Novel Form of Lipolysis Induced by Leptin*

May-Yun Wang, Young Lee, and Roger H. UngerDagger

From the Gifford Laboratories, Center for Diabetes Research and the Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and the Veterans Administration Medical Center, Dallas, Texas 75216

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hyperleptinemia causes disappearance of body fat without a rise in free fatty acids (FFA) or ketones, suggesting that leptin can deplete adipocytes of fat without releasing FFA. To test this, we measured FFA and glycerol released from adipocytes obtained from normal lean Zucker diabetic fatty rats (+/+) and incubated for 0, 3, 6, or 24 h in either 20 ng/ml recombinant leptin or 100 nM norepinephrine (NE). Whereas NE increased both FFA and glycerol release from adipocytes of +/+ rats, leptin increased glycerol release in +/+ adipocytes without a parallel increase in FFA release. In adipocytes of obese Zucker diabetic fatty rats (fa/fa) with defective leptin receptors, NE increased both FFA and glycerol release, but leptin had no effect on either. Leptin significantly lowered the mRNA of leptin and fatty acid synthase of adipocytes (FAS) (p < 0.05), and up-regulated the mRNA of peroxisome proliferator-activated receptor (PPAR)-alpha , carnitine palmitoyl transferase-1, (CPT-1), and acyl CoA oxidase (ACO) (p < 0.05). NE (100 nM) also lowered leptin mRNA (p < 0.05) but did not affect FAS, PPARalpha , ACO, or CPT-1 expression. We conclude that in normal adipocytes leptin directly decreases FAS expression, increases PPARalpha and the enzymes of FFA oxidation, and stimulates a novel form of lipolysis in which glycerol is released without a proportional release of FFA.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adenoviral transfer of the leptin gene into normal rats causes rapid loss of all visible body fat within 7 days (1). Unlike the ketotic fat loss in starvation or insulin deficiency, in which the fatty acids (FA)1 and glycerol are released proportionately from the adipocytes, hyperleptinemic fat loss is unaccompanied by elevations in plasma free fatty acid (FFA) levels or ketones or by ketonuria (2). One possible explanation for the nonketotic fat loss is that the FAs are oxidized inside the adipocytes. This idea has received further support from the demonstration that the expression of two major enzymes of long chain FA oxidation, acyl CoA oxidase (ACO), and carnitine palmitoyl transferase-1 (CPT-1) are strikingly increased in the adipocytes of hyperleptinemic rats during the disappearance of their fat (3, 4). This finding implies that experimentally induced hyperleptinemia can convert adipocytes from fat-storing cells into fat-burning cells.

Most workers in the field believe that leptin acts largely, if not exclusively, via centers in the hypothalamus, suppressing appetite by inhibiting orexic factors such as neuropeptide Y (5) and by increasing thermogenesis via sympathetic innervation of brown adipose tissue (6). It seemed possible, therefore, that the disappearance of the fat of white adipocytes might be the result of leptin-induced, adrenergically mediated activation of lipolysis. Yet there is evidence consistent with the possibility that, at least at high concentrations, leptin can act directly on tissues independently of hypothalamic mediation. First, leptin receptors (OB-R), including the full-length isoform, OB-Rb, are expressed in white adipocytes (10). Second, leptin has been shown in vitro to reduce the expression of lipogenic enzymes in preadipocytes (8) and to increase glycerol release from mature adipocytes (7). Third, if the fat depletion caused by hyperleptinemia is, in fact, caused by norepinephrine through stimulation of sympathetic centers in the hypothalamus, it would be accompanied by a concomitant increase in plasma FFA levels (9), which did not occur in the hyperleptinemic rats. For these reasons, we suspected that the fat loss of hyperleptinemia involved a novel type of leptin-mediated lipolysis that was independent of catecholamines. The following study was designed to test this possibility.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adipocyte Isolation and Culture-- Isolation of adipocytes from lean (+/+) and obese (fa/fa) Zucker rats was performed as described previously (9). Briefly, minced epididymal fat pads were digested at 37 °C for 2-3 h in a buffer containing type II collagenase (1 mg/ml), albumin (3.5%), and glucose (0.55 mM). The digestion mixture was swirled and poured through 100-µm nylon mesh into 50-ml conical polypropylene tubes. Cells were washed three times with Krebs-Ringer bicarbonate buffer (pH 7.4) containing 5% albumin and cultured for 0, 3, 6 and 24 h at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, antibiotics (penicillin and streptomycin) and with or without recombinant leptin (kindly provided by Dr. Gayle Yamamoto of Zymogenetics, Inc., Seattle, WA) or norepinephrine.

