Hypoglycemia after Mitral Valve Repair in Dogs.

Hypoglycemia has not been previously reported as a postoperative complication of mitral valve repair (MVR) in dogs; however, the authors have encountered cases of hypoglycemia after MVR. This study aimed to determine the incidence of hypoglycemia in dogs after MVR and investigate its causes. Blood glucose levels were measured at multiple timepoints in dogs undergoing MVR. Simultaneously, insulin and glucagon blood concentrations in dogs with hypoglycemia preoperatively and postoperatively were compared to verify the physiological responses to hypoglycemia. Furthermore, risk factors for hypoglycemia, using variables selected based on the characteristics of MVR and dogs undergoing MVR, were examined prospectively. The incidence of hypoglycemia after MVR was 14.2%, and plasma glucagon concentrations increased in these dogs (mean: 260 pg/mL and 644 pg/mL pre- and postoperatively, p < 0.001), whereas serum insulin concentrations decreased (median: 0.50 ng/mL and 0.29 ng/mL pre- and postoperatively, p = 0.002). Therefore, hyperinsulinemia or hypoglucagonemia is unlikely to be the cause of postoperative hypoglycemia. The identified risk factors for hypoglycemia included low body weight and asymptomatic myxomatous mitral valve disease. Monitoring blood glucose levels after MVR should be included in the standard hospitalization plan to prevent hypoglycemic emergencies in dogs.

Effects of low-protein diet and feed restriction on mRNA expression of cationic amino acid transporters in porcine skeletal muscles.

We investigated the effects of a low-protein diet and feed restriction on the mRNA expression of cationic amino acid transporters (CATs) in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pigs. Eighteen piglets were divided into three groups: a control (CP21%), low-protein diet (LP, CP16%), and feed-restricted diet (FR, CP21%, 76% feed intake of control pigs) groups. The expression levels of CAT-1 in the LD and BF muscles of LP pigs were higher than that of control pigs, whereas that of FR pigs showed no difference. The CAT-2A expression levels in the RH muscle of FR pigs were higher than that of control pigs. The free lysine concentrations in all muscles of LP and FR pigs were lower than that of control pigs. To examine the factors that affect CATs mRNA expression, we evaluated the effects of lysine, arginine, insulin-like growth factor-I, and dexamethasone on the expression of CATs in C2C12 myotubes. CAT-1 expression levels increased in lysine and/or arginine deprivation. We show that CAT-1 and CAT-2A expression levels in skeletal muscles differ in response to dietary treatments and CAT-1 expression in skeletal muscles appears to increase in response to low free lysine concentrations.

Isolation and Genome Sequencing of Hepatitis E Virus Genotype 1 Imported from India to Japan.

Hepatitis E virus (HEV) is the causative agent of viral hepatitis E. In Japan, HEV genotype 3 (G3) and G4 are predominantly detected, while G1, mainly imported from countries in continental Asia, is rare. In the present study, we detected a G1 HEV strain in a patient who visited Japan from India. When PLC/PRF/5 cells (subclone 4-21) were inoculated with a stool suspension from this patient, accumulation of HEV RNA was observed in the spent culture medium, indicating that HEV had been successfully isolated from this specimen. A nearly complete HEV genome was obtained by RT-PCR amplification. Phylogenetic analyses revealed that the newly isolated HEV strain, designated 9HE36c, belongs to subtype 1g of HEV G1.

Circadian rhythms of the mRNA abundances of clock genes and glucose transporters in the jejunum of weanling-growing pigs.

