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Karolinska Institutet, Clinical Research Center and Department of Medicine, Karolinska University Hospital, SE-141 86 Huddinge, Stockholm, Sweden
Address all correspondence and requests for reprints to: Peter Arner, Professor, M.D., Ph.D., Department of Medicine, Karolinska University Hospital Huddinge, SE-141 86 Huddinge, Stockholm, Sweden. E-mail: peter.arner{at}medhs.ki.se.
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Abstract |
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Objective: The objective of this study was to investigate fat cell size and leptin production in the basal state after long-term steady-state weight reduction to the nonobese state.
Design: This prospective case-control study had a duration of 3 ± 1 (mean ± SD) yr.
Patients: Twenty-five obese women (cases) were studied. Each case was compared with a control subject matched for age, sex, and body mass index (BMI) at nadir of weight for the cases.
Setting: This study was conducted at Karolinska University Hospital (Stockholm, Sweden).
Intervention: The subjects were followed until they reached a steady-state weight reduction after lifestyle modification or bariatric surgery (cases). Treatment target was the nonobese state (BMI < 30 kg/m2). Subcutaneous adipose tissue secretion of leptin, serum leptin levels, and fat cell volume were determined after an overnight fast.
Results: Ten obese women (40%) reached the nonobese state. This was accompanied by marked decreases in fat cell volume, leptin secretion, and serum leptin concentrations (P < 0.0001). The postobese cases had 43% smaller fat cell volume (P = 0.0008), 68% lower adipocyte leptin production (P = 0.001), and 54% lower serum leptin levels (P = 0.0007) than control subjects, despite almost identical percent body fat in the two groups. Fat cell volume, but not percent body fat or BMI, was directly proportional to leptin secretion and serum leptin concentrations.
Conclusion: Adipose tissue hyperplasia (too many small fat cells) and low leptin production resulting in relative hypoleptinemia in the fasting (basal) state are common features of the postobese state in women.
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Introduction |
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The relationship between leptin levels and the energy balance is complex as reviewed (9, 10, 11). Leptin stimulates energy expenditure and inhibits appetite. Humans lacking functional endogenous leptin develop severe obesity that can be successfully treated with exogenous leptin administration (12, 13). However, most obese subjects are hyperleptinemic, indicative of a leptin-resistant state. Indeed, exogenous leptin doses producing a circulating leptin concentration more than 10 times the normal give only a small average decrease in the body weight of obese subjects (14). On the other hand, the decrease in leptin after weight loss is disproportional to changes in adiposity (15, 16, 17, 18), and low-dose leptin has significant effects on energy expenditure in body weight-reduced subjects (5). As discussed, the latter data suggest that relative leptin deficiency is present in the body weight-reduced state (10, 19). Although energy expenditure decreases after weight reduction (2), and the decrease in leptin during energy restriction is related to appetite (16), there is no direct proof of leptin deficiency after weight reduction, and the possible mechanism remains unknown.
To determine whether leptin production in the basal state and adipose tissue cellularity become normal or not when obesity is successfully treated to the nonobese (i.e. normal) state, we conducted a prospective and controlled study on obese women who were followed for up to 5 yr until they reached a new steady-state weight after bariatric surgery or lifestyle modification. These individuals were compared with subjects matched for body mass index (BMI) and age at the weight-reduced state. The treatment target was to reach the nonobese state (BMI < 30 kg/m2). Leptin production and serum leptin after an overnight fast were determined and set in relation to adipose tissue cellularity because there is a strong relationship between fat cell size and leptin production (6, 20, 21). The study was designed in 1997 and started in 1998. The last subject was included in 2002.
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Subjects and Methods |
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This study was designed as a prospective long-term case-control study of obese women before and after they had reached a new steady-state BMI level after weight reduction therapy. Our target was to reach the postobese state (BMI < 30 kg/m2). During 1998–2002, we recruited 25 obese otherwise healthy women with BMI of 31–50 kg/m2 and age 30–50 yr for leptin and adipose cellularity studies who were planned for nonpharmacological weight reduction therapy and defined as cases. All cases were subjected either to adjustable vertical gastric banding (n = 18) or to lifestyle modification (increased motivation, altered eating habits, increased exercise, and regular follow-up) (n = 7). The lifestyle group was followed for 3, 6, 9, and 12 months when they were reexamined. During the intermediate check-ups (3, 6, and 9 months), body weight was recorded, and the women were interviewed for eating habits, exercise habits, and motivation. All women kept to the lifestyle motivation program. At 12 months, body weight was stable (< ±1 kg) as compared with values obtained at the 9-month examination. The subjects undergoing gastric banding were followed yearly until body weight had decreased to nadir and was stabilized (<±1 kg) for at least 3 months according to self-report. Then they were reexamined.
