Liporegulation in Diet-induced Obesity

THE ANTISTEATOTIC ROLE OF HYPERLEPTINEMIA*

Young LeeDagger §, May-Yun WangDagger §, Tetsuya KakumaDagger , Zhuo-Wei WangDagger , Evelyn Babcock, Kay McCorkle||, Moritake HigaDagger , Yan-Ting ZhouDagger , and Roger H. UngerDagger ||**

From the Dagger  Gifford Laboratories, Touchstone Center for Diabetes Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8854, the  Department of Radiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9085, and the || Veterans Affairs Medical Center, Dallas, Texas 75216

Received for publication, September 19, 2000, and in revised form, November 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test the hypothesis that the physiologic liporegulatory role of hyperleptinemia is to prevent steatosis during caloric excess, we induced obesity by feeding normal Harlan Sprague-Dawley rats a 60% fat diet. Hyperleptinemia began within 24 h and increased progressively to 26 ng/ml after 10 weeks, correlating with an ~150-fold increase in body fat (r = 0.91, p < 0.0001). During this time, the triacylglycerol (TG) content of nonadipose tissues rose only 1-2.7-fold implying antisteatotic activity. In rodents without leptin action (fa/fa rats and ob/ob and db/db mice) receiving a 6% fat diet, nonadipose tissue TG was 4-100 times normal. In normal rats on a 60% fat diet, peroxisome proliferator-activated receptor alpha  protein and liver-carnitine palmitoyltransferase-1 (L-CPT-1) mRNA increased in liver. In their pancreatic islets, fatty-acid oxidation increased 30% without detectable increase in the expression of peroxisome proliferator-activated receptor-alpha or oxidative enzymes, whereas lipogenesis from [14C]glucose was slightly below that of the 4% fat-fed rats (p < 0.05). Tissue-specific overexpression of wild-type leptin receptors in the livers of fa/fa rats, in which marked steatosis is uniformly present, reduced TG accumulation in liver but nowhere else. We conclude that a physiologic role of the hyperleptinemia of caloric excess is to protect nonadipocytes from steatosis and lipotoxicity by preventing the up-regulation of lipogenesis and increasing fatty-acid oxidation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Compelling theoretical considerations coupled with corroborating experimental evidence argue against the conventional view that the physiologic role of leptin is to prevent obesity. First, plasma leptin levels of rodents and humans are low in the lean and high in the obese (1), hardly the credentials of an antiobesity hormone. Second, diet-induced obesity is not prevented in hypoleptinemic mice by restoring their plasma leptin levels to normal with recombinant leptin (2). Third, there is no evidence that overnutrition and obesity have ever posed a serious survival threat in evolution. On the contrary, the principal survival threat throughout evolution has been famine, against which obesity provides a measure of protection as the "thrifty gene" hypothesis maintains (3). Finally, it seems implausible to suggest that hormones evolve for the purpose of preventing the clinical consequences of their own deficiency. Just as insulin evolved to confer advantages in nutrient metabolism rather than to prevent diabetic ketoacidosis, leptin must have evolved, not to prevent its deficiency syndrome, obesity (4), but rather to confer a metabolic advantage that has not as yet been identified.

We previously had suggested that the metabolic advantage conferred by the hyperleptinemia of obesity might be the prevention of overaccumulation of triacylglycerols (TG)1 in nonadipose tissues (5). Clearly, leptin does have powerful antilipogenic activity in some such tissues (6). In the absence of leptin action, lipogenesis is increased and fatty-acid (FA) oxidation is reduced (7), accounting for the steatosis and lipotoxicity that occur in such circumstances (7-9). For example, in Zucker Diabetic Fatty (ZDF) rats with a loss-of-function mutation in the leptin receptors (10, 11), tissue TG ranges from 10 to 50 times the normal content (8) and is associated with functional impairment of pancreatic beta -cells (12, 13) and myocardium (9) and insulin resistance (14). Ultimately, the progressive overaccumulation of lipids causes death of cells in pancreatic islets and myocardium, resulting in diabetes and myocardial failure, which are the most serious complications of obesity. It has been proposed that the lipid overaccumulation enlarges the intracellular pool of fatty acyl-CoA beyond the oxidative requirements of the cell (15), thereby providing substrate for potentially destructive nonoxidative pathways, such as de novo ceramide formation (16) and lipid peroxidation (17, 18).

