Comparing pig breeds with genetically low and high backfat thickness: differences in expression of adiponectin, its receptor, and blood metabolites.

Here we characterized gene expressions in subcutaneous adipose tissue and blood metabolites of pigs with genetically low backfat (Landrace) and high backfat (Meishan). As pigs aged from 1 wk-to 3-mo old, mRNA levels of adipose-specific genes increased, although their gene expressions coding for major enzymes involved in lipid metabolism (lipoprotein lipase, fatty acid synthase, and hormone-sensitive lipase) did not differ between lean and fat pigs. Instead, there were significant effects for adiponectin and its receptor AdipoR1 mRNA levels between the two breeds of which respective expressions were lower and higher in Meishan by 3 mo of age. Contrary to changes in gene expressions, the concentrations of blood glucose, triglyceride (TG), and NEFA in both breeds decreased during growth, and 3-mo-old Meishan evidenced lower glucose with higher TG than the Landrace. The homeostasis model assessment insulin resistance (HOMA-IR) index was also calculated from the measurements of fasting glucose and insulin concentration, and Meishan showed a higher value than the Landrace. We next examined these differences in Landrace and Meishan crossbreds, which were phenotypically distinguishable by the backfat thickness as the former lean type and the latter fat type. As with the purebreds, high backfat Meishan crosses showed the characteristics of lower glucose and higher TG in circulating levels and also lower adiponectin transcripts in subcutaneous adipose tissue. Collectively, our results demonstrate that levels of adiponectin and its receptor gene expressions, blood glucose, blood lipids, and HOMA-IR in pigs vary between lean and fat. These observations strongly suggest the possibility that overall metabolic differences rather than adipocyte ability itself contribute to the fatness of genetically high backfat pigs.

Thiamine accumulation and thiamine triphosphate decline occur in parallel with ATP exhaustion during postmortem aging of pork muscles.

We aimed to clarify the mechanisms affecting postmortem thiamine and its phosphoester contents in major edible pork muscles, namely the longissimus lumborum (LL) in addition to vastus intermedius (VI). Metabolomic analysis by capillary electrophoresis-time of flight mass spectrometry revealed that the level of thiamine triphosphate (ThTP), approximately 1.8-fold higher in LL than in VI muscle at 0h postmortem, declined in the first 24hrs, resulting in an undetectable level at 168h postmortem in both muscles. In contrast, the thiamine content in both muscles increased after 24h postmortem during the aging process. The thiamine accumulation and ThTP decline progressed in parallel with a drastic reduction of the ATP level. The intermuscular differences in pH at 24h and in expression of thiamine transporter and thiamine pyrophosphokinase might result in delayed thiamine generation in LL. These results suggest that postmortem ATP exhaustion forced ThTP hydrolysis and further depyrophosphorylation of thiamine diphosphate in the porcine muscles, which resulted in thiamine accumulation.

Muscle type specific expression of tropomyosin isoforms in bovine skeletal muscles.

Nucleotide sequences encoding an entire coding region for bovine tropomyosin (TPM) isoforms were determined. Three TPM isoforms, TPM1, TPM2 and TPM3, were expressed in bovine skeletal muscles, and exhibited a 93.3%, 99.6% and 100% amino acid homology to the human sequence, respectively. Based on the sequences, the composition of TPM isoforms was analyzed on cDNA and protein levels from five physiologically different muscles (masseter, diaphragm, psoas major, longissimus thoracis and semitendinosus) using RT-PCR and proteome analyses. Although the content of TPM2 was constantly about 50% of the total TPM in all muscles, the contents of TPM1 and TPM3 were different in muscles according to their function in muscle contraction. In masseter, the content of TPM3 cDNA was about 50% and higher than that of other muscles. In longissimus thoracis and semitendinosus, the contents of TPM1 cDNA were 29.6% and 31.7%, respectively, which were comparatively higher than that of other muscles. The result suggests that the TPM dimer consists of the TPM2 subunit regularly and TPM1 or TPM3 depending on whether the muscle is fast or slow type, respectively.