BACKGROUND: Whether abundance of glucose transporter mRNAs in the small intestine of pigs shows circadian rhythms and its regulation by clock genes was still unknown. OBJECTIVES: We examined whether the abundance of glucose transporters and clock genes mRNAs in the small intestine of pigs shows circadian rhythms. METHODS: Twenty barrows (4 weeks old) were reared under 12 h bright and 12 h dark lighting conditions. During the 3-week feeding trial, pigs were allowed free access to feed. The abundances of the mRNA of glucose transporters (SGLT1 and GLUT2) and clock genes (Bmal1, Per1, Per2, and Cry2) in the intestine were measured at four time points (ZT2, ZT8, ZT14, and ZT20). RESULTS: In the jejunum, the abundance of SGLT1 mRNA was higher at ZT20 and ZT2 and lower at ZT8 and ZT14 (p < 0.05). The abundances of GLUT2 mRNA in the jejunum at ZTs 20 and 2 were tended to be higher than those at ZTs 8 and 14 (p = 0.05). In the jejunum, the abundance of Bmal1 mRNA was higher at ZT8 and ZT14 than at ZT20 and ZT2 (p < 0.05). Further, the abundance of Per1 mRNA at ZT2 was higher than those at the other sampling times (p < 0.05). The abundance of Per1 mRNA at ZT8 was higher than that at ZT14 (p < 0.05), while that of Per2 mRNA was higher at ZT2 than those at ZTs 20 and 14 (p < 0.05). CONCLUSION: We speculate that these circadian rhythms of abundances of glucose transporter mRNAs are regulated by the clock genes expressed in the jejunum.

Dietary lysine restriction induces lipid accumulation in skeletal muscle through an increase in serum threonine levels in rats.

We previously reported that dietary amino acid restriction induces the accumulation of triglycerides (TAG) in the liver of growing rats. However, differences in TAG accumulation in individual cell types or other tissues were not examined. In this study, we show that TAG also accumulates in the muscle and adipose tissues of rats fed a low amino acid (low-AA) diet. In addition, dietary lysine restriction (low-Lys) induces lipid accumulation in muscle and adipose tissues. In adjusting the nitrogen content to that of the control diet, we found that glutamic acid supplementation to the low-AA diet blocked lipid accumulation, but supplementation with the low-Lys diet did not, suggesting that a shortage of nitrogen caused lipids to accumulate in the skeletal muscle in the rats fed a low-AA diet. Serum amino acid measurement revealed that, in rats fed a low-Lys diet, serum lysine levels were decreased, while serum threonine levels were significantly increased compared with the control rats. When the threonine content was restricted in the low-Lys diet, TAG accumulation induced by the low-Lys diet was completely abolished in skeletal muscle. Moreover, in L6 myotubes cultured in medium containing high threonine and low lysine, fatty acid uptake was enhanced compared with that in cells cultured in control medium. These findings suggest that the increased serum threonine in rats fed a low-Lys diet resulted in lipid incorporation into skeletal muscle, leading to the formation of fatty muscle tissue. Collectively, we propose conceptual hypothesis that "amino-acid signal" based on lysine and threonine regulates lipid metabolism.

Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification.

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140-170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.

Effects of dietary lysine levels and lighting conditions on intramuscular fat accumulation in growing pigs.

This study was conducted to test our hypothesis that intramuscular fat (IMF) accumulation increases in pigs fed on a low lysine diet during the dark period than those fed on the same diet during the light period. Using barrows aged 6 weeks, we monitored whether serum glucose and insulin levels were affected by light conditions. Two diets with different levels of lysine, 0.78% (LL diet) and 1.37% (control diet) were prepared. Eight pigs were fed on the diet during the light period, while the remaining pigs were fed during the dark period. The pigs were fed either the LL diet or the control diet. Although IMF contents of Longissimus dorsi (LD) muscle were higher in the pigs fed on a LL diet (p < .05), the light conditions had no effect. Low dietary lysine caused reduction in serum glucose levels (p < .05) and serum insulin levels (p = .0613). However, they were also unaffected by the lighting conditions. To gain further insights, we determined the messenger RNA levels of insulin receptor, insulin receptor substrate 1, acetyl CoA carboxylase, and fatty acid synthase in LD and Rhomboideus muscles and in the liver.

Expression of cationic amino acid transporters in pig skeletal muscles during postnatal development.