Reverse Transcriptase PCR-- mRNA was semiquantified by reverse transcriptase-PCR. Total RNA from adipocytes was extracted using TRIzol Reagent. After treating with RNase-DNase I, reverse transcription was carried out using 1 µg of total RNA. First strand cDNA was PCR-amplified with sequences specific for leptin (5'-GGAGGAATCCCTGCTCCAGC-3' and 5'-CTTCTCCTGAGGATACCTGG-3'), ACO (5'-GCCCTCAGCTATGGTATTAC-3' and 5'-AGGAACTGCTCTCACAATGC-3'), CPT-1 (5'-TATGTGAGGATGCTGCTTCC-3' and 5'-CTCGGAGAGCTAAGCTTGTC-3'), FAS (5'-GGTTGATGGCTCACACACCT-3' and 5'-TCAACTCACTCGAGGCTCAG-3'), and PPARalpha (5'-AAGCCATCTTCACGATGCTG-3' and 5'-TCAGAGGTCCCTGAACA-GTG-3'). The conditions of PCR were as follows: denaturation for 45 s at 92 °C, annealing for 45 s at 55 °C, and elongation for 1 min at 72 °C with 30 cycles. The PCR products were subjected to electrophoresis on 1.2% agarose gel and were quantified by Southern blot analysis by means of gene-specific 32P-labeled probes for leptin (5'-CGGATACCGACTGCGTGTGTGAAATGTCAT-3'), ACO (5'-GCCTGCACTTTCTTCAGCCATCTTCAACGA-3'), CPT-1 (5'-ACTCTGGTTGGAATCTGA-CTGGGTGGGATT-3'), FAS (5'-AAGAAGCATATGGCTTCAGCTTCAG-CCTCA-3'), and PPARalpha (5'-ACTCGGTCTTCTTGATGACCTGCAC-GAGCT-3'). The Molecular Imager (Bio-Rad) was used for quanti-fication. As a control for RNA quality and quantity, beta -actin mRNA was amplified.

Glycerol and FFA Assays-- Glycerol in the medium was measured by the method of McGowen and co-workers (10). For glycerol assay, 200 µl of medium was deproteinated with 20 µl of 5 M perchloric acid. After deproteination, samples were neutralized with 10 M KOH (pH 9.5) and placed on ice for 30 min. Samples were centrifuged for 10 min at 4 °C to remove KClO4 precipitate, and glycerol was measured in the supernatant. To measure FFA, 500 µl of medium was extracted with 500 µl of chloroform, dried under N2, and measured using the method of Shimizu et al. (11).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Leptin on Leptin mRNA-- A dramatic early effect of adenovirus-induced hyperleptinemia in vivo is the disappearance of leptin mRNA from the adipocytes (4). To establish the in vitro biologic activity of the recombinant leptin, we cultured adipocytes in 20 ng/ml of recombinant leptin, which approximates the levels in hyperleptinemic rats. Leptin mRNA had declined significantly (p < 0.05) by 14, 39 and 50% of normal in 3, 6, and 24 h, respectively (Fig. 1A). In adipocytes from fa/fa rats, there was no effect on leptin mRNA (Fig. 1B). These results establish a direct action of leptin on mature adipocytes.


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Fig. 1.   The effect of 20 ng/ml of recombinant leptin (upper panels) and 100 nM norepinephrine (lower panels) on leptin mRNA in isolated adipocytes of (A and C) normal +/+ and (B and D) obese fa/fa ZDF rats. A representative blot is displayed. *, p < 0.05 (n = 3).

Norepinephrine at 100 nM concentration reduced leptin mRNA by 30% (p < 0.05) in the adipocytes of +/+ rats (Fig. 1C), confirming earlier observations in 3T3-L1 cells (12). Norepinephrine did not reduce leptin mRNA in the adipocytes of fa/fa (Fig. 1D).

