The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2299-2306

Phenotypic Consequences of a Nonsense Mutation in the Leptin Receptor Gene (fa^k) in Obese Spontaneously Hypertensive Koletsky Rats (SHROB)^1,2,3

Tatsuya Ishizuka, Paul Ernsberger, Sha Liu, David Bedol, Timothy M. Lehman, Richard J. Koletsky^*, and Jacob E. Friedman⁴

Departments of Nutrition and ^* Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The genetically obese Koletsky rat (SHROB, fa^k) has a novel point mutation of the leptin receptor at amino acid +763, resulting in a premature stop codon in the leptin receptor extracellular domain. This implies that all leptin receptor isoforms should be absent in this model. We examined the phenotypic consequences of this mutation on leptin and leptin receptor mRNA in hypothalamus and peripheral tissues from SHROB and their lean SHR littermates. Despite the mutation, mRNA for both the long (ObRa) and the short (ObRb) form were expressed at comparable levels in SHROB and SHR in brain and throughout peripheral tissues. Adipose tissue mRNA for leptin was two to three times greater in SHROB compared to SHR (P < 0.01), while circulating leptin concentration was 170 times greater than SHR littermates (P < 0.01), suggesting extreme leptin resistance in SHROB. Leptin was also detected in the cerebrospinal fluid (CSF) of SHR and SHROB (13.8 and 27.2 pmol/L, respectively); however, the CSF/plasma ratio for leptin was 32-fold greater in SHR than in SHROB. To assess the putative action of leptin and leptin receptors on insulin-mediated glucose transport, muscles from SHR and SHROB were incubated in vitro with recombinant human leptin. Leptin directly suppressed insulin-mediated glucose transport by 50% in skeletal muscle from SHR but not in obese SHROB rats lacking all forms of the leptin receptor. These results suggest that the natural leptin receptor knockout in the SHROB represents a unique rat model to define the functional role(s) of leptin in central and peripheral energy metabolism.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The obesity gene (ob) encodes a protein, leptin, which is secreted from adipose tissue and interacts with hypothalamic receptors to regulate appetite and energy expenditure (Campfield et al. 1996, Caro et al. 1996b, Hallas et al. 1995, Pelleymounter et al. 1995). Genetic defects in the leptin-hypothalamic feedback loop result in obesity and hyperinsulinemia in several rodent models: leptin deficiency in the ob/ob mouse (Zhang et al. 1994) and absent or reduced activity of the leptin receptor in the db/db mouse (Chen et al. 1996) and fa/fa rat (Chua et al. 1996), respectively. Circulating levels of leptin are very high in the last two models, consistent with oversecretion in the face of a receptor defect. These and other findings emphasize the importance of leptin in weight regulation. Levels of circulating leptin are increased in obese humans and in dietary-induced obese animals (Considine et al. 1995, Frederich et al. 1995, Maffei et al. 1995), suggesting that leptin resistance is a distinguishing feature of most obesities. The mechansims of leptin resistance are crucial to understanding human obesity. The hypothesis that leptin action is mainly limited by its transport across the blood-brain barrier has been suggested (Caro et al. 1996a). However, leptin resistance might also be a consequence of impaired signal-transduction by the leptin receptor, analagous to the post-receptor mechanisms of insulin resistance (Caro et al. 1996b, Tartaglia et al. 1997).

The obese spontaneously hypertensive Koletsky rat (SHROB)⁵ develops obesity, hyperlipidemia, hyperinsulinemia and proteinuric kidney disease (Koletsky 1972). The obese phenotype in the SHROB Koletsky rat results from a single homozygous recessive trait, originally designated f, shown to be allelic with the Zucker fatty trait (fa) but of distinct origin (Koletsky et al. 1996). The obesity mutation, designated fa^k, is a nonsense mutation of the leptin receptor gene, resulting in a premature stop codon in the leptin receptor extracellular domain at +763 (Takaya et al. 1996a, Yamashita 1997). As a consequence, none of the leptin receptor isoforms should encode a transmembrane domain necessary for signal transduction in SHROB. In contrast, the Zucker fatty (fa) rat has a missense mutation at position +269 coding for a different exon in the extracellular domain in the leptin receptor (Chua et al. 1996, Iida et al. 1996, Takaya et al. 1996b). The fa mutation reduced binding affinity of the leptin receptor (Yamashita et al. 1997), reduced its receptor density, and impaired responsiveness to exogenous leptin in fa/fa rats in vivo (Cusin et al. 1996). The obesity mutation fa^k is predicted to truncate all forms of the leptin receptor, leaving the SHROB incapable of signaling central and peripheral responses mediated by leptin. The highest levels of leptin receptors are expressed in the choroid plexus, suggesting that the receptor may mediate leptin transport from blood to cerebrospinal fluid (CSF) (Devos et al. 1996, Lynn et al. 1996). Although leptin enters the brain by a specific and saturable transport mechanism (Banks et al. 1996), the role of the leptin receptor isoforms in this process has not been clearly demonstrated.