As soon as the second examination of a case was decided, a control subject was selected for each case. The control was closely matched for the BMI and age of the case at the second examination. The controls were selected from an ongoing study of the regulation of human adipose tissue function in healthy subjects with large interindividual variation in BMI (22). From 1997 when recruitment started, we had recruited 255 control subjects. None of the controls had undergone an important weight change the last 6 months before the examination according to self-report. The nonobese controls had never had BMI > 30 kg/m2 according to self-report of maximum body weight. When selecting the control subject, the one in charge of the selection (P.L.) only had access to data on BMI, age, and waist to hip ratio. The selected controls were 15 women who were obese (BMI, 30–50 kg/m2; age, 28–60 yr) and 10 women who were nonobese (BMI, 21–29 kg/m2; age, 29–55 yr). On a subset of nine women, three biopsies were performed, two at different times during reduced steady-state weight (2.8 ± 0.7 and 3.9 ± 0.6 yr, respectively). None of the cases or control subjects was on continuous pharmacotherapy. None was completely sedentary or involved in athletic performance. The hospital’s committee of ethics approved the study. The study was explained to each subject, and informed consent was obtained.
Experimental protocol
The subjects came to the laboratory at 0700–0800 h after an overnight fast. Height, weight, and waist and hip circumferences were measured. Percent body fat was measured by bioimpedance (Tanita TBF 305, Tanita Corporation, Tokyo, Japan). In a methodological investigation, performed on a separate population of women with similar characteristics as the present population, 38 subjects (BMI, 17–41 kg/m2) were investigated, and bioimpedance data were compared with dual-energy x-ray absorptiometry (DEXA). A strong (r = 0.92) relationship between the measures was obtained. The slope was not different from 1.0, but the intercept was +5% for DEXA (P = 0.027). Among nonobese, the correlation was r = 0.93, and among obese, it was r = 0.86. A venous blood sample was obtained for the determination of plasma glucose by the hospital’s routine chemistry laboratory for determination of plasma insulin by RIA (Pharmacia, Uppsala, Sweden) and for serum leptin using the Linco RIA (Linco, St. Charles, MO) as described (21). Thereafter, an abdominal sc adipose tissue specimen was obtained under local anesthesia using a needle biopsy technique (23). One portion of adipose tissue was subjected to collagenase treatment, and the mean size and weight of the isolated fat cells were determined as previously described (24). In brief, the isolated adipocytes were filtered through a 250-µm nylon mesh and washed three times with 10–20 ml of an albumin glucose (AGB)-containing buffer solution (pH 7.4), described before (21) to remove collagenase from the medium. The cells were resuspended in the AGB solution to a final concentration of 5%. Fat cell size was determined by placing an aliquot of the cell suspension on a siliconized glass slide and measuring the diameter of 100 cells using a microscope at 256x magnification. The ocular was equipped with a scale graded in 100 parts each corresponding to 5 U (5 x 0.616 µm). The mean diameter and SD were calculated, and average fat cell weight was determined according to the formulas developed by Hirsh and Gallian (25).We have previously evaluated the method to determine fat cell size and number (26). About 300 mg of the remaining adipose tissue was cut into 
20-mg pieces and used to determine leptin release exactly as described (21). In brief, tissue was incubated in the AGB medium for 2 h at 37C in a shaking water bath with air as the gas phase. Medium was removed and analyzed for leptin. Total leptin release to the incubation medium per 2 h per 107 fat cells was calculated as described (21).
Statistical methods
The a priori planned comparison was between postobese subjects and their matched controls (target treatment analysis). We also investigated the whole participant population (intention-to-treat analysis) and a separate subgroup that underwent surgery. In total, three different group comparisons were made. P values are not adjusted for multiple testing. Values are means ± SD. They were compared by Student’s paired t test in obese before vs. after weight reduction and, unless otherwise stated, by Student’s unpaired t test in obese after weight reduction controls. Leptin values were 10 log transformed before analysis.
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Results |
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). In both groups, the average BMI of gastric banding subjects was reduced to a steady-state level after 2–5 yr. The same was true for BMI at 12 months in the lifestyle group. On average, the second biopsy in the gastric banding group was performed after 2.9 ± 1.0 yr in those not reaching target and after 2.3 ± 0.7 yr in those reaching target.
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shows the measured parameters in the participant population. In the obese, the values for all parameters were significantly lower after compared with before weight reduction (P < 0.005). When weight-reduced subjects and controls were compared, fat cell volume was 27% lower (P < 0.0002) in cases compared with controls. The fat cell size distribution plot in Fig. 2 reveals an even distribution of fat cell size in the whole participant population after intervention and their controls. As expected, the size distribution plot is slightly shifted to the left for the obese subjects after weight loss compared with their controls. Furthermore, the coefficient of variation for fat cell diameter determined in each subject as SD/mean did not differ between postobese and controls (13 ± 2 and 14 ± 2% variation, respectively). All other parameters showed no significant difference between these two groups including body fat distribution measured as waist and hip.
