Misato KOBAYASHI, 1;2; * Tamio OHNO, 3 Teruo KAWADA, 4 Hiroshi IKEGAMI, 2 Masahiko NISHIMURA, 3 and Fumihiko HORIO 1;5;y

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1 Biosci. Biotechnol. Biochem., 7 (3), , 26 Serum Adiponectin Concentration: Its Correlation with Diabetes-Related Traits and Quantitative Trait Loci Analysis in Mouse SMXA Recombinant Inbred Strains Misato KOBAYASHI, 1;2; * Tamio OHNO, 3 Teruo KAWADA, 4 Hiroshi IKEGAMI, 2 Masahiko NISHIMURA, 3 and Fumihiko HORIO 1;5;y 1 Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya , Japan 2 Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka , Japan 3 Division of Experimental Animals, Center for Promotion of Medical Research and Education, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya , Japan 4 Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto , Japan 5 Department of Food and Nutritional Sciences, College of Bioscience and Biotechnology, Chubu University, 12 Matsumoto-cho, Kasugai, Aichi , Japan Received September 2, 25; Accepted November 19, 25 Adiponectin is thought to be an important mediator of insulin sensitivity and atherosclerosis. Using mouse 19 SMXA recombinant inbred (RI) strains, a powerful tool for analyzing multifactorial genetic traits, we found relationships between serum adiponectin levels and diabetes-related traits, body mass index, and serum lipid levels, and also determined the loci controlling serum adiponectin levels by quantitative trait loci (QTL) analysis. RI strains exhibited widely ranging serum adiponectin concentration distribution patterns and diabetes-related traits. The serum adiponectin concentration showed the strongest negative correlation with fasting serum insulin concentration, but negative correlations were also observed with serum triglycerides, cholesterol, and liver weight. In contrast, neither the body mass index nor the blood glucose concentration correlated with serum adiponectin levels. These results suggest that hypoadiponectinemia might be used as a predictor of insulin resistance. In addition, two suggestive QTLs for serum adiponectin concentration were detected on Chromosome (Chr) 7, and an A/J allele at these loci was associated with elevated serum adiponectin concentrations. Identification of genes responsible for regulating the serum adiponectin concentration might lead to the development of novel treatments for patients with diabetes concomitant with hypoadiponectinemia. Key words: adiponectin; insulin resistance; diabetes; quantitative trait loci; SMXA recombinant inbred Adiponectin is composed of a carboxyl-terminal globular domain and an amino-terminal collagenous domain, 1,2) and circulates in serum as a hexamer of low molecular weight (LMW) and a large multimeric structure of medium (MMW) and high molecular weight (HMW). 3) In human serum, not only full-length adiponectin but also a cleaved fragment containing the globular head group has found. 4) Adiponectin is abundantly present in human blood (5 2 mg/ml), and concentrations of adiponectin have been reported to decrease with obesity. 5) In Pima Indians, hypoadiponectinemia is a strong predictor of the development of type 2 diabetes. 6) Genome-wide scans of human subjects have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to Chr 3q27, which contains the adiponectin gene. 7,8) A missense mutation of the adiponectin gene, which leads to reductions in the plasma adiponectin concentration, is associated with insulin resistance, diabetes, and atherosclerosis. 9) In ob/ ob mice and KK-Ay mice, animal models of obese type 2 diabetes, the serum adiponectin concentrations are low, and administration of adiponectin to these mice has been shown to improve insulin sensitivity. 1,11) Adipo- * M. Kobayashi is a Research Fellow of the Japan Society for the Promotion of Science. y To whom correspondence should be addressed. Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 12 Matsumoto-cho, Kasugai, Aichi , Japan; Tel: ; Fax: ; horiof@isc.chubu. ac.jp