The leptin receptor has two major forms resulting from alternative RNA splicing at the 3' end. Northern blot analysis has shown that the long form mRNA is present in the highest level in hypothalamus but can be detected in nearly all tissues at low levels using Rnase protection assay or polymerase chain reaction (PCR) (Lee et al. 1996, Tartaglia et al. 1995). The role of the long form of the leptin receptor in peripheral tissues is unknown but may be present in sufficient quantities to trigger important aspects of leptin receptor function. Leptin has been shown to directly inhibit insulin action in numerous tissues, such as liver (Cohen et al. 1996), pancreas (Emilson et al. 1997), white adipose tissue (Bai et al. 1996, Muller et al. 1997) and skeletal muscle (Muoio et al. 1997), suggesting the possible involvement of peripheral leptin receptors in nutrient partitioning and energy metabolism. To understand more about the impact of leptin receptors and to evaluate their role in the pathogenesis of obesity-related syndromes, we have described the phenotypic consequences resulting from a complete natural knockout of leptin receptors in the original obese Koletsky rat strain SHROB. Starting from increased leptin expression and massive expansion of adipose tissue depots, in this report we examined the expression of leptin, leptin receptor mRNA and plasma receptor binding proteins. In addition, to address the significance of peripheral leptin receptors to glucose metabolism, we determined the effects of leptin on insulin-stimulated glucose transport in skeletal muscle from rats with normal leptin receptors and in SHROB rats with the fa^k mutation.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  The SHROB (Koletsky rat) arose originally in 1970 at CWRU from mating of a female SHR and male Sprague-Dawley rat. Lean littermates from this original mating were then bred to form a closed self-sustaining colony which has been maintained by brother-sister mating for the last 26 y and at least 80 generations at CWRU. Experiments were conducted on 12-18-wk-old homozygous male and female SHROB rats (fa^k/fa^k). Age- and sex-matched hypertensive lean SHR littermates (Fa^k/fa^k or Fa^k/Fa^k) were selected as breeders based on genotyping by PCR genetic screening for the Tru-91 restriction site present in the leptin receptor gene, as outlined below. Animals were housed individually and were provided free access to food (Harlan-Teklad 8664, Madison, WI) and water. Rooms were on a 12:12 h light-dark cycle (lights on from 07:00 to 19:00) at a constant temperature of 21°C. These studies were carried out with the approval of the Case Western Reserve University Animal Care and Use Committee.

Reagents.  ¹²⁵I-Leptin, rat leptin and insulin RIA kits were obtained from Linco Research (St. Charles, MO). Recombinant human leptin was provided by Genentech, Inc. (South San Francisco, CA). This reagent has been proven to reduce food intake, and promote thermogenesis and progressive weight loss when injected into normal and ob/ob mice (Levin et al. 1996).

Genotyping.  High quality DNA for genotyping was obtained from a tail 0.5 cm biopsy. Each tail biopsy was transferred into an ependorf tube containing 1 mL of lysis buffer (100 mmol/L Tris-HCl, pH 8.0; 5 mmol/L EDTA; 0.2% SDS; 0.9 mg Proteinase K) and incubated overnight at 55°C or until digested. Then, 1 mL of phenol/chloroform (1:1) was added to digest further and incubated for 5 min at 55°C. The samples were centrifuged at 15,000 × g for 5 min and the procedure was repeated in the same tube. The precipitate was washed with 50 uL 3 mol/L sodium acetate, pH 5.2, and 1 mL cold 100% ethanol and mixed overnight on a shaker at 4°C. The pellet was centrifuged for 10 min and washed with 1 mL 70% ethanol and resupended in 48 µL double distilled H₂O. The fa_k locus was assessed with a Tru-91 restriction enzyme, after amplification with sense 5'-ATGAATGCTGTGCAGTC-3' and antisense primers 5'-AAGGTTCTTCCATTCAAT-3' which flank the restriction site at +2261, coding for amino acid 763 (see Fig. 1).

