Leptin is one of the key molecules in maintaining energy homeostasis. Although genetically leptin-deficient Lepob/Lepob mice have greatly contributed to elucidating leptin physiology, the use of more than one species can improve the accuracy of analysis results. Using the N-ethyl-N-nitrosourea mutagenesis method, we generated a leptin-deficient Lepmkyo/Lepmkyo rat that had a nonsense mutation (Q92X) in leptin gene. Lepmkyo/Lepmkyo rats showed obese phenotypes including severe fatty liver, which were comparable to Lepob/Lepob mice. To identify genes that respond to leptin in the liver, we performed microarray analysis with Lepmkyo/Lepmkyo rats and Lepob/Lepob mice. We sorted out genes whose expression levels in the liver of Lepmkyo/Lepmkyo rats were changed from wild-type (WT) rats and were reversed toward WT rats by leptin administration. In this analysis, livers were sampled for 6 h, a relatively short time after leptin administration to avoid the secondary effect of metabolic changes such as improvement of fatty liver. We did the same procedure in Lepob/Lepob mice and selected genes whose expression patterns were common in rat and mouse. We verified their gene expressions by real-time quantitative PCR. Finally, we identified eight genes that primarily respond to leptin in the liver commonly in rat and mouse. These genes might be important for the effect of leptin in the liver.
- ENU mutagenesis
- Lepmkyo/Lepmkyo rat
- Lepob/Lepob mouse
- leptin responsive gene
- microarray analysis
leptin is an adipocyte-derived hormone that regulates energy homeostasis mainly through the hypothalamus (1). In addition to food intake and energy expenditure, leptin regulates glucose and lipid metabolism (5, 13, 20). Leptin-deficient Lepob/Lepob mice are well known to have made a great contribution in the discovery of leptin, and they have been used as an animal model of obesity and obesity-associated diabetes mellitus. Although much has been learned from Lepob/Lepob mice, it is important to use other models of different species to generalize results from one species.
For the last 30 yr, many investigators have chosen to use mouse models, because the technologies of embryonic stem cells allowed the generation of knockout and knock-in mice (7). However, rats have a long history in medical research, being fundamental from drug development to advances of neuroscience and physiology. Rats are considered to be a better model than mice in their behavioral and physiological characteristics, which are more relevant to humans.
Leprfa/Leprfa Zucker rats and Leprf/Leprf Koletzky rats already exist as rat models whose lack of leptin signals is due to mutations in the leptin receptor (22, 23). The phenotypes in these rats are also identical in many aspects to those in Lepob/Lepob mice (4). However, leptin receptor mutant rats are unfit models for the study of the effect of leptin treatment. Although generation of leptin knockout rats by pronuclear microinjections of zinc finger nucleases has been reported recently (26), the investigators did not assess the effect of leptin administration. So this is the first report of leptin administration in the leptin-deficient rat.
In this study, we used the N-ethyl-N-nitrosourea (ENU) mutagenesis method to generate a leptin-deficient Lepmkyo/Lepmkyo rat that had a nonsense mutation (Q92X) in the leptin gene (17). The mutation site of Lepmkyo/Lepmkyo rat was upstream of that of R105X in Lepob/Lepob mice (28). We confirmed that Lepmkyo/Lepmkyo rats secreted no measurable leptin in the serum and that their obese phenotypes including severely fatty liver were comparable to Lepob/Lepob mice. To identify genes that respond to leptin in the liver, we compared gene expressions in the liver between Lepmkyo/Lepmkyo rats and their wild-type (WT) littermates and also between leptin-treated and saline-treated Lepmkyo/Lepmkyo rats by microarray analysis. We sorted out genes whose expression levels in the liver of Lepmkyo/Lepmkyo rats were changed from WT rats and were reversed toward WT rats by leptin treatment. Moreover, to identify genes that commonly respond to leptin in the liver of rats and mice, we did the same series of experiments with Lepob/Lepob mice. We finally identified eight genes that commonly respond to leptin in the liver of rats and mice. Using more than one species can improve the accuracy of analysis results. Thus, genes identified in this study might be important for the effect of leptin in the liver.
