Genetic dissection of the fatty liver QTL Fl1sa by using congenic mice and identification of candidate genes in the liver and epididymal fat.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a multifactorial disease caused by interactions between environmental and genetic factors. The SMXA-5 mouse is a high-fat diet-induced fatty liver model established from SM/J and A/J strains. We have previously identified Fl1sa, a quantitative trait locus (QTL) for fatty liver on chromosome 12 (centromere-53.06 Mb) of SMXA-5 mice. However, the chromosomal region containing Fl1sa was too broad. The aim of this study was to narrow the Fl1sa region by genetic dissection using novel congenic mice and to identify candidate genes within the narrowed Fl1sa region. RESULTS: We established two congenic strains, R2 and R3, from parental A/J-12SM and A/J strains. R2 and R3 strains have genomic intervals of centromere-29.20 Mb and 29.20-46.75 Mb of chromosome 12 derived from SM/J, respectively. Liver triglyceride content in R2 and R3 mice was significantly lower than that in A/J mice fed with a high-fat diet for 7 weeks. This result suggests that at least one of the genes responsible for fatty liver exists within the two chromosomal regions centromere-29.20 Mb (R2) and 29.20-46.75 Mb (R3). We found that liver triglyceride accumulation is inversely correlated with epididymal fat weight among the parental and congenic strains. Therefore, the ectopic fat accumulation in the liver may be due to organ-organ interactions between the liver and epididymal fat. To identify candidate genes in Fl1sa, we performed a DNA microarray analysis using the liver and epididymal fat in A/J and A/J-12SM mice fed with a high-fat diet for 7 weeks. In epididymal fat, mRNA levels of Zfp125 (in R2) and Nrcam (in R3) were significantly different in A/J-12SM mice from those in A/J mice. In the liver, mRNA levels of Iah1 (in R2) and Rrm2 (in R2) were significantly different in A/J-12SM mice from those in A/J mice. CONCLUSIONS: In this study, using congenic mice analysis, we narrowed the chromosomal region containing Fl1sa to two regions of mouse chromosome 12. We then identified 4 candidate genes in Fl1sa: Iah1 and Rrm2 from the liver and Zfp125 and Nrcam from epididymal fat.

Detection of differentially expressed candidate genes for a fatty liver QTL on mouse chromosome 12.

BACKGROUND: The SMXA-5 mouse is an animal model of high-fat diet-induced fatty liver. The major QTL for fatty liver, Fl1sa on chromosome 12, was identified in a SM/J × SMXA-5 intercross. The SMXA-5 genome consists of the SM/J and A/J genomes, and the A/J allele of Fl1sa is a fatty liver-susceptibility allele. The existence of the responsible genes for fatty liver within Fl1sa was confirmed in A/J-12(SM) consomic mice. The aim of this study was to identify candidate genes for Fl1sa, and to investigate whether the identified genes affect the lipid metabolism. RESULTS: A/J-12(SM) mice showed a significantly lower liver triglyceride content compared to A/J mice when fed the high-fat diet for 7 weeks. We detected differences in the accumulation of liver lipids in response to the high-fat diet between A/J and A/J-12(SM) consomic mice. To identify candidate genes for Fl1sa, we performed DNA microarray analysis using the livers of A/J-12(SM) and A/J mice fed the high-fat diet. The mRNA levels of three genes (Iah1, Rrm2, Prkd1) in the chromosomal region of Fl1sa were significantly different between the strains. Iah1 mRNA levels in the liver, kidney, and lung were significantly higher in A/J-12(SM) mice than in A/J mice. The hepatic Iah1 mRNA level in A/J-12(SM) mice was 3.2-fold higher than that in A/J mice. To examine the effect of Iah1 on hepatic lipid metabolism, we constructed a stable cell line expressing the mouse Iah1 protein in mouse hepatoma Hepa1-6 cells. Overexpression of Iah1 in Hepa1-6 cells suppressed the mRNA levels of Cd36 and Dgat2, which play important roles in triglyceride synthesis and lipid metabolism. CONCLUSIONS: These results demonstrated that Fl1sa on the proximal region of chromosome 12 affected fatty liver in mice on a high-fat diet. Iah1 (isoamyl acetate-hydrolyzing esterase 1 homolog) was identified as one of the candidate genes for Fl1sa. This study revealed that the mouse Iah1 gene regulated the expression of genes related to lipid metabolism in the liver.