View larger version (29K): [in this window] [in a new window]   Fig 1. Schematic representation of the leptin receptor cDNA (long form = ObRb and short form = Ob-Ra) in the obese SHROB fak/fak rat. The leptin receptor cDNA (+1 to +3490 base pairs) encodes a 5.1-kb mRNA encoding a receptor protein with a single transmembrane domain (TMD). The nonsense fak mutation (M) T  A stop at position +2289 within the extracellular domain corresponds to amino acid 763, while the Zucker rat (fa/fa) missense mutation is at amino acid 269. The specific nucleotide sequences used for the PCR primers for the long form (OBRb R) and short form (OBRa R) were designed corresponding to the unique C-terminal sequences (including the 3' untranslated region) produced by alternative splicing of the 3' exons of the receptor. The divergent sequences for each isoform are the result of splicing 3' exons downstream of the codon for amino acid 889 (Chen et al. 1996). The alternative splice site (A) is shown for the leptin receptor short form at position +2677 and a truncated alternative (putative soluble form) ObRe (E) is also shown.

Tissue, blood and CSF sampling.  Male SHROB and SHR between 12-18 wk of age were used in this study. Food was removed at 17:00 h, and the following afternoon the rats were anesthetized with ketamine (150 mg/kg) and acepromazine (5 mg/kg). Multiple tissues were removed and frozen immediately on dry ice. The hypothalamus, remaining brain, lung, heart, liver, spleen, kidney, muscle and adipose tissue were stored at 80°C until use. Blood samples were withdrawn from the vena cava of anesthetized animals, allowed to clot on ice and centrifuged for 10 min at 10,000 × g at 4°C, and the serum frozen at 70°C until assayed for leptin or insulin. CSF was obtained by inserting a 25-gauge butterfly needle into the cisterna magna. The needle was attached to a syringe and 100-150 µL of CSF was gently withdrawn. The samples of CSF were centrifuged to remove possible erythrocyte contamination. Samples estimated to have more than 0.01 µL of visible erythrocyte contamination were discarded. Equal volumes of CSF from three to four rats of each group were pooled, freeze-dried and reconstituted to obtain sufficient quantities for assay. The leptin concentration in CSF was assayed by spiking the sample with a known concentration of rat leptin standard and subtracting the leptin standard from the total leptin measured in the sample. Using this method, an estimated recovery of [gt]90% was obtained. Plasma insulin was measured with a radioimmunoassay kit (Linco, St. Charles, MO) using rat insulin standards and antibodies directed against rat insulin. Assays were conducted in duplicate and the intra-assay coefficient of variation was <5%. Plasma glucose was measured in whole blood by colorimetric glucose oxidase assay (One-Touch, Lifescan, Milpitas, CA).

RT-PCR.  RNA was isolated from frozen rat tissues using RNAzol B (Tel-test, Friendswood, TX). Tissue-specific expression of the leptin receptor short and long forms was evaluated by reverse transcriptase (RT)-PCR analysis of RNA isolated from tissues of SHROB and SHR rats. The positions of the primers on the ObR cDNA along with the mutation site in the SHROB are shown in Figure 1. A set of primer pairs corresponding to a segment of the leptin receptor intracellular domain was used to identify the long form. For the short form, a 5' primer corresponding to a segment of the extracellular domain was used while a 3' primer oligonucleotide primer corresponding to the alternatively spliced exon of the receptor short form was used. The reactions were performed with a sense primer (OB 5' F1 = 5'-CAGTGATATTTGGTCCTCTTC-3') and an antisense primer (OBRb R = 5'-GTTCCAAAAGCTCATCCAACCC-3') specific for long form and with a sense primer (OB5' F2 = 5'-AGTGAATGCTGTGCAGTC-3') and an antisense primer (OBRa R = 5'-TACTTCAAAGACTGTCCGCTC-3') for the short form. Reverse transcriptions were performed on 2 µg of total RNA isolated from multiple tissues obtained from SHR and SHROB. Single-strand cDNA was first reverse-transcribed with Moloney murine leukemia virus RT and Oligo dt. The reaction conditions were 94°C for 5 min followed by 34 cycles, each consisting of 1 min at 94°C, 1 min at 56°C, 3 min at 72°C, followed by single cycle extension for 10 min at 72°C, using Ampli-Taq. As a control for RNA quality and quantity, -actin mRNA was amplified from all RNA samples using oligonucleotides (5'-CGTAAAGACCTCTATTGCCAA-3' and 5'-AGCCATGCCAAATGTGTCAT-3'), based on the coding sequence of rat -actin.