2 678 M. KOBAYASHI et al. nectin-deficient mice have been shown to exhibit dietinduced insulin resistance, glucose intolerance, and increased neointimal formation in response to external vascular cuff injury. 12,13) Furthermore, putative antiatherogenic properties of adiponectin have been reported in apolipoprotein E-deficient mice, an atherosclerosis model. 14) These data suggest that adiponectin is an important mediator of insulin sensitivity and atherosclerosis. 15) Therefore, identification of the genes responsible for regulating serum adiponectin levels is considered crucial to elucidation of the pathogenesis of insulin resistance and atherosclerosis concomitant with hypoadiponectinemia; moreover, identification of these genes should help lead to the development of novel treatments for these diseases. In humans, however, it remains difficult to identify the genes that control target traits, in part because of the heterogeneity of genetic and environmental factors. Since genetic and environmental factors can be precisely controlled in analyses of inbred animal models, the use of inbred animal models is of great value for detailed characterization of the mechanisms that induce complex traits. We used mouse 19 SMXA recombinant inbred (RI) substrains established from parental SM/J and A/J strains. 16) RI strains are established by inbreeding of different sets of F 2 progeny from a cross between two inbred strains of mice. Each RI strain has a different combination of parental genomes in a homozygous state. 17) RI strains are a powerful tool not only for the analysis of single genetic traits, but also for that of multifactorial genetic traits. QTL analysis, a strategy for isolating multiple genes that individually account for small degrees of variance, was developed in order to identify chromosomal regions associated with particular traits. Using SMXA RI strains, previously we carried out QTL analyses of diabetes-related traits such as impaired glucose tolerance. 18,19) In the present study, we used 19 SMXA RI strains to help us to identify the relationship between serum adiponectin levels and other diabetes-related traits (e.g., body mass index and serum lipid levels), and these strains were also used to determine the loci responsible for the regulation of serum adiponectin levels. Materials and Methods Parental strains and SMXA RI strains. Parental SM/J, A/J, and 19 SMXA RI strains (SMXA-1, 4, 5, 8, 9, 1, 12, 14, 15, 16, 17, 18, 19, 21, 24, 25, 26, 27, and 3) were obtained from the Division of Experimental Animals, Graduate School of Medicine, Nagoya University (Nagoya, Japan). All mice were kept in a room maintained under conventional conditions at a controlled temperature of 23 3 C and a humidity of 55 5% on a 12-h light/dark cycle. After they reached 6 weeks of age, all mice were fed the following high-carbohydrate diet (g/kg feed) ad libitum for the next 11 weeks: casein, 12; carbohydrate (starch/sucrose, 1:1), 743; AIN93MX mineral mixture, 2) 35; AIN93VX vitamin mixture, 2) 1; choline chloride, 2; corn oil, 5; and cellulose powder (AVICEL type FD-11, Asahi Chemical Industry, Osaka, Japan.), 4. All procedures were performed in accordance with the Animal Experimentation Guide of Nagoya University. Analytic procedures for phenotype determination. Body weight and anal-nasal length of the mice were measured at 1 weeks of feeding the animals this highcarbohydrate diet. Body mass index (BMI) was calculated as body weight (g) divided by the square of the anal-nasal length (cm). Blood samples were obtained from the tail vein in non-fasting or fasting mice after 1 weeks of feeding. Serum samples were collected, centrifuged, and stored at 3 C until assay. The intraperitoneal glucose tolerance test (IPGTT) was performed at 1 weeks of feeding on this high-carbohydrate diet. After the mice had fasted for h, blood samples were collected at min (fasting blood glucose) from the tail vein of each mouse. Then each mouse was intraperitoneally injected with 2% glucose solution (2 g/kg body weight), and at 3 and 12 min after injection, additional blood samples were collected. Blood glucose concentrations were measured by the glucose oxidase method using a Glucose-B kit (Wako, Osaka, Japan). Serum triglycerides and cholesterol concentrations were measured by the glycerol-3-phosphate oxidase method using a Triglyceride-E kit (Wako), and by the cholesterol oxidase method using a Cholesterol-E kit (Wako) respectively. Serum immunoreactive insulin concentrations were measured by radioimmunoassay (ShionoRIA; Shionogi, Osaka, Japan) performed with rat insulin as the standard. Serum adiponectin concentrations were measured by enzyme-linked immunosorbent assay using a mouse Adiponectin ELISA Kit (Otsuka Pharmaceutical, Tokyo). Rabbit polyclonal antibody against mouse adiponectin used in this kit was generated using mouse recombinant full-length adiponectin. Quantitative trait loci analysis. The number of mice from each strain was 7 16, referring to the report by Belknap. 21) The phenotypic data collected for the SMXA RI strains (the average of each strain) were subjected to quantitative trait loci (QTL) analysis using Map manager QT b28. 22) The program is based on interval mapping using the free regression model. The strain distribution pattern (SDP) of 789 polymorphic markers reported for SMXA RI strains was used in the QTL analysis. 23) The positions of the microsatellite (Mit) markers were collected from the Ensembl Genome Browser ( in February 25. Significance was determined by 1, permutations to provide likelihood ratio statistics (LRS) that were suggestive (LRS 9.6), significant (LRS 15.3), or highly significant (LRS 19.). 24) LOD scores were obtained by dividing the LRS by ) In accordance with the guidelines reported by Lander and Kruglyak, 26) signifi-