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species.

Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal-newly-hatched IgY transfer are controlled by a single receptor.

Mimicking seasonal changes in light-dark cycle and ambient temperature modulates gut microbiome in mice under the same dietary regimen.

To better adapt to seasonal environmental changes, physiological processes and behaviors are regulated seasonally. The gut microbiome interacts with the physiology, behavior, and even the diseases of host animals, including humans and livestock. Seasonal changes in gut microbiome composition have been reported in several species under natural environments. Dietary content significantly affects the composition of the microbiome, and, in the natural environment, the diet varies between different seasons. Therefore, understanding the seasonal regulatory mechanisms of the gut microbiome is important for understanding the seasonal adaptation strategies of animals. Herein, we examined the effects of changing day length and temperature, which mimic summer and winter conditions, on the gut microbiome of laboratory mice. Principal coordinate analysis and analysis of the composition of microbiomes of 16S rRNA sequencing data demonstrated that the microbiomes of the cecum and large intestine showed significant differences between summer and winter mimicking conditions. Similar to previous studies, a daily rhythm was observed in the composition of the microbiome. Furthermore, the phylogenetic investigation of communities by reconstruction of unobserved states predicted seasonal changes in several metabolic pathways. Changing day length and temperature can affect the composition of the gut microbiome without changing dietary contents.

C3H/HeNSlc mouse with low phospholipid transfer protein expression showed dyslipidemia.

High serum levels of triglycerides (TG) and low levels of high-density lipoprotein cholesterol (HDL-C) increase the risk of coronary heart disease in humans. Herein, we first reported that the C3H/HeNSlc (C3H-S) mouse, a C3H/HeN-derived substrain, is a novel model for dyslipidemia. C3H-S showed hypertriglyceridemia and low total cholesterol (TC), HDL-C, and phospholipid (PL) concentrations. To identify the gene locus causing dyslipidemia in C3H-S, we performed genetic analysis. In F2 intercrosses between C3H-S mice and strains with normal serum lipids, the locus associated with serum lipids was identified as 163-168 Mb on chromosome 2. The phospholipid transfer protein (Pltp) gene was a candidate gene within this locus. Pltp expression and serum PLTP activity were markedly lower in C3H-S mice. Pltp expression was negatively correlated with serum TG and positively correlated with serum TC and HDL-C in F2 mice. Genome sequencing analysis revealed that an endogenous retrovirus (ERV) sequence called intracisternal A particle was inserted into intron 12 of Pltp in C3H-S. These results suggest that ERV insertion within Pltp causes aberrant splicing, leading to reduced Pltp expression in C3H-S. This study demonstrated the contribution of C3H-S to our understanding of the relationship between TG, TC, and PL metabolism via PLTP.

Ternatin, a cyclic peptide isolated from mushroom, and its derivative suppress hyperglycemia and hepatic fatty acid synthesis in spontaneously diabetic KK-A(y) mice.

(-)-Ternatin is a highly methylated cyclic heptapeptide isolated from mushroom Coriolus versicolor. Ternatin has an inhibitory effect on fat accumulation in 3T3-L1 adipocytes. [D-Leu(7)]ternatin, a ternatin derivative, also inhibited fat accumulation in 3T3-L1 cells, although the effectiveness of [D-Leu(7)]ternatin was lower than that of ternatin. In this study, we investigated the effects of ternatin and [D-Leu(7)]ternatin on obesity and type 2 diabetes in KK-A(y) mice, an animal model for spontaneously developed type 2 diabetes. We continuously administered ternatin (8.5 or 17 nmol/day) or [D-Leu(7)]ternatin (68 nmol/day) to mice via a subcutaneous osmotic pump. Unexpectedly, neither ternatin nor [D-Leu(7)]ternatin affected body weight or adipose tissue weight in KK-A(y) mice. In contrast, it was demonstrated that both ternatin and [D-Leu(7)]ternatin suppress the development of hyperglycemia. In liver, the SREBP-1c mRNA level tended to be lower or significantly decreased in mice treated with ternatin or [D-Leu(7)]ternatin, respectively. Moreover, we found that ternatin directly lowered the SREBP-1c mRNA level in Hepa1-6 hepatocyte cells. This study showed that ternatin and [D-Leu(7)]ternatin each had a preventive effect on hyperglycemia and a suppressive effect on fatty acid synthesis in KK-A(y) mice.

