Acute stimulation of leptin concentrations in humans during hyperglycemic hyperinsulinemia. Influence of free fatty acids and fasting

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Abstract

OBJECTIVE: To assess the acute regulation of leptin concentrations by insulin, glucose and free fatty acids (FFAs).

DESIGN: Four protocols: saline control experiment (CON); hyperglycemic clamps (8.3 mmol/l, 120 min) after an overnight fast (12 FAST); after a 36 h fast (36 FAST); and after a 36 h fast during which Intralipid/heparin was given over the last 24 h (36 FAST+FFA).

SUBJECTS: Lean, young, healthy volunteers; control group (n=6), experimental group (n=6).

MEASUREMENTS: Serum leptin concentrations.

RESULTS: Glucose and insulin concentrations were similar during the three clamp protocols. Average FFAs during the last 60 min of the clamp were 671±68 µM (CON),109±15 µM (12 FAST), 484±97 µM (36 FAST) and 1762±213 µM (36 FAST+FFA). Leptin concentrations decreased similarly during 36 FAST and 36 FAST+FFA. Leptin concentrations at 120 min (expressed as percentage of mean basal value) were 0.82±0.02 (CON), 0.93±0.08 (12 FAST) (P=0.29), 1.19±0.06 (36 FAST) (P<0.01) and 1.44±0.12 (36 FAST+FFA) (P<0.01).

CONCLUSION: During a one-day fast leptin concentrations decrease regardless of maintainance of an isocaloric balance. During acute hyperinsulinemic hyperglycemia leptin concentrations increase only after a preceding fast. This increase was most pronounced during simultaneous elevation of FFAs. Overall, our findings are compatible with the hypothesis that leptin secretion may be coupled to triglyceride synthesis rather than to the absolute lipid content of the adipocyte.

Introduction

The OB gene product leptin is thought to be an adipocyte-derived signal contributing to the regulation of body weight and satiety.1,2,3 During a short-term fast circulating leptin concentrations decrease.4,5 Interestingly, this decrease is disproportionate to the percentage of body fat lost. Moreover, after a 4 day fast followed by an additional 6 day fast during which a glucose infusion provided only 338 kcal/day, leptin concentrations increased by 80% in the first 24 h.6 Consequently, it has been suggested that the serum leptin concentration reflects ongoing triglyceride synthesis or glucose uptake by fat cells rather than the actual lipid content of the adipocyte.7

In addition to the long-term regulation by body fat mass, there is increasing evidence that leptin secretion is also under acute hormonal control. Humoral factors stimulating leptin secretion include glucocorticoids8 and insulin in particular under eu- or hyperglycemic clamp conditions.9,10,11 Fatty acids have been shown to inhibit leptin secretion in vitro12,13 but not in vivo.14 In studies in humans, hormones and/or substrates used to experimentally stimulate leptin secretion in vivo were raised to supraphysiological concentrations9 or administered over a prolonged period.15

Therefore, we performed the following studies to assess under which conditions leptin concentrations are acutely (ie<2 h) stimulated by physiological increases in hormone and substrate concentrations. In addition to a saline control experiment (CON), hyperglycemic clamps (8.3 mmol/l, 120 min) were performed after an overnight fast (12 FAST), after a 36 h fast (36 FAST) and after a 36 h fast during which Intralipid/heparin was given over the last 24 h (36 FAST+FFA).

Methods

Subjects

After approval of the protocol by the local ethical committee and obtaining informed written consent we studied a control group (n=6) who underwent a saline control experiment and an experimental group (n=6) who underwent three hyperglycemic clamps in random order approximately 1 week apart. Subjects characteristics are shown in Table 1. Prior to the study, all subjects had their medical history taken and underwent a physical examination, a routine blood test and an ECG. Subjects had been instructed to maintain their usual diet before the study.

Table 1 Subjects characteristics

Experimental protocols

For the experiments subjects were admitted to the university's research unit in the morning after an overnight fast and indwelling catheters were inserted into an antecubital vein for infusions and a dorsal hand vein in a retrograde fashion which was kept in a heated chamber for arterialized blood sampling. In every protocol baseline blood samples were obtained at 7.30 h (equals −30 min) and at 8.00 h experiments were started. Four experimental protocols were performed in random order no more than 4 weeks apart.