Northern blot analysis.  Total RNA (20 µg) was isolated and size-fractionated in 0.9% agarose-formaldehyde gels and transferred to Gene-screen plus membrane filters. After washing with 2× SSC (0.3 mol/L NaCl, .03 mol/L sodium citrate), the filter was dried and baked for 2 h at 80°C. Membranes were hybridized with a leptin receptor antisense riboprobe obtained by RT-PCR as outlined above. A plasmid containing the OB Rb fragment was linearized with the enzyme Sac I, and antisense RNA was transcribed in vitro with T7 polymerase (MAXI script translation kit, Ambion, Austin, TX). Hybridization was carried out overnight at 42°C in 50% formamide, 5× SSPE (1× SSPE, 0.18 mol/L NaCl, 10 mmol/L NaHPO₄ and 1 mmol/L EDTA, pH 7.7), 2× Denhardt's solution, 100 mg/L yeast tRNA and 10% dextran sulfate. For leptin mRNA, RNA from multiple tissues from SHR and SHROB were run on a 0.9% agarose-formaldehyde gel, transferred to Gene-screen (Dupont, Boston, MA) membranes and hybridzed overnight with a leptin cDNA obtained by PCR from genomic DNA as outlined previously (Friedman et al. 1997a). The cDNA was labeled with [-³²P]dCTP to a specific activity of 8.2 MBq/µg DNA using a random-primed labeling kit according to the manufacturer's instructions (Boehringer Mannheim). After hybridization, the filters were washed at 55°C in 1:5 dilution of SSC containing 0.1% SDS and exposed overnight using X-omat film (Kodak, Rochester, NY). The image intensity of the autoradiogram was determined using a Sci-Scan 5000 laser densitometer (U.S. Biochemical, Cleveland, OH). Subsequent hybridization to -actin was used to correct for differences in RNA content/loading, and the results were expressed as a ratio relative to -actin.

Plasma leptin binding proteins.  One microliter of plasma from SHR, SHROB and Sprague-Dawley rats deprived of food overnight was incubated with 8 µL (0.005 kBq) ¹²⁵I-labeled rat leptin in 1 mL loading buffer (450 mmol/L Tris-HCl, 12% glycerol, 4% SDS, 2.5 mg/L Commassie blue G, 2.5 g/L Phenol red) at 37°C overnight and electrophoresed under nonreducing conditions in a 5% Tris-glycine gel. To test the specificity of ¹²⁵I-leptin binding to plasma, samples were incubated with 1 µg of recombinant leptin. The gel was dried under vacuum and subjected to autoradiography using Kodak XAR X-ray film (Rochester, NY).