Low Ascorbic Acid Intake Induces Inflammatory Changes in Intestine and Liver of ODS Rats.

We previously demonstrated that ascorbic acid (AsA) deficiency, caused by an AsA-free diet, induces inflammatory changes in the liver and intestine of osteogenic disorder Shionogi (ODS) rats that cannot synthesize AsA. However, whether low AsA intake induces inflammatory changes remains unknown. Here, we assessed the inflammatory changes in ODS rats caused by low AsA intake and compared them to ODS rats that were fed a diet supplemented with sufficient amounts of AsA (300 mg/kg). Male ODS rats (12-wk-old) were fed an AsA-free diet (0 ppm group), AsA 20 mg/kg diet (20 ppm group), AsA 40 mg/kg diet (40 ppm group) or AsA 300 mg/kg diet (300 ppm group) for 22 d. The hepatic mRNA levels of acute phase proteins, including C-reactive protein (CRP) and haptoglobin, were higher in the 0 and 20 ppm groups, than in the 300 and 40 ppm groups, but were not significantly higher in the 20 ppm group. Serum CRP concentrations were significantly higher in the 0 and 20 ppm groups than in the 300 and 40 ppm groups. Jejunal and ileal interleukin-1ß (IL-1ß) mRNA levels were higher in the 0 and 20 ppm groups than in the 300 ppm group. Jejunal and ileal IL-6 mRNA levels tended to be higher in the 0 and 20 ppm groups than in the 300 ppm group. Furthermore, the portal IL-6 concentration gradually increased with decrease in the AsA intake. Thus, inflammatory changes could occur in both AsA-deficient ODS rats and ODS rats with low AsA intake.

Coffee and caffeine improve insulin sensitivity and glucose tolerance in C57BL/6J mice fed a high-fat diet.

We have previously demonstrated that coffee and caffeine ameliorated hyperglycemia in spontaneously diabetic KK-A(y) mice. This present study evaluates the antidiabetic effects of coffee and caffeine on high-fat-diet-induced impaired glucose tolerance in C57BL/6J mice. C57BL/6J mice fed a high-fat diet were given regular drinking water (control group), or a 2.5-fold-diluted coffee or caffeine solution (200 mg/L) for 17 weeks. The ingestion of coffee or caffeine improved glucose tolerance, insulin sensitivity, and hyperinsulinemia when compared with mice in the control group. The adipose tissue mRNA levels of inflammatory adipocytokines (MCP-1 and IL-6) and the liver mRNA levels of genes related to fatty acid synthesis were lower in the coffee and caffeine groups than those in the control group. These results suggest that coffee and caffeine exerted an ameliorative effect on high-fat-diet-induced impaired glucose tolerance by improving insulin sensitivity. This effect might be attributable in part to the reduction of inflammatory adipocytokine expression.

Involvement of thyrotropin in photoperiodic signal transduction in mice.

Local thyroid hormone catabolism within the mediobasal hypothalamus (MBH) by thyroid hormone-activating (DIO2) and -inactivating (DIO3) enzymes regulates seasonal reproduction in birds and mammals. Recent functional genomics analysis in birds has shown that long days induce thyroid-stimulating hormone production in the pars tuberalis (PT) of the pituitary gland, which triggers DIO2 expression in the ependymal cells (EC) of the MBH. In mammals, nocturnal melatonin secretion provides an endocrine signal of the photoperiod to the PT that contains melatonin receptors in high density, but the interface between the melatonin signal perceived in the PT and the thyroid hormone levels in the MBH remains unclear. Here we provide evidence in mice that TSH participates in this photoperiodic signal transduction. Although most mouse strains are considered to be nonseasonal, a robust photoperiodic response comprising induced expression of TSHB (TSH beta subunit), CGA (TSH alpha subunit), and DIO2, and reduced expression of DIO3, was observed in melatonin-proficient CBA/N mice. These responses could not be elicited in melatonin-deficient C57BL/6J, but treatment of C57BL/6J mice with exogenous melatonin elicited similar effects on the expression of the above-mentioned genes as observed in CBA/N after transfer to short-day conditions. The EC was found to express TSH receptor (TSHR), and ICV injection of TSH induced DIO2 expression. Finally, we show that melatonin administration did not affect the expression of TSHB, DIO2, and DIO3 in TSHR-null mice. Taken together, our findings suggest that melatonin-dependent regulation of thyroid hormone levels in the MBH appears to involve TSH in mammals.

