Kan Sato

Cold‐induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle

Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non‐shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold‐acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441–444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the effects of cold acclimation on the fatty acid‐induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study differences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold‐acclimated chickens. The results obtained here show that suppression of palmitate‐induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold‐acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold‐acclimated chickens was also found. Taken together, the present studies on cold‐acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.

Broiler chickens (Ross strain) lack insulin-responsive glucose transporter GLUT4 and have GLUT8 cDNA

Identification of insulin-responsive glucose transporter proteins, GLUT4 and GLUT8, was attempted in chickens that characteristically are hyperglycemic and insulin resistant. Northern blot analysis using rat GLUT4 cDNA probe and RT-PCR using primers designed against the conserved regions in mammalian GLUT4 cDNA were not successful in identifying GLUT4 homologue(s) in various chicken tissues. Furthermore, GLUT4 homologues could not be detected in chicken tissues by genomic Southern blot analyses using a rat GLUT4 cDNA probe. These data, therefore, suggest that the GLUT4 homologous gene is deficient in chicken tissues. However, GLUT8, another insulin-responsive glucose transporter in the blastocyst, was identified with the aid of RACE (rapid amplification of cDNA ends) reactions in the chicken testis. Chicken GLUT8 was composed of 1449 bp with a coding region for a 482 amino acid protein. The deduced amino acid sequence was 58.8, 56.3, and 56.8% identical with human, rat, and mouse GLUT8, respectively. By RT-PCR, GLUT8 mRNA expressions were detected in chicken brain, kidney, adrenal, spleen, lung, testis, and pancreas; and barely detectable in skeletal muscle, liver, adipose tissue, and heart. Here we firstly report that GLUT8 was identified in chickens, while GLUT4, a major insulin-responsive transporter in mammals, is deficient in these animals. We propose the hypothesis that the hyperglycemia and insulin resistance observable in chickens is associated with their possible deficiency of GLUT4.

Possible role for avPGC‐1α in the control of expression of fiber type, along with avUCP and avANT mRNAs in the skeletal muscles of cold‐exposed chickens

Peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α), a transcriptional coactivator, plays a role in mitochondrial biogenesis, muscle fiber specialization, and adaptive thermogenesis. Because of an absence of brown adipose tissue, the skeletal muscle tissue in chickens serves as an important source of thermogenesis to counter the cold. The present experiments were conducted (i) to clone the cDNA of PGC‐1α homologs from chicken skeletal muscle and to examine alterations to PGC‐1α mRNA expression in the skeletal muscles of cold‐exposed chickens, (ii) to study the effect of cold‐acclimation on the metabolic fiber phenotype of typically fast‐glycolytic (type IIB) pectoralis muscles, and (iii) to compare avANT and avUCP mRNA expression in control and cold‐exposed chickens. Results show that the cloned avPGC‐1α cDNA encodes a 796 amino‐acid protein (GenBank Accession No. AB170013) showing 84% identity with rodent PGC‐1α cDNA. Exposure of chickens to a cold environment resulted in the prompt upregulation of avPGC‐1α expression, which preceded increments in avUCP and avANT expression in skeletal muscle mitochondria. Consistent with the morphological appearance of muscles, an increase in the number of fast‐oxidative‐glycolytic (type IIA) fibers in the pectoralis muscle, which contains exclusively type IIB fibers in control chickens, was observed in cold‐acclimated chickens. These findings provide novel information about possible regulatory pathways in avian skeletal muscle during thermogenesis.

Persistent hypoglycemia induced by continuous insulin infusion in broiler chickens

1. Persistent hypoglycaemia was experimentally induced by insulin infusion to improve understanding of the regulatory mechanisms of blood glucose concentrations specific to chickens. 2. An osmotic minipump containing bovine insulin was implanted to deliver insulin in vivo at a constant rate (11.25 to 45 U/kg BW/d) for 5 d in 4-week-old broiler chickens force-fed a maintenance diet once a d. Birds infused with the highest dose of insulin died within 3 to 4 d. 3. In chickens continuously infused with insulin at 22.5 U/kg BW/d, fasting glucose concentrations in plasma determined every 3 h during the 3rd day of infusion were consistently and significantly lower than in controls. 4. Continuous infusion of insulin at 22.5 U/kg BW/d induced persistent hypoglycaemia (almost one-half the normal blood glucose concentration) lasting for at least 4 d in broiler chickens. 5. Insulin infusion did not significantly change plasma NEFA or protein concentrations and increased plasma GOT activity only at 1 of the daily experimental sampling points.