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. As in the whole participant population, all parameters decreased markedly after weight reduction (P < 0.005). When reaching target, the postobese group had values for serum leptin, adipocyte leptin production, and fat cell volume that were 54, 68, and 43% lower, respectively, than in controls (P = 0.0007, 0.001, and 0.0008, respectively), although cases and controls had almost identical percent body fat, BMI, and body fat distribution.
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). As can be seen (comparing with data in Table 1
), they are slightly more obese than the control group but otherwise do not differ in baseline characteristics. The outcome of this comparison was the same as in all postobese (Table 2).
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by comparing body fat percentage or fat cell volume, on the one hand, and leptin secretion or serum leptin, on the other hand (Fig. 3). When mean values for obese before and after weight reductions to the nonobese state and for controls were plotted together, there was a near-linear relationship between fat cell volume and serum leptin or adipocyte leptin secretion. However, this linear relationship was not present when percent body fat or BMI (graph not shown) was related to serum leptin or leptin secretion. The linear relationship between leptin secretion and fat cell volume in individual subjects is demonstrated in Fig. 4, showing the postobese women before and after weight reduction plus their control subjects.
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).
On nine of the obese undergoing gastric banding, it was possible to make three biopsies, two at different times during the period of reduced steady-state weight (Fig. 5). When examined at 2.8 ± 0.7 and 3.9 ± 0.6 yr, BMI, body fat percentage (data not shown), fat cell volume, and serum leptin and adipocyte leptin production were reduced as compared with before gastric banding. However, the values at examinations two and three did not differ between each other.
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Discussion |
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In the whole study group (those not reaching treatment target plus those becoming postobese), weight reduction decreased serum leptin to levels slightly lower than in controls, whereas adipocyte leptin production was not significantly different from control subjects. However, these leptin results are difficult to interpret because some of the obese controls might have weighed differently previously, although no important changes in body weight were recorded at least 6 months before the investigation. Furthermore, data in the whole group of cases are obscured by findings in those obese subjects who lost very little weight.
The differences in leptin between weight-reduced cases, and their controls were observed despite the fact that the two groups had similar body fat content and BMI. We estimated body fat by bioimpedance, which might be less accurate than more elaborate methods. However, the bioimpedance method is accurate when nonobese subjects are studied and is therefore relevant for the postobese cases and their controls. Furthermore, we found strong correlations between bioimpedance and DEXA, which is the golden standard method. Although there is a slight underestimation of body fat with bioimpedance as compared with DEXA in our methodological investigation the slope of the relationship between the two measures is near 1.0, indicating that the underestimation of body fat by bioimpedance is constant over the whole range of bodyweights that were examined. Finally, we obtained similar results using body fat or BMI.
A previous cross-sectional study on circulating leptin in the fasting state showed an increased hormone concentration among postobese subjects (6). However, this could probably be explained by more body fat (and 2 kg/m2 higher BMI) among the postobese than their control subjects.
One might argue that our postobese subjects were not in steady state at the second examination and therefore had leptin deficiency after an overnight fast. However, a negative energy balance is excluded as an important factor because body weight during the 3 months before the second investigation in the postobese was constant (<±1kg weight change). Furthermore, it is apparent that the magnitude of leptin reduction is constant as long as body weight remains reduced at a steady-state level. Obese, who were examined at two different occasions more than 1 yr apart in a steady-state body weight-reduced condition, had almost identical leptin production and serum leptin concentrations at the two examinations. On the other hand, it is possible that the weight-reduced women, even though in energy balance, consumed significantly fewer calories to maintain their reduced body weight than the control subjects. It has been reported that 5 yr after weight reduction (average = 30 kg), female subjects enrolled in the National Weight Control Registry who are maintaining a reduced body weight consumed only 1306 kcal/d, 26% less than the energy consumption of women of comparable age reported in the National Health and Nutrition Examination Survey III (27). Thus, a difference in energy intake and expenditure between the subjects in the weight-reduced and control groups could play a role in the disturbed relationship between leptin and BMI/body fat. Because of logistic and cost barriers, we were unable to record energy intake, physical activity, and metabolic rate in this study.
We also observed that the size of abdominal sc fat cells is much smaller in weight-reduced subjects than in control subjects who have the same BMI and percent body fat as the weight-reduced cases. Subcutaneous fat is by far the largest adipose region and, thus, represents most of the measured body fat. Furthermore, the variations in fat cell size within this depot are relatively small, as reviewed (28). It is therefore most likely that body weight-reduced obese subjects have more sc fat cells than matched control subjects (in a steady state for body weight), i.e. they have adipose hyperplasia. Hyperplasia could be a primary defect among the obese, or it could have developed during weight gain and remained after weight reduction.