Glucose transport assay.  The effect of leptin on glucose transport was measured in isolated rat epitrochlearis muscles as described previously (Friedman et al. 1997b) with certain modifications. Rats were deprived of food overnight and anesthetized with ketamine (150 mg/kg) and acepromazine (5 mg/kg), and the epitrochlearis muscle with tendon attached was isolated and removed from both forelimbs. The muscles were preincubated at 29°C in 2 mL of Krebs-Henseleit bicarbonate (KHB) buffer containing 1% BSA, 39 mmol/L mannitol, 1 mmol/L pyruvate and leptin at 0, 3, 30 or 100 nmol/L. The samples were gassed continuously with 95% CO₂/5% CO₂ in a shaking water bath at 60 cycles per min. After 1 h muscles were transferred to fresh incubation media with either 0 or 20 U/L bovine insulin and incubated for an additional 30 min. After incubation the muscles were transferred to transport assay medium containing KHB with 10 g/L BSA, 8 mmol/L 3-O-methyl-D-glucose, 1 mmol/L pyruvate, 9.25 MBq/mmol 3-O-[³H]MG, 31 mmol/L mannitol and 370kBq/mmol [1-¹⁴C]mannitol containing leptin and incubated at 37°C for 10 min. Muscles were then rinsed in KRB at 0°C for 10 min to reduce extracellular label. The tissues were then quickly removed, trimmed of connective tissue, blotted on gauze, and immediately freeze clamped. Frozen muscles were weighed and digested in 0.5 mL 1 mol/L KOH for 30 min at 70°C and neutralized with 0.5 mL 1 mol/L HCl. A 0.3-mL aliquot of the supernatant was added to 5 mL of Cryoscint liquid scintillation fluid (ICN, Costa Mesa, CA). The specific activity of the incubation media was obtained using 50-µL samples obtained from each well. The incubation media samples were added to 950 µL 1 mol/L KOH-HCl solution similar to the muscle digest, and all samples were counted for radioactivity in a Beckman LS 8100 Liquid Scintillation Counter with dual quench correction. The rate of 3-O-[³H]MG transport was expressed in nanomoles per milligram wet tissue per 10 min, after correction for extracellular 3-O-[³H]MG. The differences between insulin-stimulated SHR vs. SHROB were analyzed by one-way ANOVA. A second one-way ANOVA was used to compare the insulin-stimulated transport rate with insulin + leptin treatment.

Statistical analysis.  Results are presented as means ± SEM for the indicated number of rats. Comparisons between SHR and SHROB were made using Student's unpaired t test. Statistical significance was set at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

Metabolic profile of SHROB and SHR rats.  Table 1 summarizes the metabolic phenotypes of the rats used in these studies. SHROB were 67% heavier than SHR. The Lee index, an established measure of rodent obesity, was greater in SHROB. The body mass index, used to assess human obesity, was 66% greater in SHROB. Daily energy intake from the standard nonpurified diet was 38% greater in SHROB relative to SHR, but intake per unit of body weight did not differ. Marked fat deposition occurs throughout the body in SHROB, especially in the subscapular region, which exceeded 40 g in SHROB but cannot be accurately estimated in SHR littermates. Retroperitoneal, gonadal (epididymal/endometrial) and mesenteric fat pads were heavier in SHROB than in SHR by three- to eightfold. The liver was also enlarged and mottled by visible lipid deposition.

View larger version (11K): [in this window] [in a new window]   Fig 2. Plasma and CSF leptin concentrations in SHROB and SHR. Data for plasma leptin are mean ± SE from male SHR (n = 21) and SHROB (n = 17) at 12-16 wk of age. CSF leptin concentrations are the results of pooled samples containing equal volumes of CSF from four rats in each group. The ratio of CSF to plasma leptin concentrations was multiplied by 100 for convenience. Please note that plasma leptin levels are expressed on a logarithmic scale. *P < 0.001, SHROB > SHR.

View larger version (32K): [in this window] [in a new window]   Fig 3. Autoradiograph of Northern blot reflecting levels of leptin mRNA in subcutaneous adipose tissue from lean SHR and obese SHROB rats at 16 wk of age. Total cellular RNA was isolated from adipose tissue and leptin, and -actin mRNA was detected as described under Experimental procedures. A representative hybridization from a Northern blot of three different rats is shown and ethidium bromide staining of RNA included. A quantification of results from seven SHR and eight SHROB rats from each group is presented relative to hybridization to -actin. Data are mean ± SEM.*P < 0.05, SHROB > SHR.

Serum insulin levels in starved rats were elevated by >20-fold in the SHROB than in the lean SHR, and differences in postprandial levels were proportionately greater. This degree of hyperinsulinemia implies extreme insulin resistance, but SHROB were not hyperglycemic. Postprandial blood glucose levels were marginally but significantly elevated in SHROB, denoting slight glucose intolerance. There was a 100% elevation in total cholesterol and a nearly eightfold increase in serum triglycerides.