The cationic amino acid transporter (CAT) protein family transports lysine and arginine in cellular amino acid pools. We hypothesized that CAT expression changes in pig skeletal muscles during rapid pig postnatal development. We aimed to investigate the tissue distribution and changes in the ontogenic expression of CATs in pig skeletal muscles during postnatal development. Six piglets at 1, 12, 26, 45, and 75 days old were selected from six litters, and their longissimus dorsi (LD), biceps femoris (BF), and rhomboideus (RH) muscles, and their stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, and cerebrum were collected. CAT-1 was expressed in all the 12 tissues investigated. CAT-2 (CAT-2A isoform) expression was highest in the skeletal muscle and liver and lowest in the jejunum, ileum, kidney, and heart. CAT-3 was expressed mainly in the colon and detected in the jejunum, ileum, and cerebrum. The CAT-1 expression was higher in the skeletal muscle of day 1 pigs than in that of older pigs (P < 0.05). The CAT-2 mRNA level was lowest at day 1, but increased with postnatal development (P < 0.05). There was no significant change in CAT-1 expression among the LD, BF, and RH during postnatal development (P > 0.05); however, there was a change in CAT-2 expression. The CAT-2 expression was highest in the LD of 12-, 26-, 45-, and 75-day-old pigs, followed by the BF and RH (P < 0.05). These results suggest that CAT-1 and CAT-2 play different roles in pig skeletal muscles during postnatal development.

Changes in muscle fiber type and expression of mRNA of myosin heavy chain isoforms in porcine muscle during pre- and postnatal development.

The purpose of this study is to elucidate developmental changes in muscle fiber type in the pig during pre- and postnatal development. For this purpose, we performed a histochemical analysis for myosin adenosine triphosphatase activity to assess muscle fiber type and determined abundances of messenger RNA (mRNA) of myosin heavy chain (MHC) isoforms. Samples of Longissimus dorsi (LD) muscle were taken from fetuses on day 90 of the fetal stage. Further, samples of LD, Rhomboideus and Biceps femoris (B. femoris) muscles were taken from pigs when they were 1, 12, 26, 45 or 75 days old. Expression of MHC 2b mRNA in the LD and the B. femoris muscles rapidly and considerably increased from the late fetal stage to the early postnatal stage and this increase was associated with the development of type 2b fibers at least in the LD muscle. As shown by the rapid and considerable changes in expression of MHC 2b mRNA, it seems that a certain plasticity of muscle fiber type still remains in this developmental stage.

Comparison of atrogin-1/MAFbx mRNA expression in the gizzards of egg- and meat-type chickens.

We examined atrogin-1/MAFbx mRNA expression in the smooth muscle of gizzards from egg- and meat-type chickens. Gizzard weight relative to body weight was significantly lower in the meat-type chickens than in the egg-type at 14 d of age. In contrast, the level of atrogin-1/MAFbx mRNA in the gizzard was significantly higher in the meat-type chickens than in the egg-type chickens. Thus atrogin-1/MAFbx mRNA expression in the smooth muscle of the gizzard was higher in meat-type chickens than in egg-type chickens, in contrast to its expression in the skeletal muscles.

Compensatory growth of C2C12 myotubes induced by the combined effect of lysine sufficiency and modulation of IGF-I and glucocorticoid levels.

We have reported that a change from a lysine-deficient diet to a lysine-sufficient diet induced compensatory growth in rats and pigs. The aim of the present study was to determine whether compensatory growth of C2C12 myotubes occurs only by sufficiency of lysine or also by the synergic effect of sufficiency of lysine and modulation of the levels of insulin-like growth factor-I (IGF-I) and glucocorticoid in a medium. The results provide the first evidence of compensatory growth of C2C12 myotubes induced by sufficiency of a single amino acid in combination with modulation of the levels of IGF-I and glucocorticoid.

Atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, is highly expressed in the smooth muscle of the chicken gizzard.

This study was conducted to investigate the expression of atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, in the smooth muscle of the chicken gizzard. Atrogin-1/MAFbx mRNA expression was detected in the skeletal muscle, heart (cardiac muscle), gizzard (smooth muscle), brain, and liver of chicks, with highest expression in the smooth muscle of the gizzard. The expression of atrogin-1/MAFbx mRNA in the smooth muscle of the gizzard was increased by fasting (24 h), and this increase was reduced by refeeding (2 h). These results indicate that atrogin-1/MAFbx mRNA is highly expressed in the smooth muscle of the chicken gizzard, and that the expression of it is regulated by nutritional conditions.

Effects of low protein diet and low protein diet supplemented with synthetic essential amino acids on meat quality of broiler chickens.