It is very likely that the findings with fat cell size and leptin among postobese subjects are linked. Cross-sectional studies of man and mice show that the positive correlation between adipocyte leptin production (mRNA and protein secretion) and fat cell volume is not disrupted by adipocyte hypertrophy in obesity (21, 29). When we compared obese before and after weight reduction with a postobese state with the controls, a linear relationship between fat cell volume and basal adipocyte leptin production was observed among all three groups of data. The same tight relationship was observed between fasting circulating leptin levels and fat cell size among the three groups. No such relationship was observed between leptin and body fat content or BMI. It is unlikely that leptin regulates adipocyte cellularity. Indeed, obese mice lacking functional leptin have as large fat cells as mice with dietary-induced obesity and functional leptin (29). Therefore, it is very probable that hypoleptinemia in the fasting state is secondary to decreased leptin production that, in turn, is secondary to small fat cells among the postobese. Importantly, leptin production is much higher in sc adipose tissue than in visceral adipose tissue, as reviewed (30). Therefore, a change in basal adipocyte leptin production in sc fat tissue, which also is the body’s largest adipose region, has a paramount influence on the fasting circulating leptin level. We cannot, however, exclude that other factors not measured here such as leptin clearance from the circulation have contributed to the disrupted relationship between leptin and BMI/body fat.
We only studied basal in vitro leptin secretion from adipocytes incubated in the absence of insulin and the fasting circulating leptin level. Leptin is subjected to diurnal variations and is dependent on insulin effects after meals (9, 10). The proportional amplitude (percent increase of diurnal leptin levels) is blunted in obese subjects compared with that in lean subjects (31). The effect of insulin-stimulated adipocyte glucose metabolism is likely to contribute to increased leptin secretion (32, 33). However, large adipocytes are more resistant to insulin action than are small adipocytes (34). Therefore, circulating insulin levels during postabsorptive hyperinsulinemia might be similar in postobese and control subjects. In the latter, insulinized situation leptin secretion from the relatively larger fat cells of the control subjects could be lower than leptin secretion from the relatively smaller fat cells of the postobese, thus opposite to the investigated fasting state.
A major problem with obesity treatment is the frequent regain of lost weight after antiobesity therapy. The mechanisms behind this weight gain are not well understood but could involve leptin deficiency in the fasting state causing decreased energy expenditure and increased appetite. The reverse pattern is seen in patients with anorexia nervosa, where initially hypoleptinemia after refeeding to target weight is followed by hyperleptinemia, resulting in decreased appetite and increased energy expenditure (35). Studies of leptin deficiency due to lipodystrophy suggest that leptin levels around 4ng/ml might be a threshold below which leptin supplementation might have positive biological effects (36). Four of our 10 postobese women had leptin levels equal to or below this threshold. Thus, as many as 40% postobese women might have clinically significant leptin deficiency in the fasting state presuming our cohort is representative for the common cases of obesity. Furthermore, the 4 ng/ml threshold from the studies on lipodystrophy (36) could be different in our obese, postobese, and control subjects. It is possible that obese subjects over time adapt to high leptin concentrations and that a decrease of circulating leptin after weight loss might induce hunger and lower metabolic rate even if they are not below the above-suggested threshold. On the other hand, fasting leptin is probably only important for the energy balance between meals and overnight.
The importance of the proportional amplitude of the diurnal leptin pattern in the regulation of energy balance is suggested by the results of a study in which the subjects consumed an ad libitum low-fat diet for 12 wk. Although there was a reduction of absolute plasma leptin levels, the decreases of body weight and fat mass were well correlated with the effects of the low-fat/high-carbohydrate diet to increase or maintain the proportional amplitude of the 24-h leptin pattern (37). Unfortunately, we had no resources to measure the diurnal leptin profile during a day of standardized meals in this study.
Adipose hyperplasia could be another important factor behind the difficulty for obese to maintain their body weight in the weight-reduced state. Hypercellularity was present in the whole study group of weight-reduced obese, regardless of whether they became postobese or not. It is possible that the small fat cells of weight-reduced women have a more pronounced ability to accumulate lipids than the larger cells of control subjects.
We made two subgroup analyses in addition to the main analysis. However, the findings with leptin and fat cell volume will remain statistically significant after adjustment for multiple testing.
In conclusion, weight reduction of obese subjects leads to a hyperplastic sc adipose tissue (many small fat cells despite a normal fat mass), which probably explains the low basal leptin production and thereby the relative reduction in fasting leptin concentration in the circulation in relation to body fat content in the postobese state.
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Acknowledgments |
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Footnotes |
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First Published Online August 30, 2005
Abbreviations: AGB, Albumin glucose; BMI, body mass index; DEXA, dual-energy x-ray absorptiometry.
Received March 17, 2005.
Accepted August 22, 2005.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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