If the foregoing abnormalities develop in the absence of leptin action, it follows that leptin must be able to prevent them. Certainly hyperleptinemia induced by adenoviral transfer of the leptin gene has remarkable lipopenic and antilipogenic activity in tissues of normal rats, down-regulating the expression of genes involved in lipogenesis while up-regulating those genes involved in beta -oxidation and thermogenesis (19). Although they are consistent with putative antisteatotic activity of hyperleptinemia, such studies do not prove that the actual physiologic role of adipocyte-derived hyperleptinemia in obesity is to prevent the ectopic accumulation of TG in nonadipose tissues. This study was designed to test this premise.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals without Leptin Action-- Three groups of rodents were employed. Obese homozygous (fa/fa) ZDF-Drt rats, which are unresponsive to leptin because of a loss-of-function mutation in their leptin receptor (10, 11), and lean wild-type (+/+) ZDF controls were bred in our laboratory from ZDF/Drt-fa (F10) rats purchased from Dr. R. Peterson (University of Indiana School of Medicine, Indianapolis, IN). Two groups of mice, C57BL/6J-ob/ob and C57BL/KS-J-db/db, and their wild-type controls, C57BL/6J +/+ and C57BL/KS-J +/+ mice, were purchased from the Jackson Laboratory (Bar Harbor, ME).

Animals with Leptin Action-- To achieve diet-induced obesity in normal rats, Harlan Sprague-Dawley rats, purchased from Charles River Laboratories (Raleigh, NC) were employed. They were housed in individual metabolic cages (Nalgene, Rochester, NY) with a constant temperature and 12 h of light altering with 12 h of darkness. Body weight and food intake were measured weekly. Initially, all rats were fed standard chow (Teklad 4% mouse/rat diet, Teklad Madison, WI) ad libitum and had free access to water. At 4 weeks of age they either continued on this diet, which contains 24.8% protein, 4% fat, and 3.94 Kcal/g, or they were switched to a high fat diet (Purina Test Diet, Purina Mills, Inc., Richmond, IN) containing 60% fat, 7.5% carbohydrate, 24.5% protein, and 6.7 Kcal/g to produce diet-induced obesity.

Adenovirus Transfer of OB-Rb cDNA to Liver of fa/fa ZDF Rats-- In in vivo experiments containing a total of 1 × 1012 plaque-forming units of recombinant adenovirus containing the cDNA of the leptin receptor OB-Rb (AdCMV-OB-Rb) or as a control beta -galactosidase (AdCMV-beta -galactosidase), prepared as described previously (20), were infused into conscious animals over a 10-min period through polyethylene tubing (PE-50, Becton Dickinson) previously anchored in the left jugular vein of 9-week-old ZDF fa/fa rats under sodium pentobarbital anesthesia (20).

Expression of Wild-type and Mutated OB-Rb in Liver and Hypothalamus of fa/fa Rats-- To compare the expression of wild-type OB-Rb in fa/fa rats with mutated OB-Rb, total RNA of rat liver and hypothalamus were extracted using TRIzol reagent (Life Technologies, Inc.). Reverse transcription of total RNA was carried out after treating RNA samples with RNase-free DNase I. The first strand cDNA was then used to PCR-amplify an OB-RbcDNA fragment with OB-Rb-specific primers encompassing the region with the fa/fa mutation as described previously (11). The conditions of the 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. The amplified PCR products were digested with MspI at 37 °C 1 h and then run on a 1.2% agarose gel.

Northern Blot Analysis-- Total RNA was extracted by the TRIzol isolation method, and Northern blot analysis was carried out as described previously (21). cDNA probes for the oxidative enzymes, acyl-CoA oxidase (ACO) and liver-carnitine palmitoyl- transferase-1 (LIVER-CPT-1), were prepared by reverse transciptase-PCR using the following primers: ACO-sense (amino acids 2891-2910), 5'-GCCCTCAGCTATGGTATTAC-3' and ACO-antisense (amino acids 3505-3524), 5'-AGGAACTGCTCTCACAATGC-3' (GenBankTM accession number J02752); and LIVER-CPT-1-sense (amino acids 3094-3113), 5'-TATGTGAGGATGCTGCTTCC-3' and LIVER-CPT-1-antisense (amino acids 3703-3722), 5'-CTCGGAGAGCTAAGCTTGTC-3' (GenBankTM accession number L07736). The DNA fragment excised after digesting pAC CMV-OB-Rb (13) with KpnI/HindIII restriction enzymes hybridizes only the intracellular domain of OB-Rb was also used as a probe of OB-Rb. The hybridization signals were analyzed by Molecular Imager GS-363 (Bio-Rad). Values were normalized to the signal generated with an 18 S ribosomal RNA (rRNA) gene probe.