Protease activity higher in postmortem water buffalo meat than Brahman beef.

We previously demonstrated that postmortem water buffalo meat had higher tenderness than Brahman beef. In order to explain this difference in tenderness, the objective of the current study was to investigate the protease activity in these two meats. Five female crossbred water buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native) were slaughtered at 30months of age, followed by immediate sampling of Longissimus thoracis muscle for measurement of protease activity. Results showed that buffalo meat had significantly higher protease activity compared to beef (P<0.05). Furthermore, calpain inhibitor 1, a specific inhibitor of calpains 1 and 2, was the most effective inhibitor of protease activity. There was no difference in calpastatin activity, and no major differences were observed in calpains 1, 2, and calpastatin expression by Western blotting. This study suggests that higher calpain activity in early postmortem buffalo meat was responsible for the increased tenderness of water buffalo meat compared to beef.

Difference in tenderness and pH decline between water buffalo meat and beef during postmortem aging.

The objective of this research was to determine the difference in tenderness and some characteristics of water buffalo meat and beef during postmortem aging. Five female crossbred water-buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native), were finished on the same diet for 6 months and slaughtered at 30 months of age. The muscle pH was measured at 40min, 3h, 7h, 24h, and 48h postmortem. Longissimus thoracis (LT) and semimembranosus (SM) muscles were excised at 2d postmortem, and shear force was measured at 2, 4, 7, and 14d postmortem. Glycogen and lactate concentrations were determined from 0, 2, and 4d LT samples, and myosin heavy chain type of buffalo and cattle LT was determined by ELISA methods. Myofibrillar protein degradation was also observed by SDS-PAGE and Western blotting of fast-type troponin T. Results showed that the buffalo meat had significantly lower shear force values compared to beef for LT and SM muscles, which was supported by a difference in troponin T degradation. Postmortem pH decline of buffalo meat was significantly slower than that of beef, which was confirmed by lactic acid concentrations, but was not explained by glycogen content. In addition, there was no significant difference in the ratio of slow to fast type muscle fibers in buffalo and cattle, indicating that myosin heavy chain type was not responsible for the difference in pH decline and tenderness between the buffalo meat and beef. This study demonstrated that the tenderness of water buffalo meat was superior to that of Brahman beef, which may have been due to the difference in pH decline and the subsequent effect on muscle protease activity.

Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined.

Molecular cloning, nucleotide sequencing, and affinity labeling of bovine liver UDP-glucose pyrophosphorylase.

A bovine liver cDNA encoding UDP-glucose pyrophosphorylase [EC 2.7.7.9], which catalyzes the reversible uridylyl transfer between glucose 1-phosphate and MgUTP, has been cloned by the use of oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,689 base-pair insert including the complete message for the subunit polypeptide (508 amino acid residues) of the octameric enzyme. The bovine liver enzyme shows significant sequence similarities with the enzymes from potato tuber and a slime mold, Dictyostelium discoideum, but not with the enzyme from Escherichia coli, or ADP-glucose pyrophosphorylases from rice seed and E. coli. To probe the substrate-binding site in the bovine liver enzyme, the purified enzyme was incubated with an affinity labeling reagent, uridine triphosphopyridoxal, and then reduced with sodium borohydride. The enzyme was inactivated rapidly and irreversibly by the reagent at low concentrations. The inactivation was almost completely retarded by UDP-glucose and MgUTP. Structural analysis of the labeled enzyme revealed that three lysyl residues, Lys291, Lys357, and Lys396, were modified by the reagent. The three lysyl residues are conserved at the corresponding positions in the sequence of the potato tuber enzyme, in which they have catalytically important functions. These results show that the active-site structure of bovine liver UDP-glucose pyrophosphorylase is very similar to that of the potato tuber enzyme.

