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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 3 869-872
Copyright © 1999 by The Endocrine Society

Original Studies

Can Changes in Plasma Insulin Concentration Explain the Variability in Leptin Response to Weight Loss in Obese Women with Normal Glucose Tolerance?1

Marcello Carantoni, Fahim Abbasi, Salman Azhar, Patricia Schaaf and Gerald M. Reaven

Stanford University School of Medicine, Stanford, California 94305; Geriatric Research, Education, and Clinical Center, Veterans Administration Palo Alto Health Care System, Palo Alto, California 94304; and Shaman Pharmaceuticals, Inc., South San Francisco, California 94080

Address all correspondence and requests for reprints to: Gerald M. Reaven, M.D., Shaman Pharmaceuticals, Inc., 213 East Grand Avenue, South San Francisco, California 94080-4812. E-mail: greaven{at}shaman.com


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The aim of this study was to test the hypothesis that the fall in circulating insulin concentration associated with moderate weight loss determines the associated decrease in plasma leptin concentration. For this purpose, 12 healthy, nondiabetic, obese women were studied before and after an average weight loss of 9.5 kg (11.2% of initial body weight). Plasma leptin concentrations fell from a mean (±SE) value of 35 ± 3 to 17 ± 2 ng/mL (P < 0.001) in association with the loss of weight. However, there was no correlation between the decline in leptin concentration and the associated fall in weight, body mass index, fat mass, or percent body fat. Furthermore, no correlation was seen among changes in fasting plasma glucose or insulin concentrations, the 8-h integrated plasma glucose response to breakfast and lunch, or the estimate of insulin-mediated glucose disposal. The only measured variable that correlated with the fall in plasma leptin concentration (r = 0.78; P < 0.005) was the decline in the 8-h integrated plasma insulin response after weight loss (from 304 ± 44 to 232 ± 36 µU/8 h·mL; P < 0.001). Finally, multivariate regression analysis, using various estimates of degree of obesity, insulin resistance, integrated glucose response, and integrated insulin response as dependent variables, indicated that only the insulin response was independently related to the decrease in leptin concentration (P = 0.035). The fall in integrated insulin response accounted for 66% of the variance in leptin concentrations after weight loss, and this was true no matter what the estimate of change in degree of obesity. In addition to offering an explanation for the variance in postweight loss leptin concentrations, these data provide further evidence of the importance of ambient insulin concentrations in the regulation of plasma leptin concentrations.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PLASMA LEPTIN concentrations are elevated in obese individuals and decrease after weight loss (1, 2, 3, 4). However, the fall in leptin concentration associated with weight loss in normal individuals varies considerably among individuals of similar body compositions (1, 3, 4). This latter observation raises the possibility that factors other than simply the loss of body weight may play a role in determining the change in plasma leptin concentration that occurs when obese individuals lose weight. It has been known for a long time that weight loss in obese, normal individuals is associated with an improvement in insulin-mediated glucose disposal and a decrease in the plasma insulin response to glucose (5). In this context, we have recently published evidence that the greater the plasma insulin response to an oral glucose challenge, the higher the fasting leptin concentration in normal volunteers (6). Given the above information, it seemed reasonable to initiate this study, in which we have tested the hypothesis that it is the change in circulating insulin concentrations that determines the degree to which leptin concentrations decrease after moderate weight loss in healthy, but obese, volunteers. More specifically, we predicted that the greater the decrease in ambient insulin concentrations, the more leptin concentrations would decline with weight loss.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
The experimental population consisted of 12 women, aged 29–50 yr, who responded to a newspaper advertisement indicating our interest in studying the metabolic changes related to a moderate weight loss. They were all overweight, with a body mass index that ranged from 29.2–35 kg/m2. Individuals selected for study were defined as healthy on the basis of medical history, physical exam, and normal results from routine laboratory tests and electrocardiogram and were nondiabetic after a 75-g oral glucose load (7). No subject was a smoker or was taking any drugs known to affect glucose and insulin metabolism. The study protocol was approved by the Stanford University institutional review board, and written informed consent was obtained from all subjects.

