Identification of novel non-insulin-dependent diabetes mellitus susceptibility loci in the Otsuka Long-Evans Tokushima Fatty rat by MQM-mapping method

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. We have previously identified 11 quantitative trait loci (QTLs) responsible for NIDDM susceptibility on Chromosomes (Chrs) 1, 5, 7, 8, 9, 11, 12, 14, and 16 (Nidd1–11/of for Non-insulin-dependent diabetes1–11/oletf) by using the interval mapping method in 160 F₂ progenies obtained by mating the OLETF and the Fischer-344 (F344) rats. MQM-mapping, which was applied for QTL analysis based on multiple-QTL models, is reported to be more powerful than interval mapping, because in the process of mapping one QTL the genetic background, which contains the other QTLs, is controlled. Application of MQM-mapping in the F₂ intercrosses has led to a revelation of three novel QTLs on rat Chrs 5 (Nidd12/of), 7 (Nidd13/of), and 17 (Nidd14/of), in addition to Nidd1–11/of loci. The three QTLs, together with the Nidd1–11/of, account for a total of ∼70% and ∼85% of the genetic variance of the fasting and postprandial glucose levels, respectively, in the F₂. While the OLETF allele corresponds with increased glucose levels as expected for Nidd12 and 14/of, the Nidd13/of exhibits heterosis: heterozygotes showing significantly higher glucose levels than OLETF or F344 homozygotes. There is epistatic interaction between Nidd2 and 14/of. Additionally, our results indicated that the novel QTLs could show no linkage with body weight, but Nidd12/of has an interaction with body weight. Received: 23 February 1999 / Accepted: 3 August 1999     

Identification of epistatic interactions involved in non-insulin-dependent diabetes mellitus in the Otsuka Long-Evans Tokushima Fatty rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type non-insulin-dependent diabetes mellitus (NIDDM) in humans. Our present investigation was designed to identify epistatic interactions influencing NIDDM by performing least squares analysis of variance of all pairs of informative markers in 160 F2 progenies bred from an intercross of OLETF and Fischer-344 rats. We identified four interactions between Nidd15/of (chromosome 7) and Nidd16/of (chromosome 14), Nidd15/of and Nidd17/of (chromosome 15), Nidd16/of and Nidd18/of (chromosome 15), and Nidd16/of and Nidd19/of (chromosome 17), which account for a total of approximately 40% of the genetic variation of entire glucose levels after glucose challenge in the F2. The Nidd16/of locus, which is involved in three of four digenic interactions, and the Nidd19/of are likely to correspond to Nidd2/of and Nidd14/of, NIDDM loci previously identified in the F2 by single-QTL model and multiple-QTL model, respectively, while Nidd15/of, Nidd17/of and Nidd18/of loci reflect novel NIDDM loci. An aberrant increase of the entire glucose level due to synergism occurs in the double OLETF homozygote genotype of Nidd15/of and Nidd16/of, and of Nidd16/of and Nidd19/of, as well as in the OLETF homozygote genotypes of Nidd15/of and Nidd16/of, respectively, combined with the heterozygote genotypes of Nidd17/of and Nidd18/of. These findings demonstrate that inter-allelic interactions are likely to be an important component of NIDDM susceptibility.     

Effect of leptin on insulin sensitivity in the Otsuka Long-Evans Tokushima Fatty rat

Leptin has been proposed to be a sensor of energy storage in adipose tissues, and is capable of mediating a feedback signal to the hypothalamus, which is involved in the regulation of energy homeostasis and body weight. In order to investigate the issue of whether resistance to the activity of leptin on insulin sensitivity is observed in young Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 8 weeks of age, leptin (50 nmol/kg/h) was administered intravenously for 16 h to OLETF and Long-Evans Tokushima Otsuka (LETO) (lean controls) rats, followed by a measurement of insulin-stimulated glucose uptake in hindlimb muscles during hyperinsulinemic euglycemic clamp technique. In the case of LETO rats, the administration of leptin significantly decreased plasma insulin levels prior to the clamp test, but did not change plasma glucose levels. Furthermore, leptin led to an increase in insulin-stimulated glucose uptake in hindlimb muscles. However, in the case of OLETF rats, leptin administration changed neither plasma insulin levels nor insulin-stimulated glucose uptake. These data demonstrate that OLETF rats at 8 weeks of age have already become resistant to high concentration of peripheral leptin.     

Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus

The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate in mitochondria. The PDC activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by specific kinases (PDK) and phosphatases (PDP). In this study, the regulatory mechanisms of PDC were examined in skeletal muscle of the spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rat before and after the onset of diabetes. The Long-Evans Tokushima Otsuka (LETO) rat was used as control. Plasma glucose and insulin concentrations were at normal levels in both groups at 8 weeks of age but were significantly higher in OLETF than in LETO rats at 25 weeks of age (1.2-fold for glucose and 15-fold for insulin), indicating development of diabetes in the former. Plasma free fatty acids were 1.6-fold concentrated and the skeletal muscle PDC activity state was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidation in OLETF rats even before the onset of diabetes. The PDK activity and the abundance of the PDK isoform 4 protein as well as mRNA were greater in OLETF rats at both ages. Conversely, the abundance of the PDP isoform 1 protein and mRNA was less in OLETF than in LETO rats at both ages. These results suggest that concomitant greater PDK4 and less PDP1 expression in skeletal muscle of OLETF rats before the onset of diabetes are responsible for the lowering of the PDC activity and may be related with the development of diabetes mellitus.     

Proteomic method detects oxidatively induced protein carbonyls in muscles of a diabetes model Otsuka Long-Evans Tokushima Fatty (OLETF) rat

Oxidative stress is implicated in a broad variety of chronic and acute diseases, including such age-related diseases as diabetes. To understand at the protein level cellular damage caused by the stress, we developed a proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by two-dimensional gel electrophoresis. The method, being capable of analyzing high-molecular-mass proteins as large as myosin heavy chains (molecular mass approximately 200 kDa), was applied to detecting protein carbonyls in muscles of a diabetes model Otsuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. A number of proteins, including mitochondrial ATP synthase beta-chain, desmin, actin, and myosin, were found carbonylated. Our method would provide a means toward clarifying a comprehensive view of oxidative modifications of proteins during a long progression of age-related diseases and understanding the mechanism of the onset, progression, and complication of the diseases.     

Role of TGF-beta1 in the development of pancreatic fibrosis in Otsuka Long-Evans Tokushima Fatty rats

Recently established Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of naturally occurring obesity diabetes, exhibit progressive accumulation of connective tissue in the pancreas. The present study was designed to determine the pathogenic role of transforming growth factor-beta1 (TGF-beta1) in the development of pancreatic fibrosis in OLETF rats by investigating the serial changes in the expression of TGF-beta1 and extracellular matrix (ECM) in the pancreas. Progressive proliferation of connective tissue arose from the interstitial region surrounding islets at 20 wk of age and extended to the exocrine pancreas adjacent to the islets. TGF-beta1 mRNA levels in the pancreas increased at 20 wk of age and reached a peak value at 30 wk of age. Fibronectin (FN) and procollagen types I and III mRNAs peaked at 20 wk of age and remained at higher levels than those in the nondiabetic counterparts Long-Evans Tokushima Otsuka rats until 50 wk of age. Immunoreactivities for TGF-beta1 and FN were found in islets of OLETF rats at 20 wk of age and were seen in acinar and interstitial cells at 50 wk of age. Moreover, alpha-smooth muscle actin was located at interstitial region surrounding the islets. Proliferation of the connective tissue in the pancreas of OLETF rats closely correlated with expression of TGF-beta1 and ECM. Our results suggest that the development of pancreatic fibrosis in OLETF rats extends from endocrine to exocrine pancreas and that TGF-beta1 is involved in pancreatic fibrosis of OLETF rats.     

Effect of Vaccinium ashei reade leaves on lipid metabolism in Otsuka Long-Evans Tokushima Fatty rats

The effects of blueberry leaf (BBL) on lipid metabolism were studied in obese rats. Feeding of BBL lowered levels of serum lipids and C-reactive protein and alleviated hepatic triglyceride accumulation in the rats. The hypolipidemic effect might be attributable to a reduction of lipogenesis and enhancement of lipolysis in the liver. These results suggest the use of blueberry leaf as a dietary hypolipidemic component.     

