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OBJECTIVE: Our objective was to investigate the effect of lipid-induced insulin resistance and type 2 diabetes on skeletal muscle calpain-10 mRNA and protein levels. RESEARCH DESIGN AND METHODS: In the first part of this study, 10 healthy subjects underwent hyperinsulinemic euglycemic (4.5 mmol/liter) clamps for 6 h with iv infusion of either saline or a 20% Intralipid emulsion (Fresenius Kabi AG, Bad Homburg, Germany). Skeletal muscle biopsies were taken before and after 3- and 6-h insulin infusion and analyzed for calpain-10 mRNA and protein expression. In the second part of the study, muscle samples obtained after an overnight fast in 10 long-standing, sedentary type 2 diabetes patients, 10 sedentary, weight-matched, normoglycemic controls, and 10 age-matched, endurance-trained cyclists were analyzed for calpain-10 mRNA and protein content. RESULTS: Intralipid infusion in healthy subjects reduced whole body glucose disposal by approximately 50% (P<0.001). Calpain-10 mRNA (P=0.01) but not protein content was reduced after 6-h insulin infusion in both the saline and Intralipid emulsion trials. Skeletal muscle calpain-10 mRNA and protein content did not differ between the type 2 diabetes patients and normoglycemic controls, but there was a strong trend for total calpain-10 protein to be greater in the endurance-trained athletes (P=0.06). CONCLUSIONS: These data indicate that skeletal muscle calpain-10 expression is not modified by insulin resistance per se and suggest that hyperinsulinemia and exercise training may modulate human skeletal muscle calpain-10 expression.

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AIMS: Calpain-10 and calpain-3 and the diabetes ankyrin repeat protein (DARP) have all been linked to insulin resistance and type 2 diabetes. We set out to measure the expression of these genes in human skeletal muscle and relate them to functional measurements of insulin action during fasting (which induces insulin resistance) and refeeding (which reverses it). METHODS: Ten healthy male volunteers underwent 48 h of starvation followed by 24 h of high carbohydrate refeeding. On three occasions, before and after starvation and after refeeding, subjects underwent a 16 min insulin tolerance test to quantify insulin sensitivity. Muscle biopsies were obtained before and after fasting and after refeeding for the analysis of calpain-10 and calpain-3, GLUT4 and DARP expression by Western blotting and real-time PCR. RESULTS: Fasting led to a marked reduction in whole body insulin sensitivity by approx. 45% (P<0.01) and skeletal muscle GLUT4 gene expression by approx. 40% (P<0.05). However, fasting had no effect on calpain-10 and calpain-3 mRNA or protein levels, or DARP mRNA expression. Refeeding only partly restored insulin sensitivity and GLUT4 gene expression to their pre-fast values, but did not effect the expression of calpain-10, calpain-3 or DARP. CONCLUSIONS: These findings demonstrate that in healthy non-diabetic humans induction of insulin resistance by fasting and its reversal by refeeding with a high CHO diet is mirrored by changes in skeletal muscle GLUT4 but not calpain-10 and calpain-3 expression.