All measurements were performed at the General Clinical Research Center (GCRC) of Stanford Medical Center. Volunteers were admitted to the GCRC the evening before baseline studies were performed. Weight was determined without shoes in light clothing with an electronic scale. Body mass index was calculated as weight (in kilograms) divided by height (in meters) squared. Body density was determined by underwater weighing after a 12-h fast, and body fat mass and percent were computed according to the equations of Siri (8). Resistance to insulin-mediated glucose uptake was determined by a modification (9) of the insulin suppression test as initially described by our laboratory (10, 11). Each volunteer received a continuous iv infusion of somatostatin (5 µg/min) to suppress endogenous insulin secretion. Simultaneously, insulin and glucose were infused at rates of 25 and 240 mg/m2/min, respectively. Blood was sampled for measurement of plasma glucose (12) and insulin (13) concentrations every 30 min for the first 150 min and then every 10 min until 180 min had elapsed. The mean value of the four measurements made during the last 30 min of the test was used to calculate the steady state plasma insulin (SSPI) and the steady state plasma glucose (SSPG) concentrations. As SSPI is relatively similar in all individuals, SSPG provides a measurement of insulin-mediated glucose uptake: the higher the SSPG, the more insulin resistant the research subject. On the same day, fasting blood samples for measurement of plasma leptin concentrations were collected in ethylenediamine tetraacetate tubes and immediately centrifuged, and the plasma was stored at -70 C. These samples were not thawed until leptin concentrations were measured by a commercial RIA (Linco Research, Inc., St. Louis, MO). The following day, research subjects underwent a meal tolerance test.

Each volunteer received an 8-h meal tolerance test before and after weight loss. The meal tolerance test is composed of two meals: breakfast, served immediately after fasting blood samples are drawn at approximately 0800 h, and lunch, served 4 h after breakfast. The macronutrient composition of the isocaloric breakfast and lunch was 43% carbohydrate, 15% protein, and 42% fat; breakfast met 20% and lunch met 40% of the individuals daily caloric requirement. During the 8-h test, blood was withdrawn at hourly intervals. After separation, aliquots of plasma were stored frozen for measurement of glucose and insulin concentrations. The total integrated area of the plasma concentrations during this 8-h period was used to quantify plasma insulin response by the trapezoidal method.

Weight loss began the day after the first meal tolerance test. The Harris-Benedict equation (14) was used to determine each volunteer’s total caloric requirement (basal energy expenditure x 1.5). One thousand calories was subtracted from their total caloric requirement to determine daily caloric intake during the weight loss phase of the study. No one received less than 1200 Cal/day. A commercial canned liquid nutritional formula plus two high fiber muffins per day was the diet for 9 weeks. Each volunteer came into the GCRC on Monday and Thursday for measurement of body weight and to pick up their liquid nutritional formula and fiber muffins.

At the end of the weight loss phase, each volunteer met with the research dietitian to develop an isocaloric meal plan using food and beverages based on their individual preferences. After 1 week of weight maintenance, subjects were readmitted to the GCRC to repeat all the baseline measurements.

Data were stored and analyzed using SYSTAT 6.0 Package for Windows (Systat Corp., Evanston, IL). Leptin and insulin levels had a log distribution and were analyzed after log transformation to improve normality for testing and back-transformed for presentation in tables and figures. Differences between pre- and postintervention measures of all variables were determined using a paired t test. Pearson product moment correlation coefficients and multiple regression analysis were performed to determine relationships between variables of interest. Results are expressed as the mean ± SE, and statistical significance is denoted by P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Relevant anthropometric and metabolic variables, before and after weight loss, are shown in Table 1Go. If attention is first directed to the measurements made before weight loss, it is apparent that the baseline metabolic characteristic of this group of obese individuals varied from 2-fold (fasting glucose concentration) to 5-fold (day-long plasma insulin response).


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  		Table 1. Anthropometric and metabolic variables before and after weight loss

 
The study group lost an average of 9.5 kg, or 11.2% of body weight, in response to the hypocaloric diet. The results in Table 1Go indicate that the loss in weight was accompanied by significant decreases in every variable measured, with the exception of fasting and day-long glucose concentration and SSPG concentration. However, the magnitude of change in leptin concentration was by far the most dramatic, with an average fall of 50% from baseline values.

Table 2Go summarizes the correlation coefficient between the decrease in plasma leptin after weight loss and all of the other variables measured. It is apparent that the only statistically significant relationship (r = 0.78; P < 0.005) was between the decrease in leptin and the fall in the day-long insulin response. This relationship is depicted in Fig. 1Go. Figure 1Go also emphasizes that neither the decrease in fat mass nor that in percent body fat correlated with the decrease in leptin concentration after weight loss.


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  		Table 2. Relationship between decrease in leptin concentration and the change in other variables after weight loss

 


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  		Figure 1. Relationship between the decrease ({Delta}) in the plasma leptin concentration after weight loss and the associated changes ({Delta}) in fat mass, percent body fat, and the 8-h integrated plasma insulin response to breakfast and lunch.