MATERIALS AND METHODS
Rats with a leptin gene mutation were obtained by ENU mutagenesis of F344/NSlc rats, followed by MuT-POWER (Mu Transposition POoling method With sequencER) screening on the genomic DNA of 4,608 G1 male offspring in KURMA (Kyoto University Rat Mutant Archive). ENU mutagenesis procedures, screening protocols (17), and intracytoplasmic sperm injection procedure were previously described (12). The forward primer and the reverse primer used for identifying mutation of the leptin gene were 5′-GCACCTGTTGTTCCTCTTCC-3′ and 5′-TGGAATCGTGCGGATAACTT-3′, respectively. More than six backcross generations were performed against the F344/NSlc inbred background. Male C57Bl/6J Lepob/Lepob mice were purchased from CLEA Japan (Tokyo, Japan). Rats and mice were maintained on a 10 h light/14 h dark cycle (lights on 7:00 AM, lights off 9:00 PM) and fed ad libitum standard pellet diet (MF; Oriental Yeast, Tokyo, Japan). All animal care and experiments conformed to the Guidelines for Animal Experiments at Kyoto University and were approved by the Animal Research Committee of Kyoto University. The F344-Lepmkyo rat has been deposited into the National Bio Resource Project-Rat in Japan (NBPR-Rat no. 0628) and is available from the Project (http://www.anim.med.kyoto-u.ac.jp/nbr).
Genotyping for Lepmkyo mutation.
Genotyping for Lepmkyo mutation was performed by real-time PCR system using TaqMan Sample-to-SNP kit (Applied Biosystems, Carlsbad, CA) with a specific primer pair (forward primer sequences are 5′-GTTCTCCAGGTCATGAGCTATCTG-3′ and reverse primer sequences are 5′-CTGGCAGTCTATCAACAGATCCT-3′) and TaqMan MGB probes (WT probe sequences are 5′-TTGCCTTCCCAAAACG-3′ and mutant probe sequences are 5′-TTGCCTTCCTAAAACG-3′). Genomic DNA was extracted from whole blood. The cycling conditions were 20 s at 95°C followed by 40 cycles of 3 s at 95°C and 20 s at 60°C.
Whole body composition analysis.
Twenty-week-old male rats under anesthesia were scanned from nose to anus by computer tomography with La Theta LCT-100 (Aloka, Tokyo, Japan). The X-ray source tube voltage was set at 50 kV with a constant 1 mA current. Aloka software estimated the volume of adipose tissue, bone, air, and the remainder using differences in X-ray density. Distinguishing intra-abdominal adipose tissue and subcutaneous adipose tissue was based on detection of the abdominal muscle layers. Fat weight was calculated using the commonly used density factor of 0.92 g/cm3. This method provides accurate estimation of total subcutaneous and intra-abdominal fat pads as validated by dissection (11).
Measurement of body length and rectal temperature.
Body length was measured between nose and anus in 19-wk-old male rats under anesthesia. Rectal temperature was measured with a digital thermometer (BDT-100; Bio Research Center, Tokyo, Japan) at 10:00 AM in 19 wk old male rats.
Blood was obtained from the tail vein after overnight fasting at the age of 19 wk. Plasma leptin concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit for rat leptin (Millipore, St. Charles, MO). Plasma glucose concentrations were measured by a glucose assay kit (Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin concentrations were measured by an insulin-ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan). Plasma triglyceride, nonesterified fatty acid (NEFA), and total cholesterol concentrations were measured by enzymatic kits (Triglyceride E-test Wako, NEFA C-test Wako, and Cholesterol E-test Wako, respectively; Wako Pure Chemical Industries). To measure liver triglyceride contents, we sampled livers from 19 wk old rats and immediately froze them in liquid nitrogen. Lipids were extracted with isopropyl alcohol-heptane (1:1 vol/vol). After evaporating the solvent, we resuspended lipids in 99.5% (vol/vol) ethanol, and triglyceride content was measured by an enzymatic kit (Triglyceride E-test Wako, Wako Pure Chemical Industries).