3 cant linkage was defined as statistical evidence occurring by chance in the genome scan, with a probability of 5 or less. Statistical analysis. All results are expressed as the mean SEM. The phenotypic data in SM/J and A/J mice were statistically analyzed by Student s t-test. Relationships were analyzed by simple correlation. Differences of P < :5 were regarded as significant. All of the statistical analyses were performed using StatView version 5. software (SAS Institute, Cary, NC). Results Serum adiponectin and diabetes-related traits in parental strains and 19 SMXA RI strains Measurements of serum adiponectin concentrations, BMI, nonfasting blood glucose concentration, fasting serum insulin, serum lipids (triglycerides and total cholesterol) concentrations, and liver weight were performed in parental (SM/J and A/J) strains and 19 SMXA RI strains at 1 11 weeks of feeding the mice on a high-carbohydrate diet (Table 1). The serum adiponectin concentration of SM/J mice was significantly higher than that of A/J mice. There were no differences in nonfasting blood glucose, nor in nonfasting or fasting serum insulin concentrations between SM/J and A/J mice. BMI, relative liver weight, and serum cholesterol concentrations in SM/J mice were significantly lower Correlations and QTL Mapping for Adiponectin 679 than those of A/J mice. The serum adiponectin concentration of the SMXA RI strains showed wide distributions exceeding the values of the parental strains. The range of serum adiponectin concentrations in 19 SMXA RI strains was 6.2 mg/ml (SMXA-5) 29:5 (SMXA-18) mg/ml. Such wide distributions were also observed in the other diabetes-related traits, but the range of distribution of serum cholesterol concentrations was rather small. Correlation between serum adiponectin concentration and diabetes-related traits in 19 SMXA RI strains The serum adiponectin concentration was negatively correlated with the fasting and the nonfasting serum insulin concentrations, the serum triglycerides and cholesterol concentrations, and relative liver weight (Fig. 1). The correlation (r ¼ :585, p < :1) between the fasting insulin concentration and the serum adiponectin concentration was the strongest of all the correlations observed. On the other hand, no significant correlations were observed between the serum adiponectin concentration and body weight (r ¼ :353), BMI (r ¼ :315), nonfasting blood glucose concentration (r ¼ :16), relative weight (g/1 g body weight) of subcutaneous fat (r ¼ :35), retroperitoneal fat (r ¼ :227), epididymal fat (r ¼ :176), mesenteric fat (r ¼ :66), or blood glucose concentrations during IPGTT (fasting, r ¼ :173; 3 min, r ¼ :145; 6 min, r ¼ :26; 12 min, r ¼ :188; AUC, r ¼ :228). Table 1. Values of Serum Adiponectin Concentrations and Diabetes-Related Traits in Parental Strains (SM/J and A/J) and 19 SMXA RI Mice Strains Serum adiponectin (mg/ml) BMI (g/cm 2 ) Nonfasting blood glucose (mg/dl) Fasting insulin (mg/ml) Liver weight percentage (g/1 g bw) Serum triglycerides (mg/dl) Serum cholesterol (mg/dl) SMXA-18 29:5 1:9 :26 : :36 :6 4:23 : SMXA-25 2:5 1:8 :288 : :44 :2 4:52 : SMXA-24 19:7 1:6 :296 : :55 :5 4:3 : SMXA-27 19:7 2:3 :276 : :48 :3 4:68 : SM/J 16:7 1:3 :262 : :43 :6 4:39 : SMXA-4 16:4 1: :277 : :54 :5 4:7 : SMXA-19 16:3 1: :32 : :49 :3 5:1 : SMXA-12 15:5 1: :283 : :44 :5 4:13 : SMXA-21 15:5 1:5 :345 : :77 :7 4:89 : SMXA9 13:7 1:9 :29 : :81 :14 3:68 : SMXA-8 13:4 :5 :331 : :86 :7 4:74 : A/J 13:1 :9 :297 : :47 :5 4:75 : SMXA-15 13:1 1:4 :265 : :37 :9 4:3 : SMXA-1 12:4 1:7 :246 : :39 :7 4:53 : SMXA-16 11:6 1:2 :299 : :4 :2 4:7 : SMXA-1 1:8 1:3 :31 : :64 :12 4:42 : SMXA-26 1:3 :5 :31 : :53 :4 4:29 : SMXA-17 9:1 :8 :285 : :17 :51 5:3 : SMXA-3 7:6 :3 :311 : :61 :3 4:85 : SMXA-14 7:2 :1 :283 : :2 :15 5:34 : SMXA-5 6:2 :8 :327 : :17 :2 6:11 : Data are expressed as means SEM. Statistical analysis was done among SM/J and A/J mice. Significantly different by Student s t-test (P < :5).