 
To further address the relationship between plasma leptin concentration and the other metabolic variables after weight loss, multiple regression analysis was used. The results shown in Table 3Go demonstrate that only the day-long integrated insulin response after weight loss was independently related to the decrease in plasma leptin concentration. In contrast, the decreases in fat mass, SSPG, and day-long glucose response were not related to the weight loss-associated fall in leptin concentration. The same result was noted when other estimates of weight loss were entered into the model, and this was also true of the changes in fasting plasma glucose and insulin concentrations. Finally, it is apparent from Table 3Go that the fall in day-long plasma insulin response accounted for 66% of the variance in leptin concentrations after weight loss.


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  		Table 3. Multivariate regression analysis of the relationship between the change in fasting leptin concentrations and the change in other variables after weight loss

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The specific goal of this study was to test the hypothesis that the variability in plasma leptin concentration observed after an approximately 10% fall in body weight would be a function of the associated decline in day-long plasma insulin concentrations. It is clear from the results in Table 2Go and Fig. 1Go that this was the case. Specifically, the greater the fall in circulating insulin concentration after weight loss, the greater the decrease in plasma leptin concentration. Indeed, the only variable we measured that was significantly correlated with the leptin response to weight loss was the day-long insulin response. Thus, our results are totally consistent with the hypothesis that led to the initiation of this study. The fact that Wing and associates (4) could not find a relationship between the decreases in leptin and insulin concentration after weight loss is most likely due to the fact that they only measured the changes in fasting insulin, not the day-long insulin responses to meals.

However, providing an explanation for why leptin concentrations vary so much after weight loss was not the sole purpose for carrying out the present series of observations. Based upon the results of our earlier study (6), we had concluded that in addition to its well recognized association with adiposity (1, 2, 3), leptin concentrations also varied in the general population as a function of the circulating insulin concentration. We were aware from other studies that plasma insulin concentrations can vary widely in obese individuals (5, 15, 16) and that the decrease in plasma insulin concentration after weight loss is also variable from person to person (5). Thus, the results of the current protocol also provided a way to test the more general hypothesis that the ambient insulin concentration is an important, and independent, regulator of the leptin concentration. Obviously, correlation coefficients cannot prove causality, but the results of the current interventional study provide further support for this more fundamental link between day-long plasma insulin and plasma leptin concentrations, independent of variations in adiposity.

Finally, some attention should be addressed to the fact that in our previous study (6) as well as in this one, plasma insulin concentration, not insulin resistance, was most closely associated with plasma leptin concentration. It has been well documented in nondiabetic individuals that insulin resistance and plasma insulin concentration are highly correlated (17, 18), and in this population there was also a strong correlation between SSPG and both baseline fasting insulin (r = 0.60; P < 0.05) and the 8-h integrated insulin response to meals (r = 0.88; P < 0.001). Therefore, one might have predicted that the decrease in insulin resistance after weight loss would also have contributed to the ensuing variability in the decline in leptin concentration. Indeed, this was almost certainly the case, in that the decreases in SSPG concentration after weight loss also correlated somewhat with the decline in the day-long insulin response (r = 0.47; P = 0.09). The fact that changes in ambient insulin concentration correlated to a greater degree than did the decrease in insulin resistance should not be surprising, in that the plasma insulin concentration is a function of its rates of appearance and disappearance from the vascular compartment, which, in turn, are modulated by at least four other variables: 1) degree of insulin resistance, 2) changes in plasma glucose concentrations, 3) variations in glucose-stimulated insulin secretion, and 4) differences in the efficacy of insulin catabolism. If, as has been shown previously, increases in the plasma insulin concentration stimulate adipose tissue messenger ribonucleic acid for leptin (19), it should not be surprising to find that it is circulating insulin concentration, not insulin resistance, that is most closely related to the plasma leptin concentration.

In conclusion, the results presented have shown that variation in the magnitude of the fall in plasma leptin concentration after weight loss is highly correlated with the associated decrease in the day-long plasma insulin response. Furthermore, this relationship was independent of the weight loss-related changes in various measures of adiposity. In addition to offering an explanation for the variability in postweight loss plasma leptin concentrations, these results provide further support for the view that circulating insulin concentrations are an important regulator of leptin concentrations (6, 20, 21). On the other hand, it should be emphasized that the ability of insulin to increase adipose tissue leptin synthesis and secretion is not an acute phenomenon (19, 20). The fact that there was an independent relationship between changes in the leptin concentration and the day-long insulin responses, but not with the fasting insulin concentration, raises the alternative possibility that the association between insulin and leptin concentrations may be an indirect one.