Ca2+ oscillations in response to methamphetamine in dopamine neurons of the ventral tegmental area in rats subchronically treated with this drug.

Mesolimbic dopamine neurons in the ventral tegmental area (VTA), which project to the nucleus accumbens and prefrontal cortex, play an important role in the regulation of emotion, rewarding, and cognition. The dopamine neurons in the VTA have also been implicated in schizophrenia and drug abuse. Methamphetamine (METH) can induce a schizophrenia-like psychosis. Thus, the VTA is a likely effector site for the action of METH. However, effects of METH on the mesolimbic dopamine neurons are largely unknown. We treated adult SD rats with METH (5 mg/kg/day) or saline for 7 days, isolated single VTA neurons from these treated rats, and monitored the neuronal activities by measuring cytosolic Ca2+ concentration ([Ca2+]i), which was followed by immunocytochemical identification of dopamine neurons. Acute administration of METH under superfusion conditions concentration-dependently increased [Ca2+]i in VTA dopamine neurons isolated from METH- and saline-treated rats. Furthermore, acutely administered METH induced oscillations of [Ca2+]i only in the dopamine neurons of the METH-treated group. The METH-induced [Ca2+]i oscillations were inhibited by Ca2+-free conditions and by Ca2+ channel blockers. In conclusion, subchronic METH treatment sensitizes VTA dopamine neurons to this drug, resulting in induction of [Ca2+]i oscillations. This sensitization of VTA dopamine neurons may account, at least in part, for the psycho-stimulant effects of METH, such as the dependence on and sensitization to METH.

Functional significance of colocalization of PACAP and catecholamine in nerve terminals.

Medullary neurons containing pituitary adenylate cyclase-activating polypeptide (PACAP) and noradrenalin (NA) project to the hypothalamus and they are involved in the regulation of arginine vasopressin (AVP) neurons. At the ultrastructural level, PACAP immunoreactivity was detected in the granular vesicles in catecholaminergic nerve terminals that made synaptic contact with AVP neurons. Both PACAP (at least 1 nM) and NA (at least 1 microM) induced large increases in the cytosolic Ca2+ concentration ([Ca2+]i) in isolated AVP cells. PACAP at 0.1 nM and NA at 0.1 microM had little effects, if any, on [Ca2+]i. However, when 0.1 nM PACAP and 0.1 microM NA were combined, they evoked large increase in [Ca2+]i in AVP neurons. An inhibitor of protein kinase A (PKA) completely inhibited the PACAP-induced increase in [Ca2+]i, but only partly inhibited the NA-induced increase in [Ca2+]i. In AVP cells that were prelabeled with quinacrine, PACAP and NA acted synergistically to induce a loss of quinacrine fluorescence, indicating secretion of neurosecretory granules in AVP neurons. The results suggest that PACAP and NA, coreleased from the same nerve terminals, act in synergy to evoke calcium signaling and secretion in AVP neurons, and that the synergism is mediated by the interaction between cAMP-PKA pathway an as yet unidentified factor "X" linked to L-type Ca2+ channels. The synergism between PACAP and NA may contribute to the regulation of AVP secretion under physiological conditions.

Methamphetamine induces cytosolic Ca2+ oscillations in the VTA dopamine neurons.

Methamphetamine (METH) induces a schizophrenia-like psychosis. The dopamine neurons in the ventral tegmental area (VTA) have been implicated in schizophrenia and drug abuse. The present study investigated direct effects of METH on VTA dopamine neurons. We treated adult SD rats with METH (5 mg/kg/day) or saline for 7 days, isolated single VTA neurons, and monitored neuronal activities by measuring cytosolic Ca2+ concentration ([Ca2+]i) in immunocytochemically identified dopamine neurons. Acutely administered METH increased [Ca2+]i in dopamine neurons from METH- and saline-treated rats and induced oscillations of [Ca2+]i in dopamine neurons only from METH-treated rats. The METH-induced [Ca2+]i oscillations were inhibited by Ca(2+)-free conditions and Ca2+ channel blockers. The results indicate that acute METH increases [Ca2+]i in VTA dopamine neurons and that subchronic METH treatment sensitizes them to this drug, resulting in induction of [Ca2+]i oscillations. The activation of VTA dopamine neurons may be related to psycho-stimulant effects of METH.

Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area.

The orexin-orexin receptor system has been implicated in the regulation of wakefulness/sleep states. Behavioral and psycho-stimulant effects of orexins have also been shown. Mesolimbic dopamine neurons in the ventral tegmental area (VTA) are implicated in the regulation of reward and wakefulness/sleep, In the present study, we examined the effect of orexin-A on cytosolic [Ca2+]i concentration ([Ca2+]) in the isolated rat VTA dopamine neurons. Orexin-A (10-12-10-8 M) concentration dependently increased [Ca2+]i in dopamine-containing neurons. The [Ca2+]i responses to orexin-A were inhibited under Ca2+-free conditions and by blockers of voltage-gated L- and N-type [Ca2+]i channels, nitrendipine and omega-conotoxin, respectively. The [Ca2+]i responses were also abolished by a phosphatidylcholine-specific phospholipase C inhibitor, D609, and a protein kinase C (PKC) inhibitor, calphostin C. A PKC activator, TPA, mimicked orexin-A in increasing [Ca2+]i. These results indicate that orexin-A increases [Ca2+]i in VTA dopamine neurons via phosphatidylcholine-specific PLC- and PKC-mediated activation of L- and N-type Ca2+ channels. This effect may serve as the mechanism by which orexin regulates wakefulness/sleep states and exerts its behavioral and psychostimulant effects.

Nicotine increases cytosolic Ca2+ in vasopressin neurons.

Strong immunoreactivity for neuronal nicotinic acetylcholine receptor alpha4 subunit was detected in neurons of the supraoptic nucleus (SON). At the ultrastructural level, immunoreactivity for alpha4 was detected in the post-synaptic membranes as well as in the cytoplasmic matrices in the magnocellular neurons. Nicotine (1-10 microM) increased cytosolic Ca2+ concentrations ([Ca2+]i) in isolated arginine-vasopressin (AVP)-containing neurons in the rat SON. Nicotine (10 microM) was less potent in increasing [Ca2+]i in AVP-containing neurons than noradrenaline (1 microM), a known neurotransmitter in the SON. The nicotine-induced [Ca2+]i increase in AVP-containing neurons was markedly reduced when pre-treated with a protein kinase A (PKA) blocker, H89 (40 microM). These findings suggest that nicotine, a known neurotransmitter in the SON, activates AVP-containing neurons via nicotinic acetylcholine receptor which is linked to stimulation of cAMP-PKA-regulated Ca2+ signaling pathway.

Glucose-sensitive neurons in the rat arcuate nucleus contain neuropeptide Y.

Glucose is known to regulate the activity of the hypothalamic feeding centers. Neuropeptide Y (NPY)-containing neurons in the hypothalamic arcuate nucleus (ARC) have been implicated in the stimulation of feeding. We examined the presence of glucose-sensitive neurons in the ARC and their coincidence with NPY-containing neurons. Cytosolic Ca2+ concentration ([Ca2+]i) in single ARC neurons isolated from rat hypothalamus was measured with fura-2 fluorescence imaging; the cells were then stained immunocytochemically with an anti-NPY antiserum. Lowering the glucose concentration from 10 to 1 mM increased [Ca2+]i in 36 out of 180 neurons (20%), the majority of which (34 neurons, 94%) were immunoreactive for NPY. In conclusion, the ARC contains glucose-sensitive NPY-containing neurons. The suggested role of these neurons is to transduce a reduction in the glucose concentration in the brain to the release of NPY and, subsequently, stimulation of feeding.

Noradrenaline activates vasopressin neurons via alpha1-receptor-mediated Ca2+ signaling pathway.