Genetic analysis of abdominal fat distribution in SM/J and A/J mice.

Each abdominal fat depot, such as mesenteric or epididymal, differently contributes to the development of insulin resistance. The aim of this study was to identify the genetic regions that contribute to fat accumulation in epididymal/mesenteric fat and to examine whether or not the genetic regions that affect glucose metabolism and body fat distribution are coincident. We previously mapped a major quantitative trait locus (QTL) (T2dm2sa) for impaired glucose tolerance on chromosome 2 and revealed that SM.A-T2dm2sa congenic mice showed not only glucose tolerance but also fat accumulation. In the present study, to identify the loci/genes that control the accumulation of abdominal fat, we performed QTL analyses of epididymal/mesenteric fat weight by using (A/J x SM.A-T2dm2sa)F2 mice in which the effect of T2dm2sa was excluded. As a result, two highly significant QTLs for mesenteric fat, as well as three significant QTLs for epididymal/mesenteric fat, were mapped on the different chromosomal regions. This suggests that the fat accumulations in individual fat depots are controlled by distinct genomic regions. Our comparison of these QTLs for abdominal fat distribution with those for glucose metabolism revealed that the major genetic factors affecting body fat distribution do not coincide with genetic factors affecting glucose metabolism in (A/J x SM.A-T2dm2sa)F2.

Ascorbic acid deficiency induces hepatic and intestinal expression of inflammation-related genes irrespective of the presence or absence of gut microbiota in ODS rats.

We have previously demonstrated that ascorbic acid (AsA) deficiency causes inflammatory changes in the liver and intestine in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. We have suggested that AsA deficiency increased intestinal interleukine (IL)-6 production, stimulating hepatic acute phase proteins (APPs) expression via the portal vein. In this study, we determined whether these hepatic and intestinal inflammatory changes by AsA deficiency are induced in germ-free (GF) ODS rats. For 18 days, male specific pathogen-free (SPF) ODS rats were fed the basal diet containing 600 mg AsA/kg (control group) or the AsA-free diet (AsA-deficient group) in SPF conditions, while male GF ODS rats were fed the basal diet (control group) or the AsA-free diet (AsA-deficient group) in GF conditions. Firstly, AsA deficiency significantly elevated the hepatic expression of APPs in both SPF and GF rats. In hepatic mRNA levels of some APPs, significant interaction between GF and AsA-deficiency effects was observed. Secondly, AsA deficiency elevated intestinal IL-6 and IL-1ß mRNA levels in both SPF and GF rats, and significant interaction between GF and AsA-deficiency effects was observed in these mRNA levels of jejunum and cecum. In SPF and GF rats, AsA deficiency elevated portal IL-6 concentration. These results show that AsA deficiency caused hepatic and intestinal inflammatory changes in both the GF and SPF ODS rats and indicate that AsA deficiency could directly induce intestinal inflammatory changes without the involvement of gut microbiota.

Heat Stress Causes Immune Abnormalities via Massive Damage to Effect Proliferation and Differentiation of Lymphocytes in Broiler Chickens.