12 h fast+saline (CON).

As control experiment a saline infusion was started at 0 min.

12 h fast+hgc (hyperglycemic clamp) (12 FAST).

A hyperglycemic clamp was performed after an overnight fast.

36 h fast+hgc (36 FAST).

A hyperglycemic clamp was performed after a 36 h fast.

12 h fast+24 h Intralipid+hgc (36 FAST+FFA).

This represents the continuation of protocol 2. Immediately after completion of the first hyperglycemic clamp Intralipid 10% (Pharmacia & Upjohn, Erlangen, Germany) (0.17 ml/kg h) was infused over a 26 h period together with sodium heparin at a rate of 800 IE/h (priming dose 1000 IE). The hyperglycemic clamp was performed during continued infusion of Intralipid/heparin.

Hyperglycemic clamp

After baseline samples had been obtained, a hyperglycemic clamp was performed as previously described.16 An intravenous bolus of 20% glucose over 1 min was given to instantaneously raise blood glucose to approximately 8.3 mmol/l (bolus dose (mg)=body weight (kg)×desired increase in blood glucose (mg/dl)×1.5). Subsequently, the glucose infusion was adjusted to maintain blood glucose at around 8.3 mmol/l.

Sampling and analytical procedures

Blood samples for determination of serum leptin and serum FFAs were obtained every 30 min during the hyperglycemic clamp. Samples for serum insulin were taken at the same time points and at 10 min. Serum leptin was determined by a radioimmunoassay (Linco, St Charles, MO, USA) with intra- and interassay coefficients of variation <6% and a lower detection limit of 0.5 ng/ml.17 Serum insulin was determined by a microparticle enzyme immunoassay (Abbott Laboratories, Tokyo, Japan). Free fatty acid (FFA) concentrations were determined by an enzymatic method (NEFAC kit, WACO Chemicals, Neuss, Germany). Blood glucose was determined at the bedside every 5 min using a HemoCue analyzer (HemoCue, Mission Viejo, CA, USA).

Statistical analysis

Unless stated otherwise data are expressed as mean±s.e.m. For comparing changes in leptin concentrations between different protocols the relative change from baseline at 120 min was used by which time the change from baseline was greatest in all protocols. For statistical comparisons with the saline control protocol the unpaired student's t-test (two-tailed) was used. For comparisons between the hyperglycemic clamp protocols which were performed in the same subjects the paired Student's t-test (two-tailed) was used. In addition, ANOVA was performed on leptin (120 min) and insulin (60–120 min) of the three protocols (excluding saline) to assess the differences between the experiments. A P-value of less than 0.05 was considered to be statistically significant.

Results

12 h fast+saline (CON)

During the saline infusion neither plasma glucose nor plasma insulin nor serum FFAs were significantly different from baseline. Serum leptin decreased from 5.6±0.9 ng/dl at baseline to 4.5±0.7 ng/dl at 120 min (P<0.001) (Figures 1 and2).

Figure 1
figure1

Serum leptin concentrations (absolute) during the four protocols (real-time); hgc, hyperglycemic clamp.

Figure 2
figure2

Serum leptin concentrations (relative to mean baseline), FFA, insulin and glucose concentrations during the four protocols (time axis of the hyperglycemic clamp); hgc, hyperglycemic clamp. ANOVA on leptin (12 h fast+24 h Intralipid+hgc, 36 h fast+hgc, 12 h fast+hgc), P=0.01; ANOVA on insulin (12 h fast+24 h Intralipid+hgc, 36 h fast+hgc, 12 h fast+hgc), P=0.89. *12 h fast+24 h Intralipid+hgc vs 12 h fast+saline, P<0.01; **12 h fast+24 h Intralipid+hgc vs 12 h fast+hgc, P<0.01; ***12 h fast+24 h Intralipid+hgc vs 36 h fast+hgc, P=0.15; §36 h fast+hgc vs 12 h fast+saline, P<0.01; §§36 h fast+hgc vs 12 h fast+hgc, P<0.1; #12 h fast+hgc vs 12 h fast+saline, P=0.21.