The plasma leptin concentration in food-deprived SHROB was >170-fold higher than in SHR (Fig. 2). There were no significant differences detected between lean heterozygous (Fa^k/fa^k) and lean homozygous (Fa^k/Fa^k) rats (data not shown). The average concentration of leptin in pooled samples of CSF was also higher in SHROB than in SHR. When the data were expressed as a ratio of leptin in plasma to that in the CSF, SHR had a 32-fold greater ratio compared to SHROB, suggesting reduced efficiency of transport across the blood brain barrier.

Leptin mRNA expression in SHROB and SHR.  The level of ob mRNA in subcutaneous adipose tissue was elevated twofold in SHROB compared to SHR (Fig. 3). Similar increases (two- to threefold) were found in retroperitoneal, mesenteric and subscapular adipose tissue (data not shown).

Leptin receptor mRNA expression in SHR and SHROB.  Amplification with primers specific for the OBRa and OBRb yielded amplified products of predicted size (485 bp for OBRa, 473 bp for OBRb) in a variety of tissues (Fig. 4). A high abundance of leptin receptor long form was present in the hypothalamus and whole brain at comparable levels in the SHR and SHROB. The long form was also expressed at lower levels in lung, spleen and fat tissue and is detectable in heart, liver, kidney and muscle. The shorter isoform was detectable in abundance in all tissues examined. To confirm that the leptin receptor long form was expressed in the hypothalamus of the SHROB rat, a Northern blot analysis was carried out using a riboprobe specific long form of the leptin receptor. As expected from the RT-PCR data, leptin receptor expression corresponding to the 5.1-kb mRNA was detected at comparable levels in the hypothalamus of SHR and SHROB (data not shown).

View larger version (53K): [in this window] [in a new window]   Fig 4. (A) Tissue distribution of leptin receptor ObRa (short form) and ObRb (long form) in SHR and SHROB. Amplified products were detected by RT-PCR using primer pairs described in Figure 1. RT-PCR analysis of actin mRNA is included as a control for RNA quality and amount. Lanes contain RNA amplified from the following tissues: A, hypothalamus; B, brain; C, heart; D, lung; E, liver; F, spleen; G, kidney; H, muscle; I, fat. The size of the amplified bands was estimated by alignment with 100-bp DNA ladder markers.

Radioligand binding of leptin in serum.  Under nonreducing conditions, ¹²⁵I-leptin bound to serum proteins from lean rats with apparent molecular masses of 215, 168, 139, 114, 60 and 41 kD (Fig. 5). Binding to these bands was completely displaced by addition of 1 µg unlabeled leptin. Binding of ¹²⁵I leptin to serum at 168, 139 and 114 kD was markedly elevated by four- to 19-fold in serum from SHROB compared to lean SHR or Sprague-Dawley rats.

View larger version (79K): [in this window] [in a new window]   Fig 5. Radioligand binding of 125I-leptin to plasma proteins in SHR, SHROB and Sprague-Dawley (SD) rats. One microliter of plasma was incubated with 125I-leptin at 37°C overnight and electrophoresed under nonreducing conditions in a 5% Tris-glycine gel. To test the specificity of 125I-leptin binding to plasma, additional samples were incubated with 125I-leptin + 1 µg of recombinant leptin. The gel was dried under vacuum and subjected to autoradiography using Kodak XAR X-ray film.

Effects of leptin on insulin-stimulated glucose transport.  Basal glucose transport was not affected by leptin concentrations of 3, 30 and 100 nmol/L. However, leptin inhibited maximal insulin-stimulated 3-O-methylglucose transport by 52% (P < 0.05) in muscles from SHR rats at leptin concentration of 3 nmol/L (Fig. 6). No further effects were found using leptin concentrations of 30 or 100 nmol/L. In muscle from the SHROB rat, incubation with leptin up to 100 nmol/L had no inhibitory effect on insulin-stimulated glucose transport.