Glucose tolerance test.
Intraperitoneal glucose tolerance test was performed after overnight fasting of 19 wk old male rats. Rats received 2.0 mg/g glucose by intraperitoneal injection. Blood was sampled from the tail vein before and 15, 30, 60, 90, 120 min after the glucose load.
Livers were sampled from 19 wk old rats, fixed in 10% neutrally buffered formalin, and subsequently embedded in paraffin. Histological sections of 5 μm thickness were stained with hematoxylin and eosin and examined by light microscopy.
Liver sampling for microarray analysis.
Recombinant murine leptin (generously supplied by Amgen, Thousand Oaks, CA) (1 μg/mg) or saline was intraperitoneally administered in 20 wk old Lepob/Lepob mice, Lepmkyo/Lepmkyo rats and their respective control littermates after 6 h fasting. Livers were sampled 6 h after leptin or saline administration and were frozen in liquid nitrogen and stored at −80°C until use for RNA isolation.
RNA extraction and microarray gene expression arrays.
Details for the sample preparation and microarray processing are available from http://www.affymetrix.com. RNA was prepared from liver of three male mice or rats per group using Trizol (Invitrogen, Carlsbad, CA) reagent following the supplier's protocol. The quality and the concentrations of the extracted RNA were checked using the Nano-Drop 2000 (Thermo Fisher Scientific, Yokohama, Japan). Each three RNA samples were pooled for analysis. We used 250 ng of total RNA for one cycle of cDNA synthesis. Hybridization, washing, and scanning of Affymetrix GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA) and Affymetrix GeneChip Rat Genome 230 2.0 Arrays were done according to standard Affymetrix protocols. The hybridized chips were scanned with an argon-ion laser confocal microscope (Hewlett-Packard, Palo Alto, CA). Fluorometric data were processed by Affymetrix GeneChip Command Console software and analyzed with Affymetrix Expression Console software 7. Normalization was performed with Affymetrix Microarray Suite 5.0 (MAS5.0) algorithm to obtain the signal intensity and the detection call, which shows the expression of a gene with a defined confidence level for each probe set. The mRNA expression levels for all genes on the array were scaled to a mean of 500 for each array. The detection call can be “present” when the perfect match probes are significantly more hybridized than the mismatch probes, false discovery rate (FDR) < 0.04, “marginal” for FDR > 0.04, and <0.06 or “absent,” FDR > 0.06.
Real-time quantitative RT-PCR.
