gene expression was evaluated by means of Wilcoxson's signed rank test. All statistical analyses were conducted using the Statview Statistical Package (The SAS Institute Inc., NC, USA).
Body weight and composition
Carbohydrate overfeeding for 4 days significantly increased body weight in lean and overweight subjects (Table 1).
Plasma glucose and insulin concentrations
Compared with isocaloric conditions, 4 days of carbohydrate overfeeding did not alter fasting plasma glucose nor insulin concentrations in either lean or overweight subjects (Table 2). However, fasting insulin concentrations after overfeeding was significantly higher in overweight than in lean subjects (P=0.0003). After oral glucose, the increases in plasma glucose and insulin concentrations were similar after carbohydrate overfeeding and isocaloric feeding in both groups (Figure 1). However, when compared with lean subjects, the overweight had significantly higher glycemic (P<0.05) and insulinemic (P<0.01) responses to oral glucose.
Plasma lipid parameters
Carbohydrate overfeeding significantly lowered fasting plasma FFA concentrations in both lean and overweight subjects (P<0.05, Table 3). After glucose administration, plasma FFA concentrations were suppressed in both groups of subjects under each dietary condition (Figure 2). Fasting plasma TG was significantly increased after 4 days of carbohydrate overfeeding in both groups (P<0.01). Overweight subjects had higher plasma TG values throughout the metabolic studies, both after isocaloric diet and carbohydrate overfeeding.
Substrate utilization and whole body net DNL
Changes in respiratory exchange ratio (RER) are illustrated in Figure 3. With overfeeding the RQ increased and exceeded values of 1.0 after glucose ingestion in both overweight and lean individuals, indicating that whole body net DNL was stimulated. However, compared with lean subjects, the overweight subjects had smaller increases in RER throughout the test. Although a significant increase in net DNL was observed in both groups (P<0.001), it was less in the overweight individuals than the lean (0.001, Figure 4). After 4 days of isocaloric diet, a higher rate of lipid oxidation was observed in the overweight than in lean subjects (167±32 vs 90±12 mg/kg FFM/5 h, P<0.05). After carbohydrate overfeeding, however, lipid oxidation rate was suppressed in both groups, and no statistical difference was observed (overweight vs lean: 29±8 vs 5±2 mg/kg FFM/5 h). Carbohydrate oxidation after glucose ingestion was increased in both groups after overfeeding, however overweight subjects had lower rates of oxidation compared to lean subjects (lean: 7.6±0.2 vs 10.1±0.3; obese: 5.9±0.3 vs 8.7±0.4 mmol/kg FFM/5 h). The amount of glucose stored as glycogen (ie, nonoxidative glucose disposal corrected for DNL) was higher in overweight than in lean subjects (P<0.05) (Figure 5). Overfeeding reduced net protein oxidation from 38.9±2.5 to 27.8±2.3 mg/min (P<0.001) in lean and from 44.5±4.9 to 36.1±2.7 mg/min in overweight (P<0.05).
Lipogenic gene expression
Overfeeding increased the expression of SREBP-1c mRNA by 25 and 43% and FAS mRNA by 66 and 84% in adipose tissue of overweight and lean subjects, respectively (P<0.05. in all cases) (Figure 6). The expression of ACC mRNA was not altered by overfeeding in either group. FAS mRNA levels were significantly lower in overweight than in lean individuals patients after overfeeding (P<0.001). Otherwise gene expression did not differ between overweight and lean subjects.