View larger version (24K): [in this window] [in a new window]   Fig 6. Effect of leptin on 3-O-methylglucose transport activity in epitrochlearis muscle of SHR and SHROB rats. Muscles from SHR (n = 9) and SHROB (n = 11) were preincubated in Krebs Henseleit bicarbonate buffer containing 1 mmol/L pyruvate in the absence or presence of leptin (3 nmol/L). After 1 h the muscles were transferred to identical media with the further addition of either 0 or 20 U/L bovine insulin and incubated for 30 min with 8 mmol/L 3-O-methyl-D-glucose, 1 mmol/L pyruvate, 9.25 MBq/mmol 3-O-[3H]MG, 31 mmol/L mannitol and 370 kBq/mmol [1-14C]mannitol containing leptin and incubated at 37°C for 10 min. Data are mean ± SEM. a,bLike letters indicate that means differ significantly, P < 0.05.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Two unique single-gene obesity mutations, fa and fa^k, have been identified in rats. The fa^k mutation was long known to be allelic with the Zucker fatty (fa) rat mutation, since crosses with heterozygotes carrying the fa mutation with heterozygous fa^k rats yielded obese hybrid progeny (Yen et al. 1977). However, Zucker fatty rats have a missense mutation at position +269 coding for a different exon in the extracellular domain of the leptin receptor, resulting in lower sensitivity to leptin. The fa^k mutation (sometimes referred to as "corpulent," cp) lacks all forms of the leptin receptor and is predicted to produce a truncated receptor with no transmembrane domain and no cytoplasmic signaling domain. The fa^k mutation and has been crossed into other strains such as the SHR/N-cp rat (Marette et al. 1993) and SHHF/Mcc-fa^cp rat (McCune et al. 1996). The fact that hyperglycemia is observed in other crossed strains derived from the original Koletsky rat colony suggests that diabetes is not an intrinsic function of the fa^k mutation itself but likely requires polygenic interaction with other diabetogenic modifier genes present in the background of these other strains.

We found that despite the mutation by using RT-PCR we could detect leptin receptor Ra and Rb in multiple tissues at comparable levels in SHR and SHROB. Our results are similar to those of Takaya et al. (1996a) but differ from those of Wu-Peng et al. (1997) who detected the long form Rb but suggest they were unable to detect leptin receptor Ra (short form) in obese Koletsky rats. The reasons for this discrepancy are unclear, since the single base pair change at position 763 is located upstream from the alternative splicing site for the receptors at position 889 and would not be expected to affect the level of mRNA. Our finding of similar hybridization to leptin receptor mRNA Rb in hypothalamus using solution hybridization confirms that the mutation had no effect on the level of transcript in obese Koletsky rats.

There is good evidence that leptin circulates in plasma both as a free 16-kD protein and bound to multiple serum proteins. The identities of these circulating leptin-binding proteins are not currently known, although a secreted form of the leptin receptor is a logical candidate based on the existence of secreted forms of other cytokine-like receptors. However, the presence of a soluble leptin receptor in plasma has not been reported to date. The results of our serum protein binding assay seem to suggest that a soluble receptor-ligand complex with a predicted molecular wt of 106 kDa (90 kDa receptor + 16 kDa leptin) in SHR or SD serum or 100 kDa in the SHROB is not present in the serum of these animals. The molecular weight of the [¹²⁵I]-leptin binding proteins in the SHR and SHROB were nearly identical, showing that truncation of the leptin receptor in SHROB did not cause a shift in the apparent molecular weight of serum binding proteins. However, specific antibodies to the rat leptin receptor isoforms will be needed to confirm this. There is also the possibility of multimer bands of the same protein aggregating in this type of experiment. Several publications using mouse or human plasma have shown binding proteins of similar molecular weight to those seen in our experiment. These data are consistent with the presence of multiple serum leptin binding proteins in both the SHROB and SHR. Our data also show that the capacity, or possibly the affinity, of leptin binding to serum proteins is much greater in SHROB than SHR. The identity of the leptin binding proteins has not been elucidated; however, it has been suggested that serum leptin binding may influence some aspects of leptin signaling (Sinha et al. 1996).