Broiler chickens are highly sensitive to high ambient temperatures due to their feathers, lack of skin sweat glands, and high productivity. Heat stress (HS) is a major concern for the poultry industry because it negatively affects growth as well as immune functions, which increase the potential risk of infectious disease outbreaks. Therefore, it is vital to elucidate HS's effect on the avian immune system, especially considering the global rise in average surface temperature. Our study identified a series of immunological disorders in heat-stressed broiler chickens. We exposed 22-day-old broiler chickens to a continuous HS condition (34.5 ± 0.5°C) for 14 days and immunized them with a prototype bovine serum albumin (BSA) antigen. The plasma and lymphoid tissues (thymus, bursa of Fabricius, and spleen) were harvested at the end of the experiments to investigate the induction of BSA-specific immune responses. Our results revealed that plasma titers of immunoglobulin (Ig)Y, IgM, and IgA antibodies specific for BSA were lower than those of thermoneutral chickens immunized with BSA. Furthermore, the spleens of the heat-stressed broiler chickens displayed severe depression of Bu1+ B cells and CD3+ T cells, including CD4+ T cells and CD8+ T cells, and lacked a fully developed germinal center (GC), which is crucial for B cell proliferation. These immunological abnormalities might be associated with severe depression of CD4-CD8- or CD4+CD8+ cells, which are precursors of either helper or killer T cells in the thymus and Bu1+ B cells in the bursa of Fabricius. Importantly, HS severely damaged the morphology of the thymic cortex and bursal follicles, where functional maturation of T and B cells occur. These results indicate that HS causes multiple immune abnormalities in broiler chickens by impairing the developmental process and functional maturation of T and B cells in both primary and secondary lymphoid tissues.

Ablation of Iah1, a candidate gene for diet-induced fatty liver, does not affect liver lipid accumulation in mice.

Nonalcoholic fatty liver disease (NAFLD) is a pathological condition caused by excess triglyceride deposition in the liver. The SMXA-5 severe fatty liver mouse model has been established from the SM/J and A/J strains. To explore the genetic factors involved in fatty liver development in SMXA-5 mice, we had previously performed quantitative trait locus (QTL) analysis, using (SM/J×SMXA-5)F2 intercross mice, and identified Fl1sa on chromosome 12 (centromere-53.06 Mb) as a significant QTL for fatty liver. Furthermore, isoamyl acetate-hydrolyzing esterase 1 homolog (Iah1) was selected as the most likely candidate gene for Fl1sa. Iah1 gene expression in fatty liver-resistant A/J-12SM mice was significantly higher than in fatty liver-susceptible A/J mice. These data indicated that the Iah1 gene might be associated with fatty liver development. However, the function of murine Iah1 remains unknown. Therefore, in this study, we created Iah1 knockout (KO) mice with two different backgrounds [C57BL/6N (B6) and A/J-12SM (A12)] to investigate the relationship between Iah1 and liver lipid accumulation. Liver triglyceride accumulation in Iah1-KO mice of B6 or A12 background did not differ from their respective Iah1-wild type mice under a high-fat diet. These results indicated that loss of Iah1 did not contribute to fatty liver. On the other hands, adipose tissue dysfunction causes lipid accumulation in ectopic tissues (liver, skeletal muscle, and pancreas). To investigate the effect of Iah1 deficiency on white adipose tissue, we performed DNA microarray analysis of epididymal fat in Iah1-KO mice of A12 background. This result showed that Iah1 deficiency might decrease adipokines Sfrp4 and Metrnl gene expression in epididymal fat. This study demonstrated that Iah1 deficiency did not cause liver lipid accumulation and that Iah1 was not a suitable candidate gene for Fl1sa.

Characterization of critical factors influencing gene expression of two types of fatty acid-binding proteins (L-FABP and Lb-FABP) in the liver of birds.

In avian species, two types of intracellular lipid-binding proteins are abundant in the liver, the liver fatty acid-binding protein (L-FABP) and the liver basic fatty acid-binding protein (Lb-FABP). Both FABPs are capable of forming complexes with free fatty acids and bile acids, but the functional distinction between L-FABP and Lb-FABP in avian liver is not fully understood. To gain insights into the functional distinction between L-FABP and Lb-FABP, we investigated the expression of both genes in relation to the pre- and post-hatching development, diurnal cycle and feeding state in the livers of chicken (Gallus gallus) and Japanese quail (Coturnix japonica). In chickens, the Lb-FABP mRNA was expressed only in the liver, while the L-FABP was expressed in both liver and intestinal tissues. Only small amounts of the L-FABP and Lb-FABP mRNAs were detected in the liver during chicken embryogenesis, but at the onset of hatching a dramatic increase in mRNA expression was observed for both genes, suggesting that the expression of the L-FABP and Lb-FABP genes is synchronized at developmental stages. Remarkably, the diurnal expression pattern differed between the two genes under a 16L:8D condition in sexually mature quail: L-FABP gene expression transiently increased at the end of the light cycle, whereas Lb-FABP gene expression peaked during the early part of the light cycle and gradually decreased as the dark period approached. We attempted to identify the factors regulating the diurnal gene expression pattern, and found that feeding stimulation was a critical factor inducing Lb-FABP gene expression irrespective of light condition. On the other hand, feeding stimulation only slightly stimulated expression of the L-FABP gene, and was not always its primary determinant. These results suggest that L-FABP and Lb-FABP play different roles in metabolic process during the postprandial state.