Real-time quantitative RT-PCR was performed to validate expression levels of candidate leptin-responsive genes. Single-stranded cDNA was synthesized from 1 μg of total RNA using SuperScript III First-Strand Synthesis System for RT-PCR, according to the manufacturer's instructions (Invitrogen). Quantitative RT-PCR was performed with SYBR Green (Applied Biosystems) by Applied Biosystems StepOnePlus RT-PCR System using gene-specific primer. The housekeeping rat or mouse mitochondrial subunit 18S rRNA genes were used for control and quantitative RT-PCR was performed with TaqMan (Applied Biosystems). The sequences of primers (Sigma-Genosys, Tokyo, Japan) used in the present study are as follows: rat Ccl2 (F) 5′-TAGCATCCACGTGCTGTCTC-3′, rat Ccl2 (R) 5′-CAGCCGACTCATTGGGATCA-3′, mouse Ccl2 (F) 5′-AGATGCAGTTAACGCCCCAC-3′, mouse Ccl2 (R) 5′-GACCCATTCCTTCTTGGGGT-3′, rat Cdca3 (F) 5′-AACAAGCATGTGTCTCGGGT-3′, rat Cdca3 (R) 5′-TCTTGCGCCTGTTTGAGACT-3′, mouse Cdca3 (F) 5′-CCTCGTTCACCTAGTGCTGG-3′, mouse Cdca3 (R) 5′-TTTCGTCTGCTGCGCTTAGA-3′, rat Fkbp5 (F) 5′-ACTGACTCGCCTGACACAAG-3′, rat Fkbp5 (R) 5′-GAGCGAGGTATCTGCCTGTC-3′, mouse Fkbp5 (F) 5′-CTTGGACCACGCTATGGTTT-3′, mouse Fkbp5 (R) 5′-GGATTGACTGCCAACACCTT-3′, rat Inhbb (F) 5′-TCACGGTGACAGGTGGAATG-3′, rat Inhbb (R) 5′-CCGTTTTCGGATGCGATGTC-3′, mouse Inhbb (F) 5′-TGACCCACACTAGGCGAAAC-3′, mouse Inhbb (R) 5′-CAGGCCACTCGAAGGATTGT-3′, rat Lin7a (F) 5′-AAGCCACAAGAATCCGGAGA-3′, rat Lin7a (R) 5′-CTGACGACCAGCTTCACACT-3′, mouse Lin7a (F) 5′-AGTACCAGTGCACAAGCTCC-3′, mouse Lin7a (R) 5′-CATGTCTTTCAGCCACCCCT-3′, rat C-Myc (F) 5′-ATTCCAGCGAGAGACAGAGGGAGTG-3′, rat C-Myc (R) 5′-ACGTTGAGGGGCATCGTCGTG-3′, mouse C-Myc (F) 5′-CGCGCCCAGTGAGGATATC-3′, mouse C-Myc (R) 5′-CCACATACAGTCCTGGATGAT-3′, rat Npr2 (F) 5′-TTGGGGAGAGTCTACGAGCA-3′, rat Npr2 (R) 5′-CTCGGTACGTGATCACCAGG-3′, mouse Npr2 (F) 5′-CCAGAACTGCTTAGCGGGAA-3′, mouse Npr2 (F) 5′-CTGACCATTCCGCACCTTCT-3′, rat Ppl (F) 5′-AGTGCTGCCACTCAAGTACC-3′, rat Ppl (R) 5′-CAGCTCTCCCCGTTGTTCTT-3′, mouse Ppl (F) 5′-GCATGCTGAGTGGAAGGAGT-3′, mouse Ppl (R) 5′-AAGTCTGAGTCCACCTTGCG-3′, rat 18s (F) 5′-GCAATTATTCCCCATGAACGA-3′, rat 18s (R) 5′-CAAAGGGCAGGGACTTAATCAAC-3′, probe: 5′-AATTCCCAGTAAGTGCGGGTCATAAGCTTG-3′, mouse 18s (F) 5′-CGCGCAAATTACCCACTCCCGA-3′, mouse 18s (R) 5′-CGGCTACCACATCCAAGGA-3′, probe; 5′-CCAATTACAGGGCCTCGAAA-3′.
Data are expressed as means ± SE. Comparison between or among groups was assessed by Student's t-test or ANOVA with Fisher's protected least significant difference test. P < 0.05 was considered statistically significant. The χ2-test was used for analysis of genotype and sex ratio using Microsoft Excel. P < 0.05 was considered statistically significant.
Generation of a novel genetically obese rat with a homozygous nonsense mutation in the leptin gene.