Multiplex Reverse Transcriptase PCR-- The procedure used was based on methods described by Jensen et al. (22) and O'Doherty et al. (23). Total RNA (1 µg) was treated with RNase-free DNase (Promega), and first-strand cDNA was generated with the oligo(dT) primer in the first-strand cDNA synthesis kit (CLONTECH). Multiplex reverse transcriptase-PCR was carried out in 25-µl reactions with 1.5 µl of the diluted cDNA reaction as template mixed with 23.5 µl of PCR mix containing 1.25 units of Taq polymerase and buffer (Roche Molecular Biochemicals) containing 25 µM dATP, dTTP, and dGTP, 2.5 µCi of 2500 Ci/mmol [alpha -33P]dCTP (1 CI = 37 GBq) (Amersham Pharmacia Biotech), and 5 pmol of each primer (Table I). The standard thermal cycle profile was as follows for lipogenic enzyme mRNA (FAS, ACC, and GPAT) and beta -oxidative enzyme gene mRNA (liver-CPT-1 and ACO): denaturation of 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min for 24 cycles in liver and for 26 cycles in islets.


                              
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Table I
Primers employed for multiplex reverse transcriptase-PCR

Reaction products were separated on 7 M urea, 1× TBE (0.1 M Tris base, 83 mM boric acid, 1 mM EDTA) and 6% polyacrylamide gels. Dried and PhosphorImager screens were scanned by a Molecular Imager System (GS-363). TATA box-binding protein mRNA was coamplified as an internal control, and data were expressed as ratios to its signal.

To avoid biased results caused by potential interference between individual amplicons, we analyzed the amplification kinetics of individual amplicons in reactions where several products were coamplified. Representative experiments, in which mRNA encoding lipogenic enzymes and TATA box-binding protein in pancreatic islets were simultaneously amplified, show the noncompetitive amplification of individual products and their almost identical rate of amplification as indicated by the slopes within the exponential phase observed from a linear regression analysis.

PPARalpha Immunoprecipitation-- 50 mg of liver from the rats were homogenized in 2 ml of lysate buffer with proteinase inhibitors. A total of 100 µg of protein in 0.5 ml of buffer were used for precipitation with 1:500 goat anti-PPARalpha (C-20, Santa Cruz Biotechnology, Inc. Santa Cruz, CA). Protein A beads from Amersham Pharmacia Biotech were used for binding. Immunoblotting was carried out with rabbit-anti-PPARalpha from Calbiochem at 1:1500.

Magnetic Nuclear Resonance Spectroscopy (MRS) and Magnetic Resonance Imaging-- Using the method of Stein et al. (24), proton MRS and magnetic resonance imaging data were obtained with a 4.7-T 40-cm-bore system (Omega chemical shift imaging model, Bruker Instruments, Fremont, CA) with a 6-inch-diameter birdcage coil. Anesthetized rats were placed supine within the coil and positioned in the center of the magnet. Proton spectra of the rat were resolved into water and fat resonances, the areas of which were quantified using the nuclear magnetic resonance software program NRM-1 (Tripos Associates, St. Louis, MO) assuming equal line widths for both resonances. Proton images were obtained from the abdominal region of each rat. Spin-echo transaxial images were acquired with the following parameters: two transients, recycle time = 500 ms, echo time = 16 ms, 2-mm slice thickness, 2-mm interslice gap, eight slices, a 140-mm field of vision, and a 128 × 256 matrix. Images were analyzed using NIH Image software (National Institute of Mental Health, Bethesda, MD).

TG Content of Tissues-- Animals were sacrificed under sodium pentobarbital anesthesia. Tissues were dissected and placed in liquid nitrogen. Total lipids were extracted from about 100 mg of tissue by the method of Folch et al. (25) and dried under N2 gas. TG was assayed by the method of Danno et al. (26).

Plasma Measurements-- Tail vein blood was collected in capillary tubes coated with EDTA. Plasma was stored at -20 °C. Plasma leptin was assayed using the Linco leptin assay kit (Linco Research, St. Charles, MO). Plasma glucose was measured by the glucose oxidase method using the glucose analyzer II (Beckman, Brea, CA). Plasma free fatty acids were determined using the Roche Molecular Biochemicals kit. Plasma TG levels were measured by the glycerol phosphate oxidase-Trinder triglyceride kit (Sigma).