Characterization of Growth, Fat Deposition, and Lipid Metabolism-Related Gene Expression in Lean and Obese Meat-Type Chickens.

Excessive fat deposition adversely affects poultry production. In this study, we investigated growth, fat deposition, and hepatic mRNA expression of 13 lipid metabolism-related genes in three unique breeds of meat-type chickens with distinct breed origins and genetic relationships. One was Nagoya (NAG), a native Japanese breed, whereas the others were White Plymouth Rock (WPR) and White Cornish (WC), which have been used worldwide as the parental breeds of common broiler chickens. NAG chickens were phenotypically characterized by slow growth, lean body fat, and high gizzard and liver weights. In contrast, both WC and WPR chickens were characterized by rapid growth but high percentage of subcutaneous fat and abdominal fat weight, resulting from high feed intake. Among the three breeds, WC had the highest percentage of pectoral muscle weight, whereas WPR was the most obese. Among lipid metabolism-related genes, the expression of PPARA, PPARG, and CD36 was mostly associated with obesity. These results provide basic information for quantitative trait locus (QTL) analysis related to growth and fat traits in an F2 population of the lean NAG breed and the obese WPR breed of meat-type chickens in future.

Genetic diversity of 21 experimental chicken lines with diverse origins and genetic backgrounds.

The genetic characteristics and diversity of 21 experimental chicken lines registered with the National BioResource Project of Japan were examined using mitochondrial D-loop sequences and 54 microsatellite DNA markers. A total of 12 haplotypes were detected in the 500-bp mitochondrial DNA sequences of the hypervariable segment I for 349 individuals of 21 lines. The 12 haplotypes belonged to three (A, D, and E) haplogroups, out of the eight (A‒H) common haplogroups in domestic chickens and red junglefowls. The haplogroups A and D were widely represented in indigenous chickens in the Asian and Pacific regions, and the haplogroup E was the most prevalent in domestic chickens. Genetic clustering by discriminant analysis of principal components with microsatellite markers divided 681 individuals of 21 lines into three groups that consisted of Fayoumi-, European-, and Asian- derived lines. In each of the cladograms constructed with Nei's genetic distances based on allele frequencies and the membership coefficients provided by STRUCTURE and with the genetic distance based on the proportion of shared alleles, the genetic relationships coincided well with the breeding histories of the lines. Microsatellite markers showed remarkably lower genetic heterozygosities (less than 0.1 observed heterozygosity) for eight lines (GSP, GSN/1, YL, PNP, BM-C, WL-G, BL-E, and #413), which have been maintained as closed colonies for more than 40 years (except for #413), indicating their usefulness as experimental chicken lines in laboratory animal science research.

Ascorbic acid deficiency increases hepatic expression of acute phase proteins through the intestine-derived IL-6 and hepatic STAT3 pathway in ODS rats.