By using ENU mutagenesis followed by MuT-POWER screening of the KURMA samples (17), we generated a genetically obese Lepmkyo/Lepmkyo rat with a homozygous nonsense mutation in the leptin gene. Lepmkyo mutation was a C-to-T transition at nucleotide 274 in the third exon of leptin gene, resulted to a substitution of glutamine at codon 92 by the stop codon (Q92X), which is upstream of the mutation (R105X) in Lepob/Lepob mice (28) (Fig. 1). Male and female Lepmkyo/+ rats were intercrossed to obtain WT, Lepmkyo/+, and Lepmkyo/Lepmkyo animals. There were 27 homozygous WT, 40 Lepmkyo/+, and 16 Lepmkyo/Lepmkyo rats. This ratio did not differ significantly from the expected 1:2:1 Mendelian ratio of genotypes (delivery n = 8, mean n of pups per delivery = 10.25; χ2 = 3.17, P = 0.999). The sex ratios also did not differ significantly from the expected ratio (male n = 42, female n = 40, χ2 = 2.42, P = 0.93).
Plasma leptin concentration and obese phenotypes in Lepmkyo/Lepmkyo rats.
ELISA did not detect plasma leptin in Lepmkyo/Lepmkyo rats (Fig. 2A). Serum leptin concentration in Lepmkyo/+ rats (7.81 ± 0.74 ng/ml) was slightly higher than half that of WT rats (12.17 ± 0.72 ng/ml). The body weight in Lepmkyo/Lepmkyo rats was significantly heavier than WT rats as early as 5 wk of age (Fig. 2B). The difference in body weight between WT and Lepmkyo/Lepmkyo rats steadily widened throughout the study period. The body weight in Lepmkyo/+ rats was always slightly heavier than that in WT rats although the difference was not statistically significant. As for the body length, there was no difference between WT and Lepmkyo/Lepmkyo rats (22.63 ± 0.13 cm in WT rats and 22.69 ± 0.24 cm in Lepmkyo/Lepmkyo rats, n = 7, P = 0.86), unlike Lepob/Lepob mice in which the body length is 5–10% shorter than that in lean littermates (3). The gross appearances of WT, Lepmkyo/+, and Lepmkyo/Lepmkyo rats at the age of 19 wk are shown in Fig. 2C. The body composition was examined with computer tomography (Fig. 2D). In Lepmkyo/Lepmkyo rats, the subcutaneous fat mass was nearly four times and the intra-abdominal fat mass was nearly twice of those in WT rats. Both subcutaneous fat mass and intra-abdominal fat mass in Lepmkyo/+ rats were slightly greater than those in WT rats. Daily food intake in Lepmkyo/Lepmkyo rats was increased by ∼50% compared with WT rats (Fig. 2E). The mean body temperature in Lepmkyo/Lepmkyo rats was significantly lower than that in WT rats (Fig. 2F). There was no significant difference between WT and Lepmkyo/+ rats in both food intake and body temperature.
Glucose and lipid metabolism in Lepmkyo/Lepmkyo rats.
We performed intraperitoneal glucose tolerance test (Fig. 3, A and B). Before glucose load, both plasma glucose and insulin concentrations in Lepmkyo/Lepmkyo rats were already increased when compared with those in WT rats. Plasma glucose concentration in response to the glucose load in Lepmkyo/Lepmkyo rats was also significantly higher than WT rats. Moreover, the increment of plasma insulin concentration was sustained after glucose load in Lepmkyo/Lepmkyo rats. These results indicate that the main cause of the impairment of glucose tolerance in Lepmkyo/Lepmkyo rats was insulin resistance. No significant difference between WT and Lepmkyo/+ rats in both plasma glucose and insulin concentrations was observed during glucose tolerance test.
Compared with WT rats, the fasting plasma triglyceride concentration in Lepmkyo/Lepmkyo rats was markedly elevated (Fig. 3C). The NEFA concentration was also increased in Lepmkyo/Lepmkyo rats although there was no significant difference (Fig. 3D). Plasma total cholesterol concentration was significantly increased in Lepmkyo/Lepmkyo rats (Fig. 3E). There was no significant difference between WT and Lepmkyo/+ rats in any of these plasma lipid concentrations.
Liver phenotype in Lepmkyo/Lepmkyo rats.