[3H]Palmitate Oxidation in Pancreatic Islets-- Oxidation of [3H]palmitate by islets was determined as described previously. Groups of 100-200 islets were incubated in duplicate with 1 mM 9,10-[3H]palmitate for 3 days. Palmitate oxidation was assessed by measuring tritiated water in the medium. Excess [3H]palmitate was removed by precipitating twice with an equal volume of 10% trichloroacetic acid and 2% bovine serum albumin. Supernatants in a microcentrifuge tube were placed in a scintillation vial containing unlabeled water and incubated at 50 °C for 18 h. Tritiated water was measured as described for use of [3H]glucose (27).

[U-14C]Glucose Incorporation into Lipids in Islets-- Incorporation of [U-14C]glucose (14.6 mmol/liter, PerkinElmer Life Sciences) into lipids was measured in islets as described previously in detail (28). About 200 islets were cultured for 3 days in medium containing 8 mmol/liter of glucose. After 3 days in culture, lipids were extracted from the islets according to the method of Bligh and Dyer (29), and counts incorporated into total lipid were determined.

Statistical Analyses-- All values shown are expressed as mean ± S.E. Statistical analysis was performed by two-tailed unpaired Student's t test by one-way analysis of variance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Response of Leptin Levels to Caloric Excess-- If the function of leptin during caloric excess is to minimize the accumulation of lipids in nonadipose tissues, hyperleptinemia should begin promptly at the start of overnutrition and increase progressively as the overnutrition continues. To test this theory, a group of 10 normal male Harlan Sprague-Dawley rats was fed a diet in which 60% of the calories were derived from fat. Age-matched control rats received a 4% fat diet. Both groups were observed for 70 days. Plasma leptin levels in control rats were relatively unchanged, rising by only 0.04 ± 0.002 ng/ml/day to a level of only 2.80 ± 0.77 ng/ml on the final day of the 70-day study. By contrast, in rats on a 60% fat diet, plasma leptin rose to 4.3 ± 0.2 ng/ml (p < 0.001) within 24 h and increased progressively thereafter by 0.37 ± 0.07 ng/ml/day to a level of 26 ng/ml at 70 days (Fig. 1A). In this group the rise in plasma leptin levels paralleled the expansion in body fat mass quantified by MRS (Fig. 1B); there was a highly significant correlation between body fat and the plasma leptin level (r = 0.91, p < 0.0001) (Fig. 1C). Thus, leptin levels appear to respond promptly to a caloric excess, and they increase in proportion to enlargement of the adipose mass, which is consistent with the postulated role.



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Fig. 1.   Relationship of plasma leptin to total body fat in Harlan Sprague-Dawley rats receiving diets with a fat content of 4 (triangle ) or 60% (). A, plasma leptin levels in the two groups. The inset provides a view of the daily leptin profile for the first week of the study. B, total body fat measured by MRS. Representative spectral tracings and transrenal images are displayed. C, the relationship between plasma leptin levels and body fat.

TG Deposition in Nonadipose Tissues in the Presence of Leptin Action-- If the hyperleptinemia induced by high fat feeding does in fact protect nonadipose tissues of normal rats from overaccumulation of lipids, their tissue TG content should remain low during the development of obesity despite the expansion of the adipose tissue mass and the concomitant rise in plasma lipid levels. To test this theory, we measured tissue TG content of nonadipose tissues 70 days after the start of the high fat diet at which point the total body fat measured by MRS had increased an ~150-fold above the pre-diet base line (Fig. 1B), and plasma TG and free fatty-acid levels were significantly higher (Fig. 2A). However, TG content in nonadipose tissues increased only 1-2.7-fold above the base line (Fig. 2B). Thus, nonadipose tissues of leptin-responsive hyperleptinemic rats accumulated only a small fraction of the total increase in body fat acquired over 70 days of excessive fat intake, during which time the animals had became grossly obese (Fig. 1B).