4 68 M. KOBAYASHI et al. Serum triglyceride (mg/dl) Fasting serum insulin (ng/ml) r = -.571, p <.1 r = -.512, p <.2 r = -.436, p < r = -.585, p <.1 r = -.457, p <.5 14 Serum cholesterol (mg/dl) Nonfasting serum Insulin (ng/ml) Liver (% of body weight) Fig. 1. Correlations between Serum Adiponectin Concentration and Fasting and Nonfasting Serum Insulin Concentrations, Serum Triglycerides and Cholesterol, and Percentage of Liver Weight in 19 SMXA RI Substrains. Mapping of QTL that regulate serum adiponectin levels in SMXA RI strains On Chr 7, we mapped the QTL involved in the regulation of the serum adiponectin concentration with a LOD score of 2.8 (near D7Mit25) and 2.3 (D7Mit7) (Fig. 2). Both QTL showed suggestive linkage, but not significantly. The mean value of the serum adiponectin concentration among the SMXA RI strains (13 substrains) with the SM/J allele at D7Mit25 was 11:5 :9 mg/ml, and that of the serum adiponectin concentration of the other strains (6 substrains: SMXA-9, 12, 18, 24, 25, and 27) with the A/J allele was 19:8 2:2 mg/ml. Similarly, at D7Mit7, the mean serum adiponectin concentration among the SMXA RI strains with the SM/J allele was 12:1 1:2 mg/ml (13 substrains), and that among the strains with the A/J allele was 18:6 2:5 mg/ml (6 substrains: SMXA-4, 8, 15, 18, 24, and 27). The A/J allele at these loci was associated with a higher serum adiponectin concentration. Discussion In this study of mouse SMXA RI substrains exhibiting a wide range of both serum adiponectin concentrations and other traits, the strongest correlation (a negative correlation) was detected between the serum adiponectin concentration and the fasting serum insulin concentration. The fasting serum insulin concentration appears to reflect the extent of insulin resistance, that is, fasting hyperinsulinemia associated with insulin resistance. On the other hand, the serum adiponectin concentration was correlated with neither BMI nor fat tissue weight in these SMXA-RI substrains. Thus it was found that hypoadiponectinemia correlated with hyperinsulinemia more strongly than with obesity in SMXA-RI substrains. Previous human studies have found a negative correlation between body mass index and the plasma adiponectin concentration in Japanese, Caucasian, and Pima Indian populations. 6,27) However, Abbasi and co-workers recently reported that adiponectin concentrations are more closely related to differences in insulin-mediated glucose disposal than with obesity in the American population; the present results coincide with those of that study. 28) Moreover, in our study, the serum adiponectin concentration was not correlated with either the blood glucose concentration or glucose tolerance. Previously we reported a wide distribution of diabetesrelated traits in 19 SMXA RI substrains. 18,19) In the present analyses, we observed that several strains the blood glucose concentrations and glucose tolerances of which were similar nonetheless exhibited a wide variety of serum insulin concentrations. This implies that

5 Correlations and QTL Mapping for Adiponectin LOD Fig. 2.. D7Mit74 ( 2 cm, - Mb) D7Mit297 (27.8 cm, 58.2 Mb) D7Mit25 (37 cm, 71.9 Mb) D7Mit17 (51.4 cm,11.1 Mb) D7Mit281 (52.4 cm, 14.4 Mb) D7Mit7 (54 cm, - Mb) D7Mit1 (66 cm, - Mb) LOD Score Curves for Serum Adiponectin Concentrations. The dashed line indicates the threshold of suggestive linkage. The map positions (cm and Mb) on the x-axis were obtained from the Ensembl Genome Browser ( susceptibility to diabetes results from a complex combination of insulin resistance and impaired insulin secretion in 19 SMXA-RI substrains. These results furthermore suggest that the serum adiponectin concentration reflects the extent of insulin resistance, rather than the onset of type 2 diabetes. Adiponectin circulates in serum in various forms, including full-length, globular, LMW, MMW, and HMW. Recently, the percentage of adiponectin in HMW form and the ratio of HMW=ðHMW þ LMWÞ in db/db mice was lower than in wild-type littermates. 