In Lepmkyo/Lepmkyo rats, the liver was markedly enlarged and lighter in color than that in WT rats. Histological examination of the liver showed large number of lipid droplets of various sizes in Lepmkyo/Lepmkyo rats (Fig. 3F). There was no accumulation of lipid droplets in Lepmkyo/+ rats. Consistent with these observations, both liver weight and liver triglyceride content in Lepmkyo/Lepmkyo rats were markedly increased compared with those in WT rats (Fig. 3, G and H). There was no significant difference between WT and Lepmkyo/+ rats in both liver weight and liver triglyceride content.
Identification of leptin-responsive genes in the liver by microarray analyses.
To identify leptin-responsive genes in the liver, we compared gene expressions in the liver between Lepmkyo/Lepmkyo rats and their WT littermates, as well as leptin-treated and saline-treated Lepmkyo/Lepmkyo rats, by the microarray method. In the leptin administration experiment, to avoid the effect of chronic metabolic changes related to food intake and body weight suppressions by leptin, we administered leptin and started rats fasting at the same time, 6 h before the liver sampling. Moreover, to identify genes that commonly respond to leptin in the liver in rats and mice, we did the same series of experiments with Lepob/Lepob mice. We confirmed that there was no significant difference in body weight and plasma glucose, insulin and triglyceride concentrations between leptin-treated and saline-treated Lepmkyo/Lepmkyo rats and also between leptin-treated and saline-treated Lepob/Lepob mice (data not shown).
Of 31,042 genes in the rat array chip, we excluded from analysis 18,425 genes with no nomenclature, which could not be found in the mouse array chip, and whose detection calls were not present in any of WT, Lepmkyo/Lepmkyo, and leptin-treated Lepmkyo/Lepmkyo rats. Of the remaining 12,617 genes, 177 genes whose expressions were decreased >1.6-fold in Lepmkyo/Lepmkyo rats relative to WT rats and increased >1.6-fold in leptin-treated Lepmkyo/Lepmkyo rats relative to saline-treated Lepmkyo/Lepmkyo rats were defined as leptin-upregulated genes in rat. However, 138 genes whose expression levels were <100 in either WT or Lepmkyo/Lepmkyo rats were excluded. We defined 237 genes whose expressions were increased >1.6-fold in Lepmkyo/Lepmkyo rats relative to WT rats and decreased >1.6-fold in leptin-treated Lepmkyo/Lepmkyo rats relative to saline-treated Lepmkyo/Lepmkyo rats as leptin-downregulated genes in rat. In this case, 158 genes whose expression levels were <100 in saline-treated Lepmkyo/Lepmkyo rats were excluded from leptin-downregulated genes. Furthermore, among 39 leptin-upregulated genes in rat, six genes whose expression was decreased >1.6-fold in Lepob/Lepob mice relative to WT mice and increased >1.0-fold in leptin-treated Lepob/Lepob mice relative to saline-treated Lepob/Lepob mice were defined as leptin-upregulated genes common in rat and mouse. Among 79 leptin-downregulated genes in rat, 14 genes whose expression were increased >1.6-fold in Lepob/Lepob mice relative to WT mice and decreased >1.0-fold in leptin-treated Lepob/Lepob mice relative to saline-treated Lepob/Lepob mice were defined as leptin-downregulated genes common in rat and mouse.
Expression patterns of these 20 leptin-regulated genes common in rat and mouse were examined by quantitative RT-PCR using the same RNA samples used for microarray analysis. Among six leptin-upregulated genes, Lin7a and Npr2 showed a similar expression pattern in microarray and quantitative RT-PCR analysis (Table 1, Fig. 4A). Among 14 leptin-downregulated genes in rats, Ccl2, Cdca3, Fkbp5, Inhbb, C-Myc, and Ppl showed similar expression patterns between microarray and quantitative RT-PCR analysis (Table 1, Fig. 4B). These eight genes are expected to play some role in the effect of leptin on the liver.