One possible role of leptin receptor isoforms in the choroid plexus as well as the lungs and the kidneys may be in the transport or clearance function for leptin (Caro et al. 1996b, Devos et al. 1996). We have demonstrated that despite only a two- to threefold elevation in leptin mRNA expression in fat tissue, the levels of circulating leptin were elevated nearly 170-fold in the plasma of SHROB rats. The extremely high circulating leptin concentration relative to expression in fat mass suggests that the leptin receptor mutation may impair clearance of leptin from the circulation. Surprisingly, however, the level of leptin examined in the CSF of the SHROB rat was equal or slightly greater than in the lean animals, suggesting that despite the lack of complete leptin receptor protein in SHROB, leptin penetrated the blood brain barrier. The high level of leptin receptor expression in the chroid plexus and the presence of tight junctions represent a likely barrier for leptin transport into the brain. Other areas in the brain such as the median eminance of the hypothalamus and the choroid plexus, where capillaries are fenestrated, may allow leptin to freely diffuse into the interstitial space. However, evidence suggests that the long form of the leptin receptor is in the arcuate nucleus of the hypothalamus and is therefore isolated from the peripheral circulation by the blood-brain barrier (Banks et al. 1996, Caro et al. 1996b). Furthermore, despite the presence of leptin in the brain, the levels of leptin were greatly reduced as a ratio to the plasma concentration of leptin, suggesting impaired leptin transport into the brain in SHROB.

Obese humans with genetic mutation(s) in the leptin gene have recently been described (Echwald et al. 1997, Montague et al. 1997), suggesting the pathogenesis of obesity in humans may be linked to altered expression as well as function of leptin receptor in the brain and periphery. In addition to modifying food intake, work by others suggests that leptin's effect on energy metabolism may also be mediated by sympathetic nervous system activation (Haynes et al. 1997) as well as by direct action on peripheral tissues. Recombinant leptin has recently been shown to impair metabolic actions of insulin in isolated rat adipocytes, including decreasing glucose uptake and inhibition of lipolysis and lipogenesis (Muller et al. 1997). To examine whether the inhibitory effects of leptin on insulin-stimulated glucose transport can occur in animals that are functionally null for leptin receptor isoforms, epitrochlearis muscles from SHR and SHROB were incubated in vitro with increasing concentrations of recombinant human leptin. Leptin inhibited insulin-stimulated glucose transport in intact epitrochlearis muscle from SHR rats, but not in SHROB rats, lacking leptin receptors. These results are compatible with earlier findings demonstrating that leptin inhibits glucose metabolism in freshly isolated adipocytes (Muller et al. 1997) and inhibits insulin-stimulated insulin receptor substrate 1 phosphorylation and down-regulation of phosphoenol-pyruvate carboxykinase (PEPCK) mRNA in hepatocytes (Cohen et al. 1996). Muoio et al. (1997) showed that leptin directly attenuated both the anti-oxidative and lipogenic effects of insulin by 50% in isolated soleus muscle. By virtue of its mass, skeletal muscle accounts for the majority of whole body insulin-stimulated glucose uptake and lipid oxidation. Leptin may therefore be a regulator of nutrient partioning via its action on skeletal muscle lipid and carbohydrate metabolism. Since leptin lowers triglyceride content in tissues throughout the body (Halaas et al. 1995), these findings suggest the absence of peripheral leptin receptors may contribute to increased triglyceride storage in tissues, especially adipose of SHROB.

In summary, the obesity mutation fa^k in SHROB results in marked hyperinsulinemia and obesity with especially marked accumulation of subcutaneous and intra-abdominal fat depots. The SHROB is also marked by the complete absence of leptin receptor protein, despite a normal expression of the mRNA for leptin receptors in multiple tissues. As a consequence of the fa^k mutation, there is evidence for a deficiency of leptin transport across the blood brain barrier, and the levels of serum leptin are elevated approximately 170-fold above lean controls. The absence of leptin receptors in SHROB, particularly in skeletal muscle, an important site of whole body energy expenditure, may contribute to the development of obesity, possibly by limiting energy expenditure and favoring lipid deposition in multiple tissues throughout the body. We conclude that the natural leptin receptor knockout Koletsky rat SHROB may provide a useful experimental model for understanding the physiological role of leptin receptors in regulation of central and peripheral energy metabolism.

	FOOTNOTES

Manuscript received 9 February 1998. Initial reviews completed 10 April 1998. Revision accepted 4 August 1998.

	ACKNOWLEDGMENTS

We thank Kulwant S. Aulak for helpful discussions and expert technical advice regarding preparation of leptin receptor riboprobes.

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