Noradrenaline (NA) (1-10 microM), dibutyryl-cAMP (1-5 mM), and forskolin (10-20 microM) increased cytosolic Ca2+ concentration ([Ca2+]i) in isolated arginine-vasopressin (AVP)-containing neurons in the hypothalamic supraoptic nucleus (SON). The NA-induced increase in [Ca2+]i in AVP-containing neurons was abolished by a specific alpha1-antagonist, prazosin (1 microM) and was markedly reduced when treated with a protein kinase A (PKA) blocker, H89 (40 microM). The NA-induced [Ca2+]i was not altered by a protein kinase C (PKC) inhibitor, calphostin C (0.1 microM) and a PKC activator, TPA (100 nM). In general, NA, a known neurotransmitter in the SON, activates AVP-containing neurons via alpha1-receptor which is linked to stimulation of cAMP-PKA-regulated Ca2+ signaling pathway.

Lowering glucose concentrations increases cytosolic Ca2+ in orexin neurons of the rat lateral hypothalamus.

Orexin neurons are specifically localized in and around the lateral hypothalamus (LH), a feeding center. Intracerebroventricular administration of orexin-A and -B stimulates feeding as well as arousal. However, little is known regarding the regulators of the orexin neuron activity. The neurons that are activated under low glucose conditions, glucose-sensitive neurons, are located in the LH and have been implicated in the control of feeding. The present study investigated the effect of glucose on the single orexin neurons isolated from the rat LH, by measuring cytosolic Ca(2+) concentration ([Ca(2+)](i)) by fura-2 microfluorometry followed by immunocytochemical staining with anti-orexin antiserum. A shift of glucose concentration form 8.3 to 2.8 mM in the superfusion solution increased [Ca(2+)](i) in 13 out of 32 orexin-immunoreactive LH neurons. The results demonstrate that glucose-sensitive orexin neurons are present in the LH and that these neurons may play a role in linking the metabolic state in the body to the orexigenic, and could also, awakening signaling in the brain.

The effect of leptin on feeding-regulating neurons in the rat hypothalamus.

Intense immunoreactivity for the leptin receptor was detected in the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMH), and lateral hypothalamus (LH) by immunohistochemistry. Cytosolic Ca2+ concentration ([Ca2+]i) in single neurons isolated from the ARC, VMH and LH was measured with dual wavelength fura-2 fluorescence imaging. A reduction of the superfusate glucose concentration from 10 to 1 mM increased [Ca2+]i in 21% of ARC neurons and 22% of LH neurons. Leptin at 0.1 nM inhibited the [Ca2+]i increase in 66 and 64% of these glucose-sensitive ARC and LH neurons, respectively. Inversely, 10 mM glucose increased [Ca2+]i in 49% of the VMH neurons, and 0.1 nM leptin at 1 mM glucose also increased [Ca2+]i in 84% of these glucose-responsive neurons. These results reveal that leptin inhibits the ARC and LH neurons and stimulates the VMH neurons via the leptin receptor expressed in these cells.

Amino acid sequences of multiple fast and slow troponin T isoforms expressed in adult bovine skeletal muscles.