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The calpain proteinases and their specific inhibitor calpastatin have been proposed to influence both the rates of myofibrillar protein turnover in vivo and meat tenderization postmortem. Elevated calpastatin concentrations in particular are associated with certain forms of hypertrophic growth and meat toughness. In the 5'region of the porcine calpastatin gene, there are 3 calpastatin promoters upstream of exons 1xa, 1xb, and 1u, respectively, each of which contain transcription factor-binding motifs, suggesting sensitivity to a variety of growth-promoting stimuli. This study examined the effect of the beta-adrenergic agonist clenbuterol and porcine ST (pST) treatment on calpastatin promoter usage in porcine LM in vivo using real-time PCR and also the responsiveness of transfected calpastatin promoter sequences to cyclic adenosine monophosphate (cAMP) and calcium (Ca2+)-related stimuli in reporter gene systems in cell studies. The effect of clenbuterol and pST on potential signaling pathways in vivo was also assessed by monitoring protein phosphatase 2B (calcineurin), NFATc3, calpain 3, IkappaB alpha, and NFkappaB by quantitative immunoblotting. Total calpastatin mRNA was increased by 52% (P < 0.05) after treatment with clenbuterol for 1 d and reduced by 35% (P < 0.01) after pST treatment for 7 d. Whereas clenbuterol had no significant differential effects on individual mRNA transcripts (types 1 to 3) derived from the 3 upstream promoters, pST significantly reduced all of these by 51, 39, and 40% (P < 0.001, 0.05, and 0.05), respectively. Promoter activity was increased in rat L6G8 cells transfected with a construct derived from exon 1u after treatment with dibutyryl cAMP (68%, P < 0.05) or forskolin (43%, P < 0.05), whereas 1xa activity was reduced by both of these agents (47 and 33%, respectively, P < 0.05). Treatment of cells with the calcium ionophore calcimycin reduced the activity of the 1u promoter by 40% (P < 0.01), with no effect on the other promoter constructs. Cyclosporin A had no effect on any promoter construct. The only signaling pathway component to be significantly altered by the in vivo treatments was calcineurin, which was decreased by 24% (P < 0.05) in clenbuterol-treated animals. In conclusion, 2 types of growth promoter in pigs had contrasting effects on calpastatin expression in LM. Transfected calpastatin promoters were differentially sensitive to cAMP- and Ca2+-related stimuli, in agreement with the proposed mode of action of the 2 growth promoters.

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The objective of this study was to investigate the protease family caspases in skeletal muscle and their potential contribution to postmortem proteolysis and meat tenderization. Ten Large White gilts were slaughtered, and samples of LM were taken at 0, 2, 4, 8, 16, 32, and 192 h after slaughter and immediately snap frozen in liquid nitrogen. Samples were subsequently analyzed for caspase 3/7 and caspase 9 activity, protein levels of known caspase substrates, alpha II spectrin and poly (ADP-ribose) polymerase (PARP), as well as, at 192 h, shear force. Specific degradation products of alpha II spectrin and PARP, which are known indicators of caspase activity, and apoptosis were detected on immunoblots of muscle samples taken over the postmortem period. The relationships between the changes observed in caspase activities and protein levels of PARP and spectrin across the entire postmortem conditioning period were investigated (n = 70). Protein levels of alpha II spectrin cleavage products across the conditioning period were found to correlate positively to caspase 3/7 activity (r = 0.38, P = 0.003) and caspase 9 activity (r = 0.32, P = 0.012), indicating that caspase-mediated cleavage was occurring in situ. There was a negative relationship between shear force and the 0 to 32 h ratio of caspase 3/7 (r = -0.62, P = 0.053) and caspase 9 activities (r = -0.68, P = 0.044). In addition, there was also a negative relationship between shear force and the level of the caspase-generated alpha II spectrin 120 kDa degradation product (r = -0.75, P = 0.012). The findings of this study indicate that changes in caspase activity and caspase-mediated cleavage take place in muscle during the conditioning period, and this could be associated with the development of tender meat.

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The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.

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The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule×Charolais rams. Samples taken at slaughter were frozen either in liquid N(2) for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically (r(2)=0.612 and 0.366, respectively, p<0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity (r(2)=0.725, p<0.001) and 135 kDa calpastatin (r(2)=0.228, p<0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.

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The present study was conducted to determine the effects of growth pattern on the calpain system and meat tenderization. Twenty-four Friesian calves were randomly allocated to three treatment groups: FAST (fast growth rate), SLOW (severely restricted growth rate) and ALTER (restricted growth for 30 days followed by fast growth rate). Four animals from each group were slaughtered on day 32 or 45 after altering the growth rates. Samples of M. longissimus dorsi were rapidly frozen at slaughter for protein analysis by Western blotting. Restricted growth reduced the immunoreactivity of a calpastatin band (135 kDa) measured at 24 h postmortem. Immunoreactivity associated with the large subunit of µ- or m-calpain appeared to be unaffected by growth patterns. Shear force measurements taken after 14 days of conditioning were positively related to 135 kDa calpastatin at 24 h postmortem. In this study there was no clear relationship between shear force and growth pattern.