Effects of dietary Spirulina on meat colour in muscle of broiler chickens

Abstract 1. The present study was undertaken to determine the effects of dietary spirulina on growth performance and pigmentation in the muscle of growing broiler chickens and to examine the possibility that zeaxanthin in spirulina may affect yellow colour development in the meat. 2. Twenty-four, 21-d-old, male broiler chicks were fed an experimental diet containing spirulina at 0, 40, or 80 g/ kg for 16 d. No significant differences among treatments were observed in body weights, nor weights or yields (as a percentage of body weight) for any of the selected traits, including liver, abdominal fat, kidney and Pectoralis profundus . 3. Spectrocolourimetric analyses revealed that the redness of Pectoralis superficialis, profundus and Sartorius muscles reached a maximum in chicks fed the 40 g/kg spirulina diet, while the yellowness of all fillets, including the Semitendinosus muscle, increased in a sub-linear fashion with increased spirulina in the diet. The overall correlation between the yellowness and zeaxanthin content in the Pectoralis muscle was significant. 4. This study provides the first conclusive evidence that dietary spirulina influences both the yellowness and redness of broiler flesh and that the increments in yellowness with dietary spirulina content may possibly be reflected in the common yellow pigment related to the accumulation of zeaxanthin within the flesh.

Identification of Factors Regulating Lipoprotein Lipase Catalyzed Hydrolysis in Rats with the Aid of Monoacid-Rich Lipoprotein Preparations

To identify the substrate specificity and regulatory factors in lipoprotein lipase (LPL) catalyzed hydrolysis of triacylglycerol-rich lipoprotein, monoacid-rich lipoproteins were used to study the kinetic parameters of LPL. Feeding growing rats with diets rich in palmitic acid (16:0), oleic acid (18:1) or linoleic acid (18:2) for 10 days increased the corresponding acid content in the triacylglycerols of the lipoproteins. Force-feeding the monoacid-rich triacylglycerols, particularly 16:0 or 18:1, increased the respective fatty acid content in both chylomicrons and VLDLs. Major apolipoproteins and lipid compositions were essentially similar among all lipoproteins differing in monoacid species, except for apo A-IV. The Vmax of LPL for 16:0-rich chylomicrons and VLDLs were higher than for 18:1- or 18:2-rich lipoproteins. Order parameter (S), an indicator of the surface fluidity of lipoproteins, decreased with the chain length and unsaturation of monoacid in similar manner as the Vmax. The Vmax of LPL increased linearly (P < 0.05) with an increase in either the palmitic acid content of the lipoprotein triacylglycerols or order parameter (S) of the lipoproteins. The order parameter (S) and Vmax of LPL were higher in 16:0 triacylglycerol emulsions with apo B than with 18:1 or 18:2 triacylglycerols. The apo A-IV in triacylglycerol emulsions stimulated Vmax of LPLs in the presence of apo B and apo C-II. The binding of apo A-IV to 16:0 triacylglycerol emulsions was higher than to other triacylglycerol emulsions. These findings suggest that lipoprotein catalysis by LPL is modulated by the 16:0 level in the lipoprotein triacylglycerol, which affects the surface fluidity and apo A-IV content of lipoproteins.

Octanoate inhibits very low-density lipoprotein secretion in primary cultures of chicken hepatocytes

The effects of octanoate, a medium-chain fatty acid, on very low-density lipoprotein (VLDL) secretion in primary cultures of chicken hepatocytes were compared with those of palmitate. Palmitate added to the incubation media at concentrations up to 0.36 mM increased intracellular triacylglycerol (TG) accumulation and VLDL-TG secretion in a concentration-dependent manner, whereas the addition of octanoate alone (0.21-0.6 mM) did not change these parameters. VLDL-TG secretion from hepatocytes cultured in media to which 0.6 or 1.0 mM octanoate had been added in the presence of 0.21 mM palmitate was significantly lower than that obtained under control incubation conditions (0.21 mM palmitate only). The addition of 1.0 mM octanoate to the incubation media with or without 0.21 mM palmitate decreased VLDL apolipoprotein B (apoB) secretion. These results demonstrate that the addition of octanoate to primary cultures of chicken hepatocytes reduces VLDL secretion in respect of both TG and apoB secretion. It is suggested that medium-chain fatty acids are a factor modulating VLDL secretion, which plays a key role in fat deposition in chickens.