Effect of Leptin on mRNA of Fatty Acid Synthetase-- Adenovirus-induced hyperleptinemia causes down-regulation of the mRNA of the lipogenic enzymes, acetyl CoA carboxylase and FAS. To determine whether leptin can directly down-regulate expression of the FAS gene, we measured its mRNA at 3, 6, and 24 h after culture of adipocytes in 20 ng/ml of recombinant leptin. FAS expression was reduced at all time points (p < 0.05) and at 24 h was only 10% of controls (Fig. 2A). This was by far the most dramatic of the direct effects of leptin observed in this study. There was no effect on FAS expression in fa/fa adipocytes (Fig. 2B). Norepinephrine at 100 nM concentration had no effect on FAS mRNA of adipocytes from either the wild-type (+/+) or fa/fa ZDF rats (Fig. 2, C and D).


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Fig. 2.   The effect of 20 ng/ml of recombinant leptin (upper panels) and 100 nM norepinephrine (lower panels) on mRNA of fatty acid synthetase in adipocytes isolated from (A and C) normal +/+ and (B and D) obese fa/fa ZDF rats. A representative blot is displayed. *, p < 0.05 (n = 3).

Effect of Leptin on mRNA of PPARalpha , CPT-1, and ACO mRNA in Adipocytes-- We had observed previously that adenovirus-induced hyperleptinemia dramatically up-regulates the in vivo expression of the enzymes of FA oxidation, of ACO and CPT-1 in adipocytes and of their transcription factor PPARalpha , whereas their triacyl glycerol virtually disappears (4). To determine whether this was a direct effect of hyperleptinemia upon the adipocytes, isolated adipocytes of normal (+/+) ZDF rats were cultured in recombinant leptin at a concentration of 20 ng/ml. After 6 h in culture PPARalpha mRNA had increased 50% (p < 0.05), whereas CPT-1 and ACO mRNA increased ~2-fold compared with controls (p < 0.05) (Fig. 3A). In similarly treated adipocytes isolated from obese fa/fa ZDF rats, there was no effect on the mRNA of any of the foregoing genes (Fig. 3B).


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Fig. 3.   The effects of 20 ng/ml of recombinant leptin on mRNA of PPARalpha , ACO, and CPT-1 of adipocytes isolated from normal +/+ (A) and obese fa/fa ZDF rats (B). A representative blot is displayed. *, p < 0.05 (n = 3).

The presence of 100 nM norepinephrine alone had no effect on PPARalpha mRNA and caused only a minimal increase in ACO and CPT-1 mRNA (not significant) (data not shown). Norepinephrine could not, therefore, have mediated the in vivo effects of leptin on the expression of these enzymes.

Comparative Lipolytic Effects of Leptin and Norepinephrine-- The foregoing direct effects of leptin on the expression of enzymes of fatty acid oxidation and synthesis were consistent with the hypothesis that the nonketotic fat loss of adenovirus-induced hyperleptinemia was the consequence of increased oxidation of FA within adipocytes, coupled with a reduction in lipogenesis. To determine whether the direct action of leptin involves a novel type of lipolytic action that differs from that of norepinephrine, we compared the release of glycerol and FFA from normal rat adipocytes cultured for 6 h in 20 ng/ml of leptin, in 100 nM norepinephrine, or in buffer alone. As shown in Fig. 4A, norepinephrine and leptin each significantly increased glycerol release from adipocytes isolated from +/+ normal ZDF rats (p < 0.05), confirming the observations of Siegrist-Kaiser et al. (7). However, whereas norepinephrine elicited the expected rise in FFA, leptin did not increase FFA release. In adipocytes of fa/fa rats, by contrast, leptin caused no increase in either glycerol or FA, whereas norepinephrine elicited a robust increase in both (Fig. 4B).


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Fig. 4.   Comparative effects of 20 ng/ml of recombinant leptin (black-triangle) and 100 nM norepinephrine (black-square) on the release of glycerol and FFA from the isolated adipocytes of normal +/+ (A) and obese fa/fa ZDF rats (B). *, p < 0.05 versus controls (; n = 5).

Dose-Response Characteristics of Leptin-induced Lipolysis-- The foregoing effects of leptin on lipolysis were observed at the unphysiologically high concentration of 20 ng/ml. To determine whether the effect on lipolysis was operative at more physiologic levels of leptin, a dose-response study was done (Fig. 5). Five ng/ml of leptin was the lowest concentration that stimulated release of glycerol (p < 0.05). This concentration is at the upper end of the range of plasma leptin levels of rodents and humans (13, 14). There was no effect of leptin on glycerol release from fa/fa adipocytes at any of the concentrations employed.