12 h fast+hyperglycemic clamp (12 FAST)

After an overnight fast plasma insulin increased from 37±8 pM at baseline to 188±53 pM (P<0.001) during the last 60 min of the hyperglycemic clamp. Serum FFAs decreased from 465±43 µM to 81±6 µM at 120 min (P<0.001). Serum leptin concentrations did not change significantly (from 5.2±1.18 ng/dl at baseline to 4.8±1.0 ng/dl at 120 min, P=0.29). The relative change in leptin concentrations (0.93±0.08) was also not different from the saline control experiment (0.82±0.02, P=0.21; Figures 1 and 2).

36 h fast+hyperglycemic clamp (36 FAST)

After the 36 h fast plasma insulin increased from 26±7 pM at baseline to 220±67 pM (P vs 12 FAST=0.24) during the last 60 min of the hyperglycemic clamp. Serum FFAs decreased from 908±74 to ±409±129 µM at 120 min P<0.01). Serum leptin concentrations did not change significantly (from 2.4±0.6 to 3.0±0.8 ng/dl at 120 min, P=0.11). The relative change in leptin concentrations (1.19±0.06), however, was significantly greater than that during the saline control experiment (0.82±0.02, P<0.01) and non-significantly different from the 12 h fast experiment (0.93±0.08, P=0.1).

12 h fast+24 h Intralipid+hyperglycemic clamp (36 FAST+FFA)

After the 24 h elevation of free fatty acids to approximately 2000 µM, plasma insulin increased from 24±3 pM at baseline to 241±59 pM (P vs other protocols N.S.) during the last 60 min of the hyperglycemic clamp. Serum FFAs decreased from 1969±206 to 1578±188 µM at 120 min (P<0.05). Serum leptin concentrations increased significantly from 2.5±0.1 to 3.5±0.01 ng/dl at 120 min (P<0.05). The relative change in leptin concentrations (1.44±0.12) was higher than that during the hyperglycemic clamp performed after the 12 h fast (0.93±0.06, P<0.01), but not significantly different from that after the 36 h fast (1.19±0.06, P=0.15). Compared to the saline control experiment the relative change in leptin concentrations was higher and statistically different (0.82±0.02, P<0.01) (Figures 1 and 2).

Discussion

The present studies were undertaken to enhance our understanding of the mechanisms involved in physiological short-term regulation of leptin concentrations. The hyperglycemic clamp after the overnight fast essentially did not prevent the diurnal fall in leptin concentrations. In contrast, after the 36 h fast leptin concentrations increased significantly during the hyperglycemic clamp. The most pronounced increase in serum leptin was observed after the 36 h fast complemented by the 24 h elevation of FFAs.

It has been previously reported that after a 4 day fast hyperglycemic hyperinsulinemia transiently restores baseline leptin concentrations within 24 h.6 In this study the decrease in leptin concentrations after one day of fasting was different compared to 4 days of fasting. Interestingly, in our study 50% of the decrease in leptin concentrations during the 24 h fast plus intralipid were already reversed during the 2 h hyperglycemic clamp with continued Intralipid. This suggests that a preceding fast primes the adipocyte to an acute increase in leptin secretion in response to changes in glucose availability (and presumably also energy intake).

In vitro studies have suggested that FFAs could inhibit leptin secretion12,13 while in vivo studies in humans have demonstrated no effect of elevated plasma FFAs on circulating leptin concentrations.14,18 During the Intralipid/heparin infusion in our study the increase in leptin concentrations was greater compared to the fasting only protocol where endogenous FFA concentrations were lower than during the experimental elevation. Since hyperglycemia was comparable and hyperinsulinemia not significantly different between both fasting protocols, our findings suggest that FFAs, if anything, may increase leptin concentrations under certain conditions.