29) In human subjects, Bobbert et al. found that body weight reduction results in a increase of (HMW þ MMW) and a reduction of LMW adiponectin in serum. 3) In this study, we did not quantify each form of adiponectin in serum separately. In future, we must take account of the differences in the amount of the each form of adiponectin in serum, accompanied by changes in diabetes-related traits. According to the present analysis, the mouse chromosomal regions (D7Mit297-D7Nds1: cm, Mb; D7Mit7 D7Mit1: cm, unknown 124 Mb) with a putative link to serum adiponectin concentrations are homologous to regions in human Chrs 1, 11, 15, and 16. Comuzzie and coworkers performed the first genetic analysis of human serum adiponectin concentrations, and they reported QTL for the regulation of adiponectin concentration on regions in Chrs 2, 3, 5, 1, 14, and 17 in people of northern European ancestry. 31) In Pima Indians, QTL controlling circulating adiponectin concentrations were mapped on Chrs 2, 3, 9, and 1, 32) but the regions of human chromosomes mapped in these studies are not homologous to mouse Chr 7. Several clinical studies have shown a negative correlation between serum adiponectin and serum triglyceride concentrations, and a positive correlation between serum adiponectin and serum HDL cholesterol concentration ) In addition, we also found in this study that the serum adiponectin concentration was negatively correlated with both serum triglycerides concentrations and serum total cholesterol concentrations. In humans, a recessive hypercholesterolemia locus, ARH1, has been mapped on Chr 15q25- q26, although the QTL that regulates serum adiponectin levels has not yet been mapped on human Chr ) The region of human Chr 15 that contains the ARH1 locus is homologous to mouse Chr 7. The human ARP1 gene is located near the ARH1 locus, and in mice, this gene is located on Chr 7. Liver transcription factor Arp1 regulates the expression of apolipoprotein B and other apolipoprotein genes, and it plays an important role in serum lipid metabolism. 35,36) It is possible that Arp1 serves as a positional and functional candidate gene for QTL regulating serum adiponectin concentrations in mouse SMXA RI strains. Although the nucleotide sequences of the open reading frame of Arp1 in the parental strains (SM/J and A/J mice) were examined in this study, we did not detect any differences in these sequences between these two strains (data not shown). But, we did not compare the hepatic Arp1 mrna levels of the parental strains. In a future study, we plan to measure these levels and examine the Arp1 gene as a candidate regulator of serum adiponectin levels. In this study, we found a negative correlation between the serum adiponectin concentration and relative liver weight. Increases in relative liver weight are known to imply the accumulation of lipids such as triglycerides

6 682 M. KOBAYASHI et al. and cholesterol. In fact, SMXA-5 mice, which have a high relative liver weight, exhibit the accumulation of triglycerides in the liver. 37) Thus it is speculated that the presence of a fatty liver is associated with a low concentration of serum adiponectin. In conclusion, we clearly demonstrated using mouse SMXA RI substrains that the serum adiponectin concentration is negatively correlated with the serum fasting insulin concentration, but not with obesity. In addition, the serum adiponectin concentration is negatively correlated with both serum triglycerides and serum cholesterol concentrations. It appears likely that a relatively low concentration of serum adiponectin reflects the presence of insulin resistance. Moreover, we mapped the suggestive QTLs for the control of the serum adiponectin concentration on Chr 7, and the presence of the A/J allele at these loci was found to be associated with higher serum adiponectin concentrations. 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