All microarray data have been deposited at the National Center for Biotechnology Information in the Gene Expression Omnibus database (GEO, http://www.ncbi.nlm.nih.gov/geo/). The series accession number is GSE42532. The GEO platform accession number is GPL1261 in mouse, GPL1355 in rat. The sample accession numbers are GSM 1044286, 1044287, 1044288, 1044289, 1044290, and 1044291.
Using gene-driven ENU mutagenesis, we generated leptin-deficient Lepmkyo/Lepmkyo rat. The mutation of leptin gene in Lepmkyo/Lepmkyo rats is located at nucleotide 274 in the third exon of leptin gene, generating a stop codon at amino acid 92 (Q92X), which is upstream of the mutation (R105X) in Lepob/Lepob mice (28). Leptin contains two cysteine residues, Cys96 and Cys146, which form a disulfide bond. This disulfide bond was shown to be required for the leptin action (27). Since both sites of nonsense mutation in Lepmkyo/Lepmkyo rats and Lepob/Lepob mice were projected to disrupt this disulfide bond, Lepmkyo/Lepmkyo rats were considered to have no functional leptin, as well as Lepob/Lepob mice.
The mean mutation frequency with ENU mutagenesis of our protocol was one mutation per 3.7 million base pairs (17). Although the chance for the occurrence of an unexpected mutation with a phenotypic effect is relatively small, this possibility also should be taken account for the experimental design and interpretation of the results. To eliminate mutations that might have been generated by ENU in chromosomal regions other than the Lep locus, we performed backcross more than six generations against F344/NSlc inbred background, and we always compared phenotypes between littermates to minimize the effect of possible unexpected mutation.
Lepmkyo/Lepmkyo rats showed morbid obesity with hyperphagia and low body temperature, hyperglycemia with hyperinsulinemia, dyslipidemia, and severely fatty liver. Low body temperature suggests decreased sympathetic nervous activity and basal metabolism in Lepmkyo/Lepmkyo rats. The glucose tolerance test demonstrated marked glucose intolerance with sustained hyperinsulinemia in Lepmkyo/Lepmkyo rats, suggesting insulin resistance. All these phenotypes are consistent with the lack of functional leptin and are identical to those in Lepob/Lepob mice (4). There are some existing rat models, such as Leprfa/Leprfa Zucker rats and Leprf/Leprf Koletzky rats, whose lack of leptin signals is due to mutations in the leptin receptor (22, 23). Although the phenotypes of these rats are also identical to Lepmkyo/Lepmkyo rats, they are not useful in studying the effect of leptin treatment. In this study, we used Lepmkyo/Lepmkyo rat as a leptin-treatable rat model and showed its usefulness.
Generation of leptin knockout rats by pronuclear microinjections of zinc finger nucleases had been reported recently (26). In addition to the metabolic phenotype, those leptin knockout rats with Sprague-Dawley background showed significant increase in bone mineral density and bone volume of the femur compared with WT littermates like ob/ob mice (26). However, we could not detect any significant difference in bone mineral density and gross observation of trabecular in the femur by computer tomography between Lepmkyo/Lepmkyo rats and their WT littermates (data not shown). Our result is consistent with the previous report on leptin receptor mutated fa/fa rats with Zucker background (24). These results indicate the strain difference in the regulation of bone mineral density. Furthermore, there is a previous report that bone mass in patients with congenital leptin deficiency have normal or low bone mineral density (18), which indicates the species difference. It was also reported that T-cell count was decreased in those leptin knockout rats. In all previous reports of leptin or leptin receptor deficiency in humans (9), mice (16), and rats (25), T-cell count was also decreased. As for the phenotype of T-cell, there is no difference between strains or species, although we did not count the T cells in Lepmkyo/Lepmkyo rats.