Daily exercise increases hepatic fatty acid oxidation and prevents steatosis in Otsuka Long-Evans Tokushima Fatty rats

Exercise training is commonly prescribed for treatment of nonalcoholic fatty liver disease (NAFLD). We sought to determine whether exercise training prevents the development of NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the molecular mechanisms underlying the effects of exercise on hepatic steatosis. Four-week-old OLETF rats were randomly assigned to either a sedentary control group (Sed) or a group given access to voluntary running wheels for 16 wk (Ex). Wheels were locked 2 days before euthanasia in the Ex animals, and both groups were euthanized at 20 wk old. Voluntary wheel running attenuated weight gain and reduced serum glucose, insulin, free fatty acids, and triglycerides in Ex animals compared with Sed (P < 0.001). Ex animals exhibited significantly reduced hepatic triglyceride levels and displayed fewer lipid droplets (Oil Red O staining) and reduced lipid droplet size compared with Sed. Wheel running increased by threefold the percent of palmitate oxidized completely to CO(2) in the Ex animals but did not alter AMP-activated protein kinase-alpha (AMPKalpha) or AMPK phosphorylation status. However, fatty acid synthase and acetyl-coenzyme A carboxylase (ACC) content were significantly reduced (approximately 70 and approximately 35%, respectively), and ACC phosphorylation and cytochrome c content were significantly elevated (approximately 35 and approximately 30%, respectively) in the Ex animals. These results unequivocally demonstrate that daily physical activity attenuates hepatic steatosis and NAFLD in an obese rodent model and suggest that this effect is likely mediated, in part, through enhancement of hepatic fatty acid oxidation and reductions in key protein intermediates of fatty acid synthesis.     

Mapping quantitative trait loci for cytokines in the pig

Increased disease resistance through improved general immune capacity would be beneficial for the welfare and productivity of farm animals. Cytokines are essential diagnostic parameters in veterinary practice. To identify quantitative trait loci (QTL) for cytokine levels in serum in the pig, Interferon‐gamma (IFN‐γ) and Interleukin 10 (IL‐10) levels and the ratio of IFN‐γ to IL‐10 were measured in a composite pig population, before and after challenge with modified live CSF (classical swine fever) vaccine. Through interval mapping using the variance component approach and the permutation test, 11 QTL (five for IFN‐γ, two for IL‐10 and four for the ratio of IFN‐γ to IL‐10) with significance levels of P < 0.10 were identified, of which five were significant at the P < 0.05 level. The most significant QTL (P < 0.01) was found on chromosome 16, with effect on the ratio of IFN‐γ to IL‐10. Within these QTL regions, a number of known genes were revealed and their potential relationships to the studied traits were discussed. Some of these genes may serve as candidate genes for these traits in swine.     

Consumption of pork-liver protein hydrolysate reduces body fat in Otsuka Long-Evans Tokushima Fatty rats by suppressing hepatic lipogenesis

This study was performed to examine the effect of consumption of pork-liver protein hydrolysate (PLH) on body fat accumulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a non-insulin-dependent diabetes mellitus model and in Long-Evans Tokushima Otsuka (LETO) rats as a control. Male 20-week-old OLETF and LETO rats were pair-fed either PLH or casein containing diet for 14 weeks. In the OLETF rats, dietary PLH significantly reduced the growth and weight of fat pad including perirenal and epididymal adipose tissues. Consumption of PLH markedly suppressed hepatic activities of lipogenesis enzymes such as glucose-6-phosphate dehydrogenase and fatty acid synthase and slightly elevated fecal excretion of total fat. In the LETO rats, growth and adipose tissue weight were unaffected by dietary treatment. The results suggest that PLH is a novel ingredient suppressing body fat in genetically obese rats by reducing lipogenesis.     