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The expression in porcine skeletal and cardiac muscle of calpastatin, the specific endogenous inhibitor of the calpain proteolytic system, was examined 16 h after a single dose of a specific beta(2)-agonist. Immunoblotting of extracts indicated that treatment increased skeletal calpastatin 135-kDa band intensity (P < 0.01), while in cardiac combined 145- and 135-kDa band intensity decreased (P < 0.05). Treatment increased skeletal (P < 0.01) but not cardiac calpastatin mRNA steady-state levels. Three types of cardiac calpastatin mRNA transcripts were identified by 5'-RACE. Types I and II encoded a putative XL region that originated either from exon 1x(A) or exon 1x(B), arranged in tandem. Type III predominated in skeletal muscle and originated from exon 1u, which was located 40-50 kb 3' to exons 1x(A) and 1x(B). The region 5' to exon 1u may act as an independent promoter regulated by a cAMP-dependent mechanisms, thereby explaining the differential response of calpastatin to adrenergic stimulation in cardiac and skeletal muscle.

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A novel method for the discrimination of bluefin tuna (Thunnus thynnus) from Atlantic bonito (Sarda sarda) was developed, based on species-specific amplification of a region of the mitochondrial cytochrome b gene by Polymerase Chain Reaction (PCR). The method, which uses a one-step amplification reaction, is more rapid to perform than any of the currently described techniques for species determination in fish. The species of origin of the DNA is indicated by the distinctive size of the PCR product on electrophoresis, but the test could readily be adapted to other forms of electrophoresis or fluorescence-based systems for quantification. Given the possibility of intraspecific variability in mitochondrial DNA and the consequent desirability of performing two independent tests, the new method constitutes a valuable addition to the range of tuna speciation methodologies currently available.

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In many breeds of sheep, a polymorphism at codon 136 of the prion protein gene has been shown to be strongly associated with the risk of developing scrapie. A single-step procedure for detecting this allelic variation is described here. When performed on a series of animals, the test was in complete agreement with their genotypes as had been previously determined by sequencing. The test is potentially easier and quicker to perform than any of the variety of methods that are currently used for this purpose.

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beta-Adrenergic agonists induce muscle hypertrophy in mammalian species and alter the extractable activity of calpain proteinase and its specific endogenous inhibitor calpastatin. The effects on skeletal and cardiac muscle calpastatin of continuously infusing a group of pigs for 7 days with the physiological agonist epinephrine (0.15 microg/kg/min) were examined and compared with a placebo group. Basal levels of extractable calpastatin activity were higher in cardiac than skeletal muscle and epinephrine infusion increased the extractable activity in both muscle types (P < 0.05). An anti-recombinant porcine calpastatin antiserum detected a 135-kDa band and a 145/135-kDa doublet on Western blots of skeletal and cardiac extracts, respectively. Epinephrine infusion increased the 135-kDa band in skeletal muscle (P < 0.05), while the ratio of 145/135 kDa in cardiac muscle was decreased (P < 0.05). From Northern blots, the patterns of calpastatin mRNA species were similar in the two muscle types, two major transcripts at 5.8 and 3. 2 kb in cardiac muscle, with equivalent bands in skeletal muscle of 5.4 and 2.8 kb. A faint 7.9-kb band was also detected in skeletal muscle. Epinephrine infusion had no effect on skeletal calpastatin mRNA but tended to increase the 5.8-kb mRNA in cardiac muscle (P = 0. 053). These data indicate a differential response of the two muscle types to mildly elevated plasma epinephrine concentration and the response to elevated epinephrine may be at the translational or posttranslational level. Therefore, this type of stimulus appears to be less effective at perturbing calpastatin gene transcription than certain orally administered synthetic beta-adrenergic agonists.