We investigated the effects of a low crude protein (CP) diet and a low CP diet supplemented with synthetic essential amino acids (EAA) on the meat quality of broiler chickens. Twenty-one-day-old chickens were assigned to one of three diets: control, low CP (LCP), or low CP supplemented with EAA (ELCP). The chickens received these diets for 10 days. The shear force value (SFV) and free glutamate content of the Pectoralis major muscle were measured as indicators of the meat toughness and taste. The collagen and crude fat content of the muscle and the cross-sectional area of myofibers were measured to evaluate the effects of the LCP and ELCP diets on meat toughness. The SFV of the ELCP group was 47% lower than that of the control group (P<0.01). However, the LCP diet did not affect the SFV. The collagen and crude fat content were not affected by the dietary treatment. The cross-sectional area was lower in the LCP and ELCP groups (P<0.05) than the control group. The free glutamate content of muscle was not affected by the dietary treatment. Thus, a low CP diet supplemented with EAA is an effective means of producing tender meat.

Effects of dietary low level of threonine and lysine on the accumulation of intramuscular fat in porcine muscle.

We attempted to determine whether shortage of dietary threonine enhances the accumulation of intramuscular fat (IMF) in porcine muscle. Although dietary low levels of lysine enhanced the concentration of IMF in the longissimus dorsi and rhomboideus muscles, dietary low threonine did not. We conclude that the effect of dietary threonine levels on the accumulation of IMF in porcine muscle is negligible.

Nitrogen balance during compensatory growth when changing the levels of dietary lysine from deficiency to sufficiency in growing pigs.

Two experiments were conducted to elucidate the nitrogen (N) balance of pigs exhibiting compensatory growth when changing the dietary lysine levels from deficiency to sufficiency. Experiment 1 elucidated whether pigs exhibited compensatory growth with dietary lysine sufficiency. Twenty 6-week-old males were assigned to one of two treatments: control and LC (lysine and control). Control pigs were fed a control diet throughout the 24-day experimental period, whereas LC pigs were fed a low lysine diet until day 21 of the experiment, followed by the control diet until the end of experiment. The dietary lysine sufficiency treatment induced an 80% increase in the growth rate of LC pigs (P < 0.05). Experiment 2 focused on the N balance of pigs that exhibited compensatory growth with dietary lysine sufficiency. Eighteen 6-week-old males were assigned to one of three treatments: control, LC, and LL (low lysine). LL pigs were fed a low lysine diet throughout the 24-day experimental period. Pigs that exhibited compensatory growth with dietary lysine sufficiency tended to retain a higher amount of N than control pigs (P = 0.10). These finding suggest that the compensatory growth induced in pigs by dietary lysine sufficiency was partly attributable to a higher level of N retention.

The effects of concentrations of lysine in media on differentiation of 3T3-L1 preadipocytes.

Intramuscular fat content is increased by feeding of low lysine diets in pigs. Reduction in dietary lysine intake results in low plasma lysine concentration and low cytosolic lysine concentration in skeletal muscles. From these observations, we hypothesized that low plasma lysine concentration in pigs fed on low lysine diets reduced supply of lysine from blood circulation to preadipocytes, and this limited supply of lysine might promote adipocyte differentiation in porcine muscles. In order to verify the hypothesis, we investigated the effects of low concentrations of lysine in culture medium on differentiation of 3T3-L1 preadipocytes. Low concentration of lysine suppressed lipid accumulation and messenger RNA (mRNA) expression and enzyme activity of fatty acid synthase. mRNA expressions of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were lower in cells cultured in low lysine medium. On the other hand, mRNA and protein expressions of C/EBPß and C/EBPδ were not inhibited by low concentrations of lysine in culture medium. These results indicate that low lysine concentrations in culture medium inhibit differentiation of 3T3-L1 preadipocytes through inhibiting the mRNA expressions of PPARγ and C/EBPα.

Promotion of intramuscular fat accumulation in porcine muscle by nutritional regulation.