Constructing a genetic linkage map and mapping quantitative trait loci for skeletal traits in Japanese flounder

A genetic linkage map of Japanese flounder was constructed using 165 doubled haploids (DHs) derived from a single female. A total of 574 genomic microsatellites (type II SSRs) and expressed sequence tag (EST)-derived markers (EST-SSRs) were mapped to 24 linkage groups. The length of linkage map was estimated as 1270.9 centiMorgans (cM), with an average distance between markers of 2.2 cM. The EST-SSRs were used together with type II SSR markers to construct the Japanese flounder genetic linkage map which will facilitate identify quantitative trait locus (QTL) controlling important economic traits in Japanese flounder. Thus, twelve skeletal traits at 2 years of age were measured for all DHs. Forty-one QTLs were detected on 14 linkage groups and totally account for a small proportion of phenotypic variation (4.5 to 17.3%). Most of QTLs detected distribute on linkage groups 5 (9 QTLs), 8 (9 QTLs), 9 (5 QTLs) and 20 (4 QTLs), in which, some QTLs perform the pleiotropy.     

Mapping quantitative trait loci with dominant markers in four-way crosses

 A common problem in mapping quantitative trait loci (QTLs) is that marker data are often incomplete. This includes missing data, dominant markers, and partially informative markers, arising in outbred populations. Here we briefly present an iteratively re-weighted least square method (IRWLS) to incorporate dominant and missing markers for mapping QTLs in four-way crosses under a heterogeneous variance model. The algorithm uses information from all markers in a linkage group to infer the QTL genotype. Monte Carlo simulations indicate that with half dominant markers, QTL detection is almost as efficient as with all co-dominant markers. However, the precision of the estimated QTL parameters generally decreases as more markers become missing or dominant. Notable differences are observed on the standard deviation of the estimated QTL position for varying levels of marker information content. The method is relatively simple so that more complex models including multiple QTLs or fixed effects can be fitted. Finally, the method can be readily extended to QTL mapping in full-sib families. Received: 16 June 1998 / Accepted: 29 September 1998     

Mapping quantitative trait loci for murine growth: a closer look at genetic architecture

Over 20 years ago, D. S. Falconer and others launched an important avenue of research into the quantitative of body size growth in mice. This study continues in that tradition by locating quantitative trait loci (QTLs) responsible for murine growth, such as age-specific weights and growth periods, and examining the genetic architecture for body weight. We identified a large number of potential QTLs in an earlier F2 intercross (Intercross I) of the SM/J and LG/J inbred mouse strains. Many of these QTLs are replicated in a second F2 intercross (Intercross II) between the same two strains. These replicated regions provide candidate regions for future fine-mapping studies. We also examined body size and growth QTLs using the combined data set from these two intercrosses, resulting in 96 microsatellite markers being scored for 1045 individuals. An examination of the genetic architecture for age-specific weight and growth periods resulted in locating 20 separate QTLs, which were mainly additive in nature, although dominance was found to affect early growth and body size. QTLs affecting early and late growth were generally distinct, mapping to separate chromosome locations. This QTL pattern indicates largely separate genetic and physiological systems for early and later murine growth, as Falconer suggested. We also found sex-specific QTLs for body size with implications for the evolution of sexual dimorphism.     

Theobroma cacao L.: a genetic linkage map and quantitative trait loci analysis

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 11 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC₁ generation using both single-point ANOVA and interval mapping. A minimum number of 2–4 QTLs (P<0.01) involved in the genetic expression of the traits studied was detected. Coincident map locations of a QTL for jorquette height and trunk diameter suggests the possibility of pleiotropic effects in cocoa for these traits. The combined estimated effects of the different mapped QTLs explained between 11.2% and 25.8% of the phenotypic variance observed in the BC₁ population.     