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The calpain proteolytic system was examined in the longissimus muscle (LD) of heterozygote pigs carrying a single copy of a mutation in the skeletal muscle ryanodine receptor gene (RyR1) that is associated with porcine stress syndrome and reduced meat quality. Conventional British White-type pigs (n = 30) were selected from a commercial line on the basis of slaughter weight, backfat depth, and pH at 45 min postmortem > 6.0; based on DNA analysis, 11 were heterozygous RyR1 mutants (Nn), and 19 were normal genotype (NN). The LD samples were taken from carcasses at 2, 4, and 24 h postmortem for calpain analysis with enzyme assay and immunoblotting, using specific antisera raised against recombinant polypeptides derived from calpain large subunits and calpastatin. Shear force (SF) was measured after conditioning for 8 d at 2 degrees C and did not differ between Nn and NN groups. The extractable activity of mu-calpain decreased over 24 h postmortem (P < .001), with no significant difference in activity between NN and Nn animals at any time. The activity of m-calpain also decreased with time (P < .001), but it was lower at all times in Nn than in normal genotypes (P < .001). After Western blotting, the immunoreactivity of mu- and m-calpain large subunit bands declined over 24 h postmortem (P < .001); values for mu-calpain were higher (P < .05) and for m-calpain were lower (P < .001) in heterozygotes than in normal animals at each sampling time. The calpastatin antibody detected a major band of 135 kDa that declined with time postmortem but did not differ between Nn and NN genotypes at any sampling time. These data indicate that the levels of extractable mu- and m-calpain, but not calpastatin, may be different in pigs that carry the RyR1 mutation.

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Members of the calpain proteinase family are present in all mammalian cells, although a novel calpain 94 kDa isoform is found almost exclusively in skeletal muscle. p94 is difficult to purify from muscle and recombinant p94 autolyses rapidly when expressed in COS cells. However, in vivo the enzyme may be stabilised by interaction with titin, which has two well-characterised binding sites for p94 at the N2- and M-lines. Both these titin subdomains are subject to muscle-specific alternative splicing, which could be related to p94 expression level or stability in muscles of different fibre type. In this study, porcine longissimus dorsi (LD), trapezius (TZ) and adductor longus (AL) were characterised as fast, intermediate and slow using commercially available specific anti-human fast- and slow-myosin heavy chain mAbs and also by conventional histochemistry. p94 was quantified both in whole muscle preparations and single fibres by western blotting using an anti-p94 antiserum generated by expressing a recombinant p94 sequence as a GST fusion protein antigen. SDS PAGE and immunoblotting revealed a single band of approximately 94 kDa with identical mobility in all muscle and fibre preparations. The intensity of the 94 kDa band was greater in LD (22 +/- 1.7 densitometric units mean +/- SEM, n = 3) than TZ and AL (10 +/- 2.3 and 6 +/- 0.9 units, respectively). Expressed as a ratio relative to actin immunoreactivity, p94 is present in all types of single fibres isolated from TZ, but at a significantly lower level (P < 0.01) in slow type I (0.08 +/- 0.01, n = 9), compared to fast IIA/IIB fibres (0.22 +/- 0.02, n = 26). No evidence was seen for rapid or variable rate of p94 degradation in either type of fibre. These data suggest a positive correlation between p94 expression level and fast glycolytic characteristics in porcine muscle.