Our present results corroborate earlier reports that carbohydrate overfeeding markedly stimulates whole body net DNL in healthy humans.12, 13 Recently, Aarsland et al14 proposed that the bulk of DNL during carbohydrate overfeeding took place in extrahepatic tissue. This hypothesis was further supported by our recent observation that the expression of SREBP-1c and FAS increased in adipose tissue of overfed lean humans.15
Although the glucose meals stimulated whole body net DNL in the overweight individuals, it was stimulated less than in the lean. This is, to our knowledge, the first report of decreased whole body DNL in overweight, overfed individuals. A previous study indicated that whole body net DNL was comparable in obese and lean individuals after ingestion of a single large carbohydrate meal.22 In another study,23 macronutrient disposal estimated by means of a calorimetric chamber was similar in lean and obese women during carbohydrate overfeeding. There was, however, no net DNL in either group of subjects when estimated over 24 h, even though excess carbohydrate intake amounted to 50% of energy requirements.23 Since indirect calorimetry provides estimates of net DNL (ie, total DNL in excess of fat oxidation), it is possible that our figure of net DNL reflects a higher rate of lipid oxidation rather than a decreased rate of net DNL in overweight patients. Overweight subjects, however, had impaired suppression of plasma free fatty acids after glucose loading. This may have contributed to lower net DNL in overweight subjects since fatty acids inhibit the expression of lipogenic enzymes.24
Several studies have addressed the expression of lipogenic enzymes and of the key transcription factor SREBP-1c in obese vs lean individuals. They reported that the expression of key lipogenic genes was significantly decreased in adipose tissue biopsies collected in overweight fasted individuals.16, 25 Our present protocol, by assessing the effect of overfeeding on postprandial adipose tissue lipogenic gene expression, brings new insights regarding the effects of diet on adipose tissue in overweight individuals. SREBP-1c, FAS, and ACC gene expression tended to be lower in adipose tissue obtained after a glucose meal in overweight vs lean subjects. Carbohydrate overfeeding for 4 days led to a significant stimulation of SREBP-1c and FAS mRNA in both lean and overweight subjects. Moreover, the percent stimulation was comparable in lean vs overweight subjects. The increase in ACC mRNA levels did not reach statistical significance in any of the two groups, possibly due to the too short duration of overfeeding. Although we cannot infer the rate of lipogenesis from gene expression profiles, these data strongly suggest that stimulation of adipose tissue DNL occurred in both lean and overweight subjects. In addition to a lower rate of net DNL, overweight patients also displayed a higher rate of nonoxidative glucose disposal, which corresponds essentially to net glycogen synthesis, since carbohydrates converted into lipids are computed as glucose oxidation.26 It indicates therefore that obese patients have an increased glycogen storage capacity.
The glucose load was administered based on lean body mass rather than total body weight because it is well recognized that skeletal muscle is the major locus of glycogen synthesis after carbohydrate loading.27 Consequently, we hypothesized that overweight and lean subjects would have the same absolute muscle glycogen storage capacity since they received the same glucose load per kg lean body weight. The present data do not allow us to evaluate whether the increased glycogen storage observed in overweight subjects took place in skeletal muscle (possibly due to lower fasting glycogen concentrations in overweight subjects) or in other tissues or organs). Owing to their higher fat mass, the glucose not metabolized in skeletal muscle was distributed to a larger adipose tissue mass in overweight than in lean subjects. This may possibly explain a larger storage as adipose tissue glycogen although this explanation remains hypothetical. This may also account for a lesser stimulation of DNL and lipogenic enzymes in overweight subjects on a fat mass basis. Based on this higher glycogen storage and lower DNL in overweight individuals, it can be hypothesized that whole body DNL is actually stimulated only when glycogen storage capacity is saturated.13 According to this scheme, a higher glycogen storage capacity may account for a lower net DNL in overweight individuals.
Plasma triglyceride concentrations were significantly higher in the overweight compared with the lean subjects, both under isoenergetic diet conditions and after carbohydrate overfeeding. Overfeeding increased plasma triglyceride concentrations in both groups, but the absolute increase was more important in overweight individuals. Both a stimulation of hepatic DNL and a decrease in VLDL-triglyceride clearance may be responsible for this effect of carbohydrate overfeeding.28, 29
In this study, carbohydrate overfeeding corresponded essentially to supplementary mono- and disaccharides. It has been reported, in healthy humans, that a high carbohydrate isocaloric diet stimulated hepatic DNL when carbohydrates were provided as simple sugars and disaccharides, but not as starchy foods.30, 31 The effects of simple vs complex carbohydrates on whole body- and extrahepatic DNL remain presently unknown, but it is quite possible that our observations reflect the consequences of high mono- and disaccharides intake rather than carbohydrates per se.
In conclusion, our data indicate that whole body net DNL is stimulated by 4 days of carbohydrate overfeeding in both lean and overweight individuals. In both groups, SREBP-1c and FAS gene expressions were concomitantly increased in adipose tissue biopsies, suggesting that adipose tissue was actively involved in carbohydrate-induced DNL. Whole body net DNL was, however, less in overweight patients. Furthermore, in overweight patients, hyperinsulinemia was not associated with an increased expression of lipogenic markers. This strongly argues against increased adipose tissue DNL in overweight individuals. The present data were collected after ingestion of a pure glucose meal and cannot be extrapolated to everyday conditions where mixed meals are ingested. This, nonetheless, supports the hypothesis32 that the increase in fat mass that occurs during the dynamic phase of obesity is essentially secondary to deposition of exogenous, dietary fats.
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This work was supported by a grant (# 3200-067787.02) from the Swiss Science Foundation and the Désireé and Niels de Foundation.
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