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Fig. 2.   A, comparison of the mean (± S.E.) plasma leptin, TG, and free fatty-acid (FFA) levels in normal Harlan Sprague-Dawley rats fed a diet containing either 4 (open bar) (n = 6) or 60% (shaded bar) (n = 6) fat for 10 weeks. *, p < 0.001. B, the mean (± S.E.) tissue TG content of six normal Harlan Sprague-Dawley rats on a 60% fat diet at 4 weeks of age before starting the high fat diet (open bar) and at 14 weeks of age after 10 weeks on 60% fat intake (shaded bar). TG content in islets is expressed as ng/islet. In liver, heart and skeletal muscle, it is expressed as mg/g of wet weight of tissue. Body fat as determined by MRS is expressed as g/animal (gram). *, p < 0.001.

Mechanism of Antisteatotic Protection in Liver-- In the liver, protection against steatosis might involve not only increased secretion of very low density liprotein but also enhanced FA oxidation. In the latter case, an increase in the expression of PPARalpha and its target enzymes liver-CPT-1 and ACO might be expected (30). To determine whether the in vivo protection against hepatic overaccumulation of TG in normal rats on a high fat diet is mediated by this mechanism, we semiquantified PPARalpha protein and ACO and liver-CPT-1 mRNAs in livers of normal rats receiving either a 60 or a 4% fat intake. PPARalpha protein and liver-CPT-1 mRNA were both significantly greater in the former group, but ACO mRNA was not different (Fig. 3, A and B).



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Fig. 3.   Mechanism of antisteatotic protection of liver during high fat feeding of normal rats. A, PPARalpha protein measured by immunoprecipitation in the liver of four rats that were fed a diet containing 4% fat and four rats that were fed a 60% fat diet. B, mRNA of enzymes of fatty-acid beta -oxidation, ACO and liver-CPT-1, quantified by northern hybridization. *, p < 0.01

Mechanism of the Antisteatotic Protection in Islets-- Unlike liver, islets cannot export excess FA, which may account for their vulnerability in obesity. To determine the mechanism of the protection against lipid overaccumulation that prevails early in the course of obesity, we measured the rate of oxidation of [3H]palmitate in isolated islets of Harlan Sprague-Dawley rats receiving either a 4 or 60% fat diet. Oxidation was 30% greater in pancreatic islets of rats on the 60% fat diet than in controls that were on the 4% fat diet (Fig. 4A). However, unlike in liver, no change in ACO or liver-CPT-1 could be detected by multiplex-PCR (data not shown). These findings suggest that the preexisting oxidative machinery of the islets was able to accommodate this increase in oxidation without an increase in expression of genes encoding the enzymes.



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Fig. 4.   Mechanism of antisteatotic protection of islets during high fat feeding of normal rats. A, comparison of rates of oxidation of [3H ]palmitate in rats receiving a diet containing either 4 (open bar) or 60% (shaded bar) fat. *, p < 0.01. B, comparison of the rates of incorporation of [U-14C]glucose into lipids in normal Harlan Sprague-Dawley (SD) rats on a 4 (open bar) or 60% (shaded bar) fat intake. These rates are significantly less than in islets of hyperphagic fa/fa ZDF rats on a 6% (striped bar) fat intake. *, p < 0.01; §, p < 0.05. C, comparison of expression of the lipogenic enzymes acetyl-CoA carboxylase (ACC), fatty-acid synthase (FAS), and glycerol-PO4 acyltransferase (GPAT) in islets of normal Harlan Sprague-Dawley rats that were fed either a 4 or 60% fat diet. As an internal control for enzyme mRNA, TATA box-binding protein (TBP) was employed. *, p < 0.005; ¶, p < 0.01; §, p < 0.05.

We had previously reported that in the absence of leptin activity as in fa/fa ZDF rats, increased lipogenesis was the most important single factor in the ectopic overaccumulation of lipids in islets (7, 31). Accordingly, in normal rats the high fat diet should not induce the increase in lipogenesis and lipogenic enzymes that had been observed in fat-laden islets of the leptin-insensitive fa/fa rats. As shown in Fig. 4, B and C, there was no increase in the incorporation of [14C]glucose to lipids or in the expression of lipogenic enzymes. In fact, a small but significant decrease in lipogenesis and in fatty acid synthase mRNA was evident (Fig. 4, B and C). This was in sharp contrast to the ZDF fa/fa rats in which lipogenesis was 2.5 times greater.