Mapping quantitative trait loci for longitudinal traits in line crosses

Quantitative traits whose phenotypic values change over time are called longitudinal traits. Genetic analyses of longitudinal traits can be conducted using any of the following approaches: (1) treating the phenotypic values at different time points as repeated measurements of the same trait and analyzing the trait under the repeated measurements framework, (2) treating the phenotypes measured from different time points as different traits and analyzing the traits jointly on the basis of the theory of multivariate analysis, and (3) fitting a growth curve to the phenotypic values across time points and analyzing the fitted parameters of the growth trajectory under the theory of multivariate analysis. The third approach has been used in QTL mapping for longitudinal traits by fitting the data to a logistic growth trajectory. This approach applies only to the particular S-shaped growth process. In practice, a longitudinal trait may show a trajectory of any shape. We demonstrate that one can describe a longitudinal trait with orthogonal polynomials, which are sufficiently general for fitting any shaped curve. We develop a mixed-model methodology for QTL mapping of longitudinal traits and a maximum-likelihood method for parameter estimation and statistical tests. The expectation-maximization (EM) algorithm is applied to search for the maximum-likelihood estimates of parameters. The method is verified with simulated data and demonstrated with experimental data from a pseudobackcross family of Populus (poplar) trees.     

Mapping quantitative trait loci for binary trait in the F2:3 design

In the analysis of inheritance of quantitative traits with low heritability, an F_2:3 design that genotypes plants in F₂ and phenotypes plants in F_2:3 progeny is often used in plant genetics. Although statistical approaches for mapping quantitative trait loci (QTL) in the F_2:3 design have been well developed, those for binary traits of biological interest and economic importance are seldom addressed. In this study, an attempt was made to map binary trait loci (BTL) in the F_2:3 design. The fundamental idea was: the F₂ plants were genotyped, all phenotypic values of each F_2:3 progeny were measured for binary trait, and these binary trait values and the marker genotype informations were used to detect BTL under the penetrance and liability models. The proposed method was verified by a series of Monte-Carlo simulation experiments. These results showed that maximum likelihood approaches under the penetrance and liability models provide accurate estimates for the effects and the locations of BTL with high statistical power, even under of low heritability. Moreover, the penetrance model is as efficient as the liability model, and the F_2:3 design is more efficient than classical F₂ design, even though only a single progeny is collected from each F_2:3 family. With the maximum likelihood approaches under the penetrance and the liability models developed in this study, we can map binary traits as we can do for quantitative trait in the F_2:3 design.     

Complexities in the genetic dissection of quantitative trait loci

The analysis of complex traits, including those involved in many common diseases, has encountered significant difficulties, and, despite major efforts during the past decade, has had little success. Current advances in genomics, however, promise to change this. Recently, Steinmetz et al. used a new technique to produce the first complete quantitative trait locus (QTL) analysis of a complex trait from phenotype to gene to be published entirely in a single report. This marks a significant advance over previous QTL analyses, which took several years. The work exemplifies some of the complexities of QTL mapping and demonstrates a novel method to resolve the underlying genetic architecture of QTLs in yeast. Here we discuss this work in the general context of genetic dissection of complex traits and QTLs.     

Mapping of quantitative trait loci affecting behaviour in swine

Behavioural indices in vertebrates are under genetic control at least to some extent. In spite of significant behavioural problems in farm animals, information on the genetic background of behaviour is sparse. The aim of this study was to map QTL for behavioural indices in swine under healthy conditions and after infection with Sarcocystis miescheriana , as behaviour can be significantly influenced by disease . This well-described parasite model subsequently leads to acute (day 14 p.i.), subclinical (day 28 p.i.) and chronic disease (day 42 p.i.), allowing the study and comparison of the behaviour of pigs under four different states of health or disease. The study was based on a well-described Pietrain/Meishan F₂ family that has recently allowed the detection of QTL for disease resistance. We have mapped six genome-wide significant and 24 chromosome-wide significant QTL for six behavioural indices in swine. Six of these QTL (i.e. 20% of total QTL) showed effects on behavioural traits of the healthy pigs (day 0). Some of them (QTL on SSC11 and 18) lost influence on behavioural activities during disease, while the effects of others (QTL on SSC5, SSC8) partly remained during the whole experiment, although with different effects on the distinct behavioural indices. The disease model has been of high relevance to detect effects of gene loci on behavioural indices. Considering the importance of segregating alleles and environmental conditions that allow the identification of the phenotype, we conclude that there are indeed QTL with interesting effects on behavioural indices in swine.