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Tenderization of skeletal muscle in meat animals has been closely linked to the postmortem activity of the calpain proteolytic enzyme system, which includes the specific inhibitor calpastatin. Increased understanding of the skeletal muscle-specific calpain isoform p94 has prompted suggestions as to whether it too could have a role in the tenderization process. In this study, two groups of pigs were identified in which shear force measurements after 8 d of conditioning indicated a large variation in the tenderness of longissimus muscle. The quantity of p94 in the muscle was monitored by immunoblotting, using a porcine-specific polyclonal antibody raised against a recombinant peptide fragment generated as a fusion protein. The antiserum recognized a 94-kDa protein associated with myofibrils in skeletal but not cardiac muscle, as expected for this calpain isoform, although it could not be tested with the native protein because of the extreme instability of p94. In the first experiment, the mean shear force for the tough group was 6.71 +/- .28 kg (n = 12, SEM) and that of the tender group was 3.87 +/- .12 kg (n = 12), but there was no difference in the normalized absorbance of the immunopositive 94 kDa band on Western blots from samples collected at approximately 40 min postmortem. In the second experiment, the stability of p94 in chilled carcasses was investigated over 24 h, using a further two groups of 10 tough and 10 tender pigs of mean shear force values 5.36 +/- .14 kg and 2.81 +/- .15 kg, respectively. In tough and tender animals, there was a decline (P < .05) in the 94-kDa immunostaining material of mean half-lives of 13.8 and 12.9 h, respectively, although there was considerable variability. Despite this variability in half lives and shear force values, no correlation was seen between these factors. Thus, in porcine longissimus muscle, the variability in tenderness after 8 d of conditioning cannot be attributed to an underlying variability in p94.

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Using the polymerase chain reaction (PCR) and DNA extracted from muscle, a single pair of oligonucleotide primers can yield amplification products from several members of the actin multigene family simultaneously. These multiple PCR products form species-specific "fingerprints" on gel electrophoresis which may be useful for meat authentication. However, for analysis of meat mixtures, the presence of a single band unique to a species would have many advantages over a multi-component fingerprint. A procedure is described in which primers amplify at a single actin gene locus, giving a positive band with DNA extracted from chicken and turkey, but no reaction with duck, pheasant, porcine, bovine, ovine or equine DNA. The chicken signal was clearly detectable with DNA from meat admixtures containing 1% chicken/99% lamb and from meat heat-treated at 120°C. For further discrimination, the chicken PCR product could be differentiated from turkey by restriction enzyme digestion.

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Actins constitute a family of highly-conserved multifunctional intracellular proteins, best known as myofibrillar components in striated muscle fibres. Most vertebrate genomes contain numerous actin genes with high sequence homology in protein coding regions but considerable variability in intron number and sizes. This genetic diversity can be utilised for livestock speciation purposes. The high sequence conservation has enabled a single pair of oligonucleotides to be used to prime the polymerase chain reaction (PCR) with DNA extracted from all animals so far studied. Multiple amplification products were obtained which on gel electrophoresis constituted characteristic species-specific 'fingerprints'. The patterns were reproducible, did not vary between individuals of the same breed or between different breeds within a species, and could be generated even from heat-processed muscle held at 120 degrees C for one hour. Given the capacity of PCR to amplify relatively short sequences in highly-degraded DNA, this approach may be suitable for authentication of processed meat products.

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Classical DNA fingerprinting is based on separation of DNA restriction fragments by electrophoresis and hybridisation to nucleic acid probes containing repetitive nucleotide sequences. The use of such mini- or micro-satellite probes tends to yield patterns specific to an individual rather than to a species, hence their value in forensic analysis but general unsuitability for meat speciation. In the present study, a cDNA probe based on conserved sequences contained in members of the actin multigene family has been evaluated for potential application in meat speciation. Genomic DNA was extracted from muscle and digested with BamHI before electrophoresis and hybridisation to a murine α-actin cDNA probe. Beef, pork, lamb, horse, chicken and fish DNA restriction fragments formed characteristic 'fingerprints' which were reproducible and varied sufficiently to allow discrimination even between closely-related species. However no major differences were seen between individuals of the same breed or between different breeds within a species. When DNA obtained from fresh tissue and also from meat heated at 120 °C was analysed, the gel patterns were essentially the same. An attractive feature of this approach is that it employs a single cross-reacting probe and set of conditions, and gives different patterns with all species so far studied. This simplicity suggests applications in meat speciation or related areas of biology.

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By extending functional primers attached to a solid phase and incorporating a digoxigenin label, it is possible to visualise PCR products as discrete spots on specific regions of a solid support after colorimetric detection. The technique has been used for the detection of the point mutation associated with porcine malignant hyperthermia.

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