Ectopic TG Deposition in the Absence of Leptin Action-- If the antilipogenic protection observed in normal rats during caloric excess did in fact require the action of the accompanying hyperleptinemia, rodent models with either a leptin deficiency (ob/ob mice) or a loss-of-function mutation in their leptin receptors (db/db mice and ZDF fa/fa rats) would be unprotected from lipid overaccumulation. We, therefore, measured the plasma leptin levels (Fig. 5A) and the TG content of islets, skeletal muscle, heart, and liver of these "unleptinized" rodents (Fig. 5B). Although their diet contained only 6% fat, the TG content of their nonadipose tissues ranged from ~4 to ~100-fold above normal controls on the same diet. Thus, when leptin action is lacking, protection from lipid overaccumulation in nonadipocytes is also lacking, even when the dietary fat intake is normal.



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Fig. 5.   A, comparison of mean (± S.E.) plasma leptin, TG, and free fatty acid (FFA) levels in rodents that are either leptin-deficient (ob/ob mice) or unresponsive to leptin because of loss-of-function mutation in its receptor gene (db/db mice and fa/fa rats) (shaded bar) and the corresponding wild-type (+/+) controls (open bar). *, p < 0.01. B, comparison of TG content in tissues of these rodents. All animals received a diet containing 6% fat. *, p < 0.001.

Overexpression of Wild-type OB-Rb in Livers of ZDF fa/fa Rats Prevents Steatosis-- If the marked hepatic steatosis and hypertriglyceridemia of obese ZDF fa/fa rats are the result of a lack of direct leptin action on the liver, transgenic overexpression of the wild-type leptin receptor in the liver of these leptin receptor-defective animals should protect them. Therefore, we infused into 9-week-old ZDF fa/fa rats 1012 plaque-forming units of recombinant adenovirus containing the cDNA of wild-type OB-Rb, the full-length isoform of the leptin receptor (AdCMV-OB-Rb). AdCMV-beta -galactosidase was infused into age-matched ZDF fa/fa rats as a control. The wild-type OB-Rb transgene introduced in vivo with an adenovirus vector was expressed exclusively in the steatotic liver of the ZDF fa/fa rats (Fig. 6A). None was detected in any other tissues including the hypothalamus.



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Fig. 6.   A, effect of intravenous infusion of obese ZDF fa/fa rats with AdCMV-OB-Rb or AdCMV-beta -galactosidase on the expression of wild-type receptor in the liver and hypothalamus. B, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of OB-Rb in liver and hypothalamus of a lean wild-type (+/+) ZDF rat, an untreated obese (fa/fa) ZDF rat, and an obese fa/fa rat 4 weeks after treatment with either AdCMV-beta -galactosidase or AdCMV-leptin. Whereas in the untreated and AdCMV-beta -galactosidase-treated fa/fa rat only mutated OB-Rb is present, in the AdCMV-leptin-treated fa/fa rat, the OB-Rb matches that of the +/+ rat. C, plasma TG after AdCMV-OB-Rb (black-triangle) or AdCMV-beta -galactosidase (open circle ). **, p < 0.01. D, triacylglycerol (TG) content of liver in obese ZDF fa/fa rats after treatment with AdCMV-OB-Rb (black bar) or AdCMV-beta -galactosidase (open bar). *, p < 0.05. E, heart TG and skeletal muscle TG after AdCMV-OB-Rb (black bar) or AdCMV-beta -galactosidase (open bar).

One week after treatment with AdCMV-OB-Rb plasma, TG levels of ZDF fa/fa rats declined slightly below pretreatment levels and remained significantly below the controls for 3 weeks after AdCMV-OB-Rb treatment (Fig. 5B). Liver TG content was significantly less than that of beta -galactosidase controls and untreated controls (Fig. 5C), the result of a delay in the increase in liver TG compared with the controls. TG contents of heart and skeletal muscle were unaffected (Fig. 6B). Food intake was identical in the two adenovirus-treated groups (29.8 ± 1.4 g/day versus 29.8 ± 1.5 g/day). Because the liver was the only site of expression of the normal OB-Rb in these ZDF fa/fa rats and the only site of antisteatotic action, we must assume that the elevated endogenous leptin levels, which averaged 24 ± 2 ng/ml in AdCMV-OB-Rb-treated rats and 28 ± 2 ng/ml in controls, exerted a direct antisteatotic action on the liver. This strongly implies that the function of hyperleptinemia of obesity is to prevent steatosis in tissues with functioning OB-Rb.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These findings suggest that a physiologic role of leptin during overnutrition is to protect nonadipocytes from the adverse consequences of lipid overaccumulation. This protection begins promptly at the start of overfeeding as the result of progressively increasing hyperleptinemia that continues to rise for the duration of hypernutrition. This appeared to minimize overaccumulation of lipids both by preventing the increase in lipogenesis that occurs in the absence of leptin action (31) and through the up-regulation of beta -oxidative metabolism of the surplus fatty acids (7). Whereas in the liver there was an increase in PPARalpha protein and CPT-1 mRNA, no such changes could be detected in pancreatic islets despite a 30% increase in the rate of [3H]palmitate oxidation. The greater induction of FA beta -oxidative enzymes in liver than in extrahepatic tissues confirms a recent observation by Cook et al. (31).