Effect of dietary supplementation of astaxanthin from Phaffia rhodozyma on lipopolysaccharide-induced early inflammatory responses in male broiler chickens (Gallus gallus) fed a corn-enriched diet.

Effect of dietary supplementation of astaxanthin (Ax) from Phaffia rhodozyma on lipopolysaccharide-induced inflammatory responses was investigated in male broiler chickens fed a corn-based diet. Birds (1 week of age) were fed a corn-enriched diet containing either 0 or 100 ppm Ax for 2 weeks and were intraperitoneally injected with lipopolysaccharide (LPS, 1 mg/kg body weight). Inflammatory responses were evaluated by determining changes in expression of messenger RNA (mRNA) in cytokines and mediators related to inflammatory responses (interleukin (IL)-1 beta and -6, inducible nitrite synthase (iNOS), interferon (IFN)- γ and cyclooxygenase (Cox)-2 in the liver and spleen after 2 h of LPS injection and plasma ceruloplasmin concentration as an acute phase protein. Birds fed Ax showed significantly higher iNOS mRNA expression in the liver and spleen compared to that of control birds. Ax-fed birds also showed greater increase in mRNA expression in the liver of IL-1, IL-6 and IFN-γ compared to that of control birds. The enhancing effect of Ax was further progressed when LPS was injected. No difference was found in plasma ceruloplasmin concentration between the Ax-fed group and control group. The results suggest that feeding supplementation of Ax (100 ppm) to a corn-enriched diet possibly does not have anti-inflammatory effect in male broiler chickens.

Species differences between chickens and rats in chemical properties of adipose tissue lipoprotein lipase

Chemical characterization of chicken and rat lipoprotein lipase (LPL) was carried out following purification of LPL. Molecular weight and isoelectric point of both purified enzymes were determined to be 60 KDa and pH 4, while optimum temperature and pH to yield the maximal activity were about 37 degrees C and pH 8.5. Metallic ions, NaCl and protamine sulfate reduced, and heparin increased, both LPL activities. Michaelis constants for LPLs determined with triolein emulsion as the substrate were 0.98 and 1.57, and those of Vmax were 379.2 and 181.3, in chickens and rats, respectively. Triton WR-1339 caused mixed-type inhibition in rat, but inhibited chicken LPL noncompetitively. In LPLs of chickens and rats, values of Ki were 66.7 and 36.4 with triolein emulsion as the substrate, and 832.4 and 66.0 with respective VLDL as the substrate. These results show species difference between chickens and rats in the affinity to lipoproteins of LPL and inhibition of LPL by Triton WR-1339.

SPECIES DIFFERENCES BETWEEN CHICKS AND RATS IN INHIBITION OF LIPOPROTEIN HYDROLYSIS BY TRITON WR-1339

To identify the species differences in lipoprotein hydrolysis, plasma lipoproteins and lipoprotein lipase (LPLase) prepared from both rats and chicks were incubated in vitro in the presence of Triton WR-1339 at concentrations up to 500 micrograms/ml. Rate of lipoprotein hydrolysis by LPLase declined gradually with an increase of Triton concentration irrespective of differences in sources in the lipoprotein and LPLase. At 250 micrograms/ml Triton, the lipoprotein hydrolysis was inhibited by 90% in rats but was only inhibited by < 20% in chicks. Lipoprotein hydrolysis inhibited by Triton was partly recovered by an increase of LPLase concentration, and extents of the recovery were prominent either when rat lipoprotein was provided in place of chick lipoprotein and when rat LPLase rather than chick LPLase was used as the enzyme source for each lipoprotein. These results suggest species differences between chicks and rats with regard to the affinity of Triton to LPLase and lipoprotein and/or the chemical characteristics of both LPLase and lipoprotein.