It is important to point out that insulin concentrations were not identical in the three hyperglycemic clamp protocols. Although not significantly different, insulin concentrations were highest in the 36 FAST+FFA protocol where also leptin increased most. Since insulin itself has been shown to stimulate leptin concentrations19 one might argue that some of the difference could be attributable to insulin. However, although insulin concentrations differed markedly between CON and 12 FAST, leptin concentrations were similar. Moreover, effects of insulin alone on leptin concentrations have a lag period of about 2 h19 while the divergence of the curves in the present study were evident much earlier. It thus appears unlikely that the small differences in insulin were responsible for the differences in leptin in a major way. Nevertheless, based on our data alone a partial effect of the higher insulin concentrations on leptin cannot be entirely excluded.

Our data are compatible with a previously proposed hypothesis regarding the nutrient regulation of leptin secretion. According to this hypothesis leptin concentrations reflect the rate of triglyceride synthesis rather than the energy balance per se.7 Thirty-six hour fasting represents a state of ongoing lipolysis and triglyceride depletion. In this sense prior triglyceride depletion would set the stage for accelerated resynthesis under the hyperglycemic clamp conditions, explaining the acute stimulation of leptin concentrations. A coupling of leptin secretion with triglyceride synthesis would also explain the observation that increased availability of FFAs resulted in a further increase of leptin secretion by accelerating triglyceride synthesis.20,21

It is of note that leptin concentrations decreased during the fast regardless of energy supplementation by intralipid and glucose (18 kcal/kg 24 h). It has been previously proposed that not so much the provision of energy but the presence of insulin-stimulated glucose metabolism is critical for sustaining leptin secretion.7 Insulin and glucose before the hyperglycemic clamps were low in the two fasting protocols and both are key factors for stimulation of triglyceride synthesis in the adipocyte.22,23 Thus, in line with the concept outlined above, circulating leptin concentrations might represent a mirror image of insulin and glucose stimulated triglyceride synthesis in the adipocyte.

The fact that leptin concentrations in the fed state are relatively resistant to any acute stimulation (including hyperinsulinemia, hyperglycemia or increased plasma FFA) suggests a robust set point of the individual adipocyte lipid storing capacity and leptin secretion rate. It also implies that in pathological conditions such as obesity a profound disturbance of the equilibrium must be present to reset basal leptin concentrations to the high concentrations seen in this condition.3

In conclusion, a hyperglycemic hyperinsulinemic clamp does not acutely prevent the diurnal decrease in leptin concentrations. In addition, during a one-day fast leptin concentrations decrease regardless of maintenance of an isoenergetic balance by lipid infusion. Moreover, during acute hyperinsulinemic hyperglycemia leptin concentrations increase only after a preceding fast. The fact that this stimulation was most pronounced during simultaneous elevation of FFAs suggests that FFAs may increase leptin concentrations under certain circumstances (eg elevated insulin and glucose). Finally, our findings are compatible with the hypothesis that leptin secretion is closely coupled to triglyceride synthesis rather than the absolute lipid content of the adipocyte or the whole body energy balance.

References

  1. 1

    Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM . Positional cloning of the mouse obese gene and its human homologue Nature 1994 372: 425–432.

  2. 2

    Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F . Effects of the obese gene product on body weight regulation in ob/ob mice Science 1995 269: 540–543.

  3. 3

    Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF . Serum immunoreactive-leptin concentrations in normal-weight and obese humans New Engl J Med 1996 334: 292–295.

  4. 4

    Weigle DS, Duell PB, Connor WE, Steiner RA, Soules MR, Kuijper JL . Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels J Clin Endocrinol Metab 1997 82: 561–565.

  5. 5

    Boden G, Chen X, Mozzoli M, Ryan I . Effect of fasting on serum leptin in normal human subjects J Clin Endocrinol Metab 1996 81: 3419–3423.

  6. 6

    Grinspoon SK, Askari H, Landt ML, Nathan DM, Schoenfeld DA, Hayden DL, Laposata M, Hubbard J, Klibanski A . Effects of fasting and glucose infusion on basal and overnight leptin concentrations in normal-weight women Am J Clin Nutr 1997 66: 1352–1356.

  7. 7

    Coleman RA, Herrmann TS . Nutritional regulation of leptin in humans Diabetologia 1999 42: 639–646.