   Footnotes
 
1 This work was supported by grants from the NIH (RR-00070 and HL-80506), the American Diabetes Association (Mentor Award), and the office of Research and Development: Medical Research Service, Department of Veteran Affairs. Back

Received September 9, 1998.

Revised December 9, 1998.

Accepted December 14, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Maffei M, Halaas J, Ravussin E, et al. 1995 Leptin levels in human and rodent: measures of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med. 11:1155–1161.
  2. Lonnqvist F, Arner P, Nordfors L, Schalling M. 1995 Overexpression of the obese (ob) gene in adipose tissue of human obese subjects. Nat Med. 1:950–953.[CrossRef][Medline]
  3. Considine RV, Sinha MK, Heiman ML, et al. 1996 Serum immunoreactive-leptin concentration in normal weight and obese humans. N Engl Med. 334:292–295.[Abstract/Free Full Text]
  4. Wing RR, Sinha MK, Considine RV, Lang W, Caro JF. 1996 Relationship between weight loss maintenance and changes in serum leptin levels. Horm Metab Res. 28:698–703.[Medline]
  5. Olefsky JM, Reaven GM, Farquhar JW. 1974 Effects of weight reduction on obesity: studies or carbohydrate and lipid metabolism. J Clin Invest. 53:64–67.
  6. Carantoni M, Abbasi F, Azhar S, et al. 1998 Plasma leptin concentrations do not appear to decrease insulin-mediated glucose disposal or glucose-stimulated insulin secretion in women with normal glucose tolerance. Diabetes. 47:244–247.[Abstract]
  7. National Diabetes Data Group. 1979 Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes. 28:1039–1057.[Medline]
  8. Siri WE. 1961 Body composition from fluid spaces and density: analysis of methods. In: Brozek J, Henschel A, eds. Techniques for measuring body composition. Washington DC: National Academy of Science; 223–244.
  9. Harano Y, Ohgaku S, Hidaka H, et al. 1977 Glucose, insulin and somatostatin infusion for the determination of insulin sensitivity. J Clin Endocrinol Metab. 45:1124–1127.[Abstract]
  10. Shen S-W, Reaven GM, Farquhar J. 1970 Comparison of impedance to insulin-mediated glucose uptake in normal subjects and in subjects with latent diabetes. J Clin Invest. 49:2151–2160.
  11. Greenfield M, Doberne L, Kramer F, Tobey T, Reaven GM. 1981 Assessment of insulin resistance with the insulin suppression test and the euglycemic clamp. Diabetes. 30:387–393.[Abstract]
  12. Kadish AK, Litle RL, Sternerg JC. 1963 A new and rapid method for determination of glucose by measurement of rate of oxygen consumption. Clin Chem. 14:116–131.
  13. Hales CN, Randle PJ. 1963 Immunoassay of insulin with insulin-antibody precipitate. Biochem J. 88:137–146.[Medline]
  14. Harris JA, Benedict FG. 1919 A biometric study of basal metabolism in man. Washington DC: Carnegie Institute of Washington; publication 279.
  15. Zavaroni I, Bonini L, Fantuzzi M, Dall’Aglio E, Passeri M, Reaven GM. 1994 Hyperinsulinemia, obesity, and syndrome X. J Intern Med. 235:51–56.[Medline]
  16. Ferrannini E, Natali A, Bell P, Cavallo-Perin P, Lalic N, Mingrone G, on behalf of the European Group for the Study of Insulin Resistance (EGIR). 1997 Insulin resistance and hypersecretion in obesity. J Clin Invest. 100:1166–1173.[Medline]
  17. Hollenbeck CB, Chen N, Chen Y-DI, Reaven GM. 1984 Relationship between plasma insulin response to oral glucose and insulin-stimulated glucose utilization in normal subjects. Diabetes. 33:460–463.[Abstract]
  18. Hollenbeck CB, Reaven GM. 1987 Variations in insulin-stimulated glucose uptake in healthy individuals with normal glucose tolerance. J Clin Endocrinol Metab. 64:1169–1173.[Abstract]
  19. Kolaczynski JW, Nyce MR, Considine RV, et al. 1996 Acute and chronic effect of insulin on leptin production in humans: studies in vivo and in vitro. Diabetes. 45:699–701.[Abstract]
  20. Boden G, Chen X, Kolaczynski JW, Polansky M. 1997 Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects. J Clin Invest. 100:1107–1113.[Medline]
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