In islets the antilipogenic action of hyperleptinemia appears to be at least as important as the increase in FA oxidation in protecting islets from the lipid overload. When leptin action is lacking as in hyperphagic fa/fa ZDF rats, the fat-laden islets have a high rate of [14C]glucose incorporation into lipids in association with increased PPARgamma , acetyl CoA carboxylase, and fatty acid synthase expression on a 6% fat intake (32). By contrast, in normal rats receiving the 60% fat diet, these rates remained in the low normal range and the lipogenic rate declined. When the antilipogenic effect of leptin is lacking, lipogenesis is excessive and is not reduced by lipid excess, as in normal islets (30,32).

The most compelling evidence in support of the antisteatotic role for leptin was the in vivo demonstration in leptin-unresponsive fa/fa ZDF rats that transgenic overexpression of the wild-type receptor in their livers prevented the severe hepatic steatosis and hypertriglyceridemia that otherwise occurred. These findings are congruent with earlier evidence of the antisteatotic action of recombinant leptin (33) and of transplanted fat tissue in "fatless" mice with congenital lipodystrophy (34). Furthermore, in our experiments the wild-type leptin receptors were expressed only in the liver and not in the hypothalamus or anywhere else. Therefore, it follows that the endogenous hyperleptinemia of those obese fa/fa rats must have acted directly via the transgenic OB-Rb to prevent the lipid overaccumulation.

The prompt rise of plasma leptin levels on the very first day of the high fat diet and their high degree of correlation with the expanding body fat are all consistent with the response of an antilipogenic hormone with a physiologic liporegulatory mission, namely to maintain FA homeostasis in nonadipocytes during overnutrition. This protection may account for the fact that in hyperleptinemic rats and humans the lipotoxic complications of diet-induced obesity do not appear until late in life when leptin effectiveness wanes (35, 36). When leptin is absent as in congenital generalized lipodystrophy (33) or when leptin receptors are congenitally defective as in ZDF rats, these complications appear in severe form early in life.

It should be emphasized that we do not suggest that the direct antisteatotic activity ascribed to the endogenous hyperleptinemia of obesity occurs in normal lean animals. It appears to be a factor only during overnutrition when plasma leptin levels approach or exceed the threshold for transport across the blood-brain barrier, which is probably in the vicinity of 10 ng/ml (37). In the absence of overnutrition, plasma levels are below 5 ng/ml, and leptin action is presumed to be largely on the hypothalamic centers for control of food intake and thermoregulation (38).


    ACKNOWLEDGEMENTS

We thank Susan Kennedy for superb secretarial services. We also thank Drs. Cai Li and Daniel Foster for critical review of this manuscript. We thank Dr. Per Bo Jensen for expert help with multiplex reverse trnscriptase-PCR.


    FOOTNOTES

* This work was supported in part by the Department of Veterans Affairs Institutional Support (SMI 821-109), National Institutes of Health Grant DK02700-37, National Institutes of Health Juvenile Diabetes Foundation Diabetes Interdisciplinary Research Program, and Novo-Nordisc Corporation.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.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: Ctr. for Diabetes Research, University of Texas Southwestern Medical Ctr., 5323 Harry Hines Blvd., Dallas, TX 75390-8854. Tel.: 214-648-3488; Fax: 214-648-9191; E-mail: runger@mednet.swmed.edu.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008553200


    ABBREVIATIONS

The abbreviations used are: TG, triacylglycerol; FA, fatty acid; ZDF, Zucker Diabetic Fatty; PCR, polymerase chain reaction; AdCMV, adenocytomegalovirus; ACO, acyl-CoA oxidase; liver-CPT-1, liver-carnitine palmitoyltransferase-1; PPAR, peroxisome proliferator-activated receptor; MRS, magnetic nuclear resonance spectroscopy.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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