Besides the antiobesity effect, leptin has a wide range of metabolic effects including an insulin-sensitizing action. However, the molecular mechanism underlying metabolic effects of leptin is not well understood. We and others have demonstrated that leptin effectively improves insulin sensitivity accompanied by a dramatic reduction of fat content in the liver and skeletal muscle in patients with lipodystrophy in which severely fatty liver and excess fat accumulation in the skeletal muscle frequently develop (5, 6, 8, 15). In this study, to investigate the molecular mechanism by which leptin reduces fat content in the liver, we used two different species models with leptin deficiency, Lepmkyo/Lepmkyo rats and Lepob/Lepob mice.
Some microarray analyses on leptin effects in the liver using Lepob/Lepob mice have been reported (2, 14, 19). In these studies, leptin was administrated daily or continuously and the sampling time point was a few days or more after the beginning of leptin treatment. Under these conditions, the investigators could not separate the secondary effects of chronic metabolic changes, such as body weight reduction and improvement of insulin resistance and dyslipidemia, from the primary effects of leptin. In this study, we sampled livers 6 h after leptin administration under fasting. We confirmed that there was no significant difference in body weight and plasma glucose, insulin and triglyceride concentrations between leptin-treated and saline-treated Lepmkyo/Lepmkyo rats and also between leptin-treated and saline-treated Lepob/Lepob mice. Of course, 6 h after leptin administration is also the limitation of this study. It might be too early to detect leptin-responsive genes. However, we placed priority on excluding secondary effects. As a result, the profile of leptin-responsive genes in the previous report is quite different from that in the present study. In all these previous reports, expression of metabolism-related genes was detected. On the other hand, in our study, all genes in which we detected change of expression were not known for their relationship with glucose or lipid metabolism in the liver. The genes identified in our study are early responsive ones. Identification of such genes might unveil the mechanism of leptin action that has been unknown so far. The mechanistic study of the genes we have identified here is an issue for the future.
In conclusion, we generated leptin-deficient Lepmkyo/Lepmkyo rats by gene-driven ENU mutagenesis. Taking advantage of leptin-treatable Lepmkyo/Lepmkyo rats, we compared gene expressions in the liver not only between Lepmkyo/Lepmkyo and WT rats, but also between leptin-treated and saline-treated Lepmkyo/Lepmkyo rats, to identify leptin-responsive genes in the liver by microarray analysis. Taking Lepmkyo/Lepmkyo rats together with the Lepob/Lepob mice, we finally identified two leptin-upregulated genes, lin-7 homolog A (Lin7a) and natriuretic peptide receptor 2 (Npr2), and six leptin-downregulated genes, chemokine (C-C motif) ligand 2 (Ccl2), cell division cycle associated 3 (Cdca3), FK506 binding protein 5 (Fkbp5), inhibin beta B (Inhbb), myelocytomatosis oncogene (C-Myc), and periplakin (Ppl). Although, little is known about the physiological significance of most of these genes in energy metabolism in the liver, the analysis of these genes will bring further understanding of leptin physiology in the future.
This work was supported by research grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, the apanese Ministry of Health, Labor and Welfare, Uehara Memorial Foundation, Grant-in-aid for Industrial Technology Research Grant Program in 2008 from the New Energy and Industrial Technology Development Organization of Japan (TM:08A02004a), and The European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. HEALTH-F4-2010-241504 (EURATRANS).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: M.A.-A., K.E., and K.N. conception and design of research; M.A.-A., K.E., C.E., T.M., A.T., and T.T. performed experiments; M.A.-A. and K.E. analyzed data; M.A.-A., K.E., T.M., T.K., Y.Y., D.A., S.Y.-K., T. Sakai, and K.H. interpreted results of experiments; M.A.-A. prepared figures; M.A.-A. and K.E. drafted manuscript; M.A.-A., K.E., and T. Serikawa edited and revised manuscript; T. Serikawa and K.N. approved final version of manuscript.
We thank Keiko Hayashi for technical assistance. The authors also acknowledge Yoko Koyama for secretarial assistance.
- Copyright © 2013 the American Physiological Society