Multiple nucleotide sequences of complementary DNA (cDNA) of bovine troponin T (TnT) isoforms expressed in the adult skeletal muscles were determined to facilitate the elucidation of the TnT degradation progress during postmortem aging of muscles. Fresh muscle samples were excised from the lingual, masseter, pectoralis, diaphragm, psoas major, longissimus thoracis, spinnalis, semitendinosus, semimembranosus, and biceps femoris muscles of three Holstein cows within 1 h of slaughter. Complementary DNA fragments of fast and slow TnT isoforms expressed in each muscle were amplified by reverse-transcribed PCR. Consequently, four major fragments of fast TnT and two fragments of slow TnT, all of which contained the complete coding region, were obtained. The sequence determination of these fragments revealed that at least eight and two isoforms were generated by the alternative splicing from bovine fast and slow TnT messenger RNA, respectively. In the fast TnT isoforms, five small variable exons were observed; three of these five exons were in the amino (N)-terminal region. The calculated molecular weight of fast and slow TnT isoforms ranged from 29,816 to 32,125 and from 30,166 to 31,284, respectively. The deduced amino acid sequences revealed that the N-terminal region of all the TnT isoforms was extremely glutamic acid-rich. Reverse-transcribed PCR analysis revealed that expression of each of these isoforms was distributed in a fast or slow muscle-specific manner. Given that TnT degradation has been reported to accompany a decrease in glutamic acid content in the conventional 30-kDa degradation product, the sequence data suggested that the 30-kDa fragment seem to be generated by the proteolytic removal of the glutamic acid-rich N-terminal ends. The multiplicity of TnT isoforms may result in a complicated pattern of TnT degradation on SDS-PAGE gel during beef aging.

Molecular characteristics and site specific distribution of the pigment of the silky fowl.

Silky fowl, a breed of chicken, is hyperpigmented in its various internal tissues. The pigment was extracted from various tissues of two strains of Silky fowl to determine its molecular structure and internal distribution. Analysis by infrared spectroscopy showed two spectrum patterns of the pigment in Silky fowl; one is from ovary and testis, the other is from periosteum and feather. The difference between the two spectra is possibly due to the sulfur contents of melanin. Especially, the spectra of the pigments from feather and periosteum shared the characteristics of synthesized melanin spectrum in common, which indicates that the melanocytes dispersed in these tissues were functionally the same. According to our quantitative analysis, the tissues examined were classified significantly in the order of the pigment content (p<0.05): periosteum > gonads (ovary or testis) = trachea > or = heart, liver, gizzard, cecum, muscles (Pectoralis and Supracoracoideus) and skin. In addition, the specific regions of embryonic neural crest derived cells, such as cardiac artery and various parts of cephalic tissues, were found to be locally hyperpigmented. These data suggest that hyperpigmentation (fibromelanosis) in Silky fowl chicken occurs in a tissue- and organ-specific manner, which is strongly related to neural crest cell development. It is hypothesized that neural crest cells of the bird, containing melanocyte progenitors, acquire unusual ability to differentiate into melanocytes excessively, and to extend the distribution of their descendant along the destinations of neural crest derivatives.

Myosin heavy chain isoforms expressed in bovine skeletal muscles.

Nucleotide sequences including the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from bovine adult skeletal muscles. The deduced amino acid sequences were 1940, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. Like other mammalian MyHC isoforms, the bovine MyHC isoforms had homologous sequences except for substitutions concentrated on the loop 1, loop 2, and light chain binding regions. RT-PCR amplifications showed that the adult bovine skeletal muscles expressed the MyHC-2a, -2x, and -slow isoforms but no -2b isoform. The absence of the MyHC-2b isoform and substitutions on the loop2 region could explain some differences in meat quality between beef and pork.

Differences in molecular structure among the porcine myosin heavy chain-2a, -2x, and -2b isoforms.

Full coding regions for fast type myosin heavy chain (MyHC) isoforms were sequenced from a porcine skeletal muscle to analyze sequence diversity relating to the contractile properties of muscle fibers. An approximately 6-kb fragment for each MyHC was amplified through RT-PCR using isoform type-specific primers, which were designed in the 5' and 3' non-coding regions of the porcine MyHCs. The lengths of deduced amino acid sequences were 1939, 1939, and 1937 for the porcine MyHC-2a,-2x, and-2b, respectively. The entire amino acid sequences were highly conserved among the three MyHCs, except for the 50/20 k junction region (loop 2) which would weakly bind actin molecules. The porcine MyHC-2b possessed different amino acids from MyHC-2a and-2x, in loop1 and ELC binding region. The sequence data suggested the diversity of contractile properties among the porcine MyHC isoforms.