  8. 8

    Miell JP, Englaro P, Blum WF . Dexamethasone induces an acute and sustained rise in circulating leptin levels in normal human subjects Horm Metab Res 1996 28: 704–707.

  9. 9

    Kolaczynski JW, Nyce MR, Considine RV, Boden G, Nolan JJ, Henry R, Mudaliar SR, Olefsky J, Caro JF . Acute and chronic effects of insulin on leptin production in humans: studies in vivo and in vitro Diabetes 1996 45: 699–701.

  10. 10

    Saad MF, Khan A, Sharma A, Michael R, Riad-Gabriel MG, Boyadjian R, Jinagouda SD, Steil GM, Kamdar V . Physiological insulinemia acutely modulates plasma leptin Diabetes 1998 47: 544–549.

  11. 11

    Utriainen T, Malmstrom R, Mäkimattila S, Yki-Järvinen H . Supraphysiological hyperinsulinemia increases plasma leptin concentrations after 4 h in normal subjects Diabetes 1996 45: 1364–1366.

  12. 12

    Rentsch J, Chiesi M . Regulation of ob gene mRNA levels in cultured adipocytes FEBS Lett 1996 379: 55–59.

  13. 13

    Deng C, Moinat M, Curtis L, Nadakal A, Preitner F, Boss O, Assimacopoulos Jeannet F, Seydoux J, Giacobino JP . Effects of beta-adrenoceptor subtype stimulation on obese gene messenger ribonucleic acid and on leptin secretion in mouse brown adipocytes differentiated in culture Endocrinology 1997 138: 548–552.

  14. 14

    Peino R, Fernandez Alvarez J, Penalva A, Considine RV, Rodriguez-Segade S, Rodriguez-Garcia J, Cordido F, Casanueva FF, Dieguez C . Acute changes in free fatty acids (FFA) do not alter serum leptin levels J Endocrinol Invest 1998 21: 526–530.

  15. 15

    Boden G, Chen X, Kolaczynski JW, Polansky M . Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects J Clin Invest 1997 100: 1107–1113.

  16. 16

    Pimenta W, Korytkowski M, Mitrakou A, Jenssen T, Yki-Järvinen H, Evron W, Dailey G, Gerich J . Pancreatic beta-cell dysfunction as the primary genetic lesion in NIDDM JAMA 1995 273: 1855–1861.

  17. 17

    Ma Z, Gingerich RL, Santiago JV, Klein S, Smith CH, Landt M . Radioimmunoassay of leptin in human plasma Clin Chem 1996 42: 942–946.

  18. 18

    Stumvoll M, Fritsche A, Tschritter O, Lehmann R, Wahl HG, Renn W, Häring H . Leptin levels in humans are acutely suppressed by isoproterenol despite acipimox-induced inhibition of lipolysis, but not by free fatty acids Metabolism 2000 49: 335–339.

  19. 19

    Dagogo-Jack S, Fanelli C, Paramore D, Brothers J, Landt M . Plasma leptin and insulin relationships in obese and nonobese humans Diabetes 1996 45: 695–698.

  20. 20

    Coppack SW, Persson M, Judd RL, Miles JM . Glycerol and nonesterified fatty acid metabolism in human muscle and adipose tissue Am J Physiol 1999 276: E233–E240.

  21. 21

    de la Llera M, Glick JM, Rothblat G . Mechanism of triglyceride accumulation in rat preadipocyte cultures exposed to very low density lipoprotein J Lipid Res 1981 22: 245–253.

  22. 22

    Green H, Kehinde O . An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion Cell 1975 5: 19–27.

  23. 23

    Saggerson ED, Greenbaum AL . The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue Biochem J 1970 119: 193–219.

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Acknowledgements

We are indebted to Sabine Wolff for her excellent technical help. This study was in part supported by a grant from the Deutsche Forschungsgemeinschaft (DFG), Stu 192-2/1 and by a grant from the European Community (QLRT-1999-00674).

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Correspondence to M Stumvoll.

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Keywords

  • obesity
  • free fatty acids
  • insulin
  • glucose
  • triglyceride synthesis

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