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Fig. 5.   Leptin dose-response curve for glycerol release from isolated adipocytes of normal +/+ (open circle ) and fa/fa () ZDF rats (n = 3). Incubation time was 24 h.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These results provide evidence for multiple direct actions of leptin on mature adipocytes in vitro. Previously, leptin had been shown to reduce expression of acetyl CoA carboxylase and FAS in 3T3-L1 cells (8) and to increase glycerol release from normal adipocytes (7). In the present study, the expression of CPT-1 was increased ~2-fold above the base-line level by 20 ng/ml of leptin, whereas that of the lipogenic enzyme, FAS, was lowered by ~90%. It is possible that the up-regulation of ACO and CPT-1 mRNA was mediated by the increase in expression of their transcription factor, PPAR-alpha , which rose 50%. The mechanism by which leptin up-regulates PPARalpha is not known.

The changes in mRNA induced by recombinant leptin in isolated adipocytes correspond qualitatively with all those observed previously in vivo in adipocytes of rats with adenovirus-induced hyperleptinemia (4). Because norepinephrine failed to induce any of these changes in the cultured adipocytes, it appears that they are the consequences of direct action of leptin on adipocytes rather than of norepinephrine. It should be stressed, however, that direct effects of leptin on adipocytes were at a concentration at 20 ng/ml, which is far above the physiologic levels in normal humans or rodents. A small but significant increase in glycerol was observed in rat adipocytes in response to 5 ng/ml (p < 0.05), which is in the upper end of the physiologic range (13, 14). However, Siegrist-Kaiser et al. (7) reported an effect at 1.8 ng/ml on glycerol release, which is within the physiologic range. Irrespective of the physiologic relevance of these direct effects of leptin upon adipocytes, they do suggest that the pharmacologic strategy of reducing adipocyte fat content by means of direct lipolytic actions of supraphysiologic levels of leptin may be useful (4).

In the earlier reports (7, 15) indicating that leptin increases glycerol release from rodent adipocytes, FFA had not been measured. The finding here that leptin-induced glycerol release is unaccompanied by FFA release explains the in vivo observation that fat loss occurring during adenovirus-induced hyperleptinemia is unaccompanied by a rise in plasma FFA, ketonemia, and ketonuria (2). This and the up-regulation of ACO and CPT-1 support the idea that the FFA are oxidized inside the adipocytes rather than exported to the liver for oxidation to ketoacids. This may provide a valuable therapeutic advantage for leptin treatment of massive obesity, because it permits the rapid removal of fat without the ketoacidosis and hyperuricemia that otherwise complicate rapid weight loss induced by diet restriction.

The mechanism of leptin-induced lipolysis remains to be elucidated. Clearly it requires a functional leptin receptor, because it was completely absent in adipocytes of fa/fa ZDF rats with defective OB-R, in which norepinephrine elicited a relatively normal lipolytic response. Leptin signal is believed to be transduced via the STAT/JAK pathway (16). It will be of interest to determine whether leptin increases cAMP and whether, like norepinephrine, it activates the hormone-sensitive lipase (17).

    ACKNOWLEDGEMENT

We thank Tess Perico for secretarial support.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK02700-37, the National Institutes of Health/Juvenile Diabetes Foundation Diabetes Interdisciplinary Research Program, Department of Veterans Affairs Institutional Support Grant SMI 821-109, Sankyo Co., Ltd., Tokyo, Japan, and Novo Nordisk A/S, Dagsvaerd, Denmark.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Center for Diabetes Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8854. Tel.: 214-648-6742; Fax: 214-648-9191; E-mail: Runger{at}mednet.SWmed.edu.

    ABBREVIATIONS

The abbreviations used are: FA, fatty acid; FFA, free fatty acid; ACO, acyl CoA oxidase; CPT-1, carnitine palmitoyl transferase-1; PCR, polymerase chain reaction; FAS, fatty acid synthase; PPARalpha , peroxisome proliferator-activated receptor-alpha ; ZDF, Zucker diabetic fatty.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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