Recently, pork with marbling has received attention as good quality pork and scientists are required to develop methods to produce pork with reasonable amounts of intramuscular fat (IMF). The aim of this review is to describe studies relevant to promotion of IMF accumulation in porcine muscle by nutritional regulation. The main focus is on effect of dietary lysine levels. First, we found dietary low lysine up-regulated glucose transporter protein 4 messenger (m)RNA expression in Longissimus dorsi (L. dorsi) and Rhomboideus muscles. In addition, the proportion of oxidative fiber of both muscles was also enhanced by dietary low lysine. Because it has been observed that higher oxidative capacity is associated with higher IMF content, we hypothesized that dietary low lysine would promote IMF accumulation. Further, higher mRNA abundance of peroxisome proliferator-activated receptor γ, a master regulator of adipogenesis, in both muscles induced by dietary low lysine, supported this hypothesis. Indeed, IMF content of L. dorsi muscle of finishing pigs given a low lysine diet for 2 months until reaching the market weight was twice that of pigs given a control diet. Possible underlying mechanisms of IMF accumulation in porcine muscle and future perspectives are also discussed in this review.

Muscle protein metabolism during compensatory growth with changing dietary lysine levels from deficient to sufficient in growing rats.

Livestock and laboratory animals show compensatory growth when they are fed ad libitum following a period of restriction feeding. Lysine is a major limiting essential amino acid in the diets both for humans and animals. We hypothesized that changing dietary lysine levels from deficient to sufficient induced compensatory growth in young rats. We elucidated the effect of lysine sufficiency on the dynamics of hormones, relevant to muscle protein synthesis and degradation, insulin-like growth factor-I (IGF-I) and corticosterone, and on the expression of proteolytic-related genes in skeletal muscle during compensatory growth. Lysine sufficiency where the dietary lysine level was increased from 0.46% to 1.30% after 2 wk of subjecting the rats to the lower lysine level induced 80% enhancement of growth rate of rats. During compensatory growth with the lysine sufficiency, fractional muscle protein synthesis rates were higher whereas fractional muscle protein degradation rates were lower than those of the control group (p<0.05). After lysine sufficiency, the expression of atrogin-1/MAFbx mRNA was decreased in gastrocnemius muscle (p<0.05). With the lysine sufficiency, serum IGF-I concentration increased (p<0.05) whereas serum corticosterone decreased (p<0.05). These findings suggest that compensatory growth with lysine sufficiency is due to a change of hormone levels before and after changing diets, resulting in incrementation of protein synthesis and suppression of protein degradation of skeletal muscle.

Comparison of proteolytic-related gene expression in the skeletal muscles of layer and broiler chickens.

Previous studies have shown that the muscle protein degradation rates of broiler are lower than those of layer chickens. In this study, we assessed proteolytic-related gene expression in the pectoralis muscle of layer and broiler chickens. The mRNA levels of atrogin-1/MAFbx and proteasome C2 subunit, but not those of ubiquitin, m-calpain large subunit, cathepsin B, or caspase-3, were lower in the skeletal muscle of the broilers than in the layers at 7 and 14 d of age. We infer that the lower muscle protein degradation of broilers than of layers at least partly relates to lower mRNA expression of atrogin-1/MAFbx and proteasome C2 subunit in the skeletal muscle of broilers.

Differential regulation of porcine hepatic IGF-I mRNA expression and plasma IGF-I concentration by a low lysine diet.

The influence of dietary lysine on hepatic insulin-like growth factor-I (IGF-I) gene expression and plasma IGF-I level was investigated. Two male 6-wk-old pigs from each of six litters were used. Each littermate was assigned to one of two diets, control or low lysine (LL), that were isoenergetic and similar in protein content and provided 14.3 MJ digestible energy/kg for both diets, 185 g protein/kg for the control diet and 180 g protein/kg for the LL diet. The control diet contained all essential amino acids in the recommended amounts, including 11.5 g lysine/kg. The LL diet was similar but contained only 7 g lysine/kg. Pigs were pair-fed these diets for 3 wk. Growth rates and feed efficiencies of pigs fed the LL diet were significantly lower than those of pigs fed the control diet (P < 0.01). Plasma IGF-I levels in pigs fed the LL diet were 52% lower than in those fed the control diet (P < 0.01), and the LL group also had lower plasma IGF-binding protein-3 (IGFBP3) levels (P < 0.05). Despite the strikingly lower plasma IGF-I in pigs fed the LL diet, hepatic IGF-I mRNA abundance did not differ between the two treatment groups. We conclude that the reduction in plasma IGF-I caused by reduced dietary lysine may have been due in part to suppression of post-transcriptional events in IGF-I expression. The lower plasma IGFBP3 in pigs fed the LL diet suggests that increased clearance rates of circulating IGF-I may have been involved in this response.