We have previously shown that ascorbic acid (AsA) deficiency elevates hepatic expression of acute phase proteins (APPs), inflammatory markers, in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. However, the precise mechanisms of this elevation are unknown. Signal transducer and activator of transcription 3 (STAT3) is one of the transcription factors inducing the expression of APPs and is activated by several cytokines including interleukin-6 (IL-6). The aim of this study was to determine whether AsA deficiency stimulates hepatic STAT3 activation and increases intestinal production of proinflammatory cytokines such as IL-6. Male ODS rats (6 weeks old) were fed either a basal diet containing 300 mg AsA/kg (control group) or an AsA-free diet (AsA-deficient group) for 18 days. AsA deficiency gradually and simultaneously elevated both mRNA levels of APPs (haptoglobin, α1-acid glycoprotein, C-reactive protein and α2-macroglobulin) and nuclear level of phosphorylated STAT3 (activated STAT3) in the liver. These results showed that the AsA-deficiency-induced expression of hepatic APPs is stimulated by proinflammatory cytokines activating STAT3. On day 14, AsA deficiency significantly elevated IL-6 mRNA level in the ileum and the concentration of IL-6 in portal blood. Furthermore, the portal concentration of IL-6 positively correlated with hepatic mRNA levels of STAT3-regulated genes. These findings suggest that IL-6, produced in the intestine as a result of AsA deficiency, is recruited to the liver via the portal vein and contributes to hepatic STAT3 activation and the elevated expression of APPs.

Exclusion of polymeric immunoglobulins and selective immunoglobulin Y transport that recognizes its Fc region in avian ovarian follicles.

In avian species, blood immunoglobulin (Ig) Y, the equivalent to mammalian IgG, is selectively incorporated into ovarian follicles, but other classes, IgA and IgM, are much less abundant in the follicles. Several mammalian Igs, including IgG and IgA, are also incorporated into ovarian follicles when administered to birds. To clarify the Ig structure required for incorporation into ovarian follicles, Ig uptakes were determined after the intravenous injection of chicken and human Igs. Three chicken Igs (cIgY, cIgA and cIgM) and two human IgAs (monomeric hIgA and polymeric hIgA) were labeled with digoxigenin, and their uptakes into quail (Coturnix japonica) egg yolks were determined by ELISA and SDS-PAGE. The uptake of cIgY was the highest among the three cIgs (22% of injected cIgY was recovered from egg yolks). Chicken IgA was efficiently incorporated into egg yolk when it formed a monomeric state. Pentameric IgM was untransportable into egg yolk. We also found that the uptake of monomeric hIgA was much more efficient than that of polymeric hIgA. These results suggest that the retention of the monomeric form contributes to the efficient transport of Igs into ovarian follicles. On the other hand, Ig uptakes among monomeric Igs nevertheless differed; for example, a time-course analysis showed that the rate of monomeric cIgY uptake was approximately eight times faster than that of monomeric hIgA. The injection of cIgY fragments Fc, Fab and F(ab')(2) resulted in the largest uptake of Fc fragment, with the same level as that of cIgY. These results suggest the presence of a selective IgY transport system that recognizes its Fc region in avian ovarian follicles.

Dietary Intake of Ascorbic Acid Attenuates Lipopolysaccharide-Induced Sepsis and Septic Inflammation in ODS Rats.

The aim of this study was to verify the protective effects of ascorbic acid (AsA) against lipopolysaccharide (LPS)-induced sepsis. The study was conducted using osteogenic disorder Shionogi (ODS) rats, which are unable to synthesize AsA. Male ODS rats (6 wk old) were fed either an AsA-free diet (AsA-deficient group), a diet supplemented with 300 mg/kg AsA (control group), or a diet supplemented with 3,000 mg/kg AsA (high-AsA group) for 8 d. On day 8, all the rats were intraperitoneally injected with LPS (15 mg/kg body weight). Forty-eight hours after the injection, the survival rates of the rats in the control (39%) and the high-AsA (61%) groups were significantly higher than that in the AsA-deficient group (5.5%). Next, we measured several inflammatory parameters during 10 h after administering LPS. At 6 h, elevated serum levels of markers for hepatic and systemic injuries were suppressed in rats fed AsA. Similarly, 10 h after LPS injection, the elevation in the serum levels of markers for renal injury were also suppressed proportionally to the amount of AsA in the diet. The elevated serum concentrations of TNFα and IL-1ß by LPS in the AsA-deficient group decreased in groups fed AsA. Hematic TNFα mRNA levels at 6 h after the LPS injection were also lowered by feeding AsA. These results demonstrated that the dietary intake of AsA improved the survival rates and suppressed the inflammatory damage, in a dose-dependent manner, caused during sepsis induced by LPS in ODS rats.