Immunohistochemical identification and fiber type specific localization of protein kinase C isoforms in equine skeletal muscle

OBJECTIVE: To investigate whether protein kinase C (PKC) isoforms are expressed in equine skeletal muscle and determine their distribution in various types of fibers by use of immunofluorescence microscopy. ANIMALS: 5 healthy adult Dutch Warmblood horses. PROCEDURE: In each horse, 2 biopsy specimens were obtained from the vastus lateralis muscle. Cryosections of equine muscle were stained with PKC isoform (alpha, beta1, beta2, delta, epsilon, or zeta)-specific polyclonal antibodies and examined by use of a fluorescence microscope. Homogenized muscle samples were evaluated via western blot analysis. RESULTS: The PKC alpha, beta1, beta2, delta, epsilon, and zeta isoforms were localized within the fibers of equine skeletal muscle. In addition, PKC alpha and beta2 were detected near or in the plasma membrane of muscle cells. For some PKC isoforms, distribution was specific for fiber type. Staining of cell membranes for PKC alpha was observed predominantly in fibers that reacted positively with myosin heavy chain (MHC)-IIa; PKC delta and epsilon staining were more pronounced in MHC-I-positive fibers. In contrast, MHC-I negative fibers contained more PKC zeta than MHC-I-positive fibers. Distribution of PKC beta1 was equal among the different fiber types. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that PKC isoforms are expressed in equine skeletal muscle in a fiber type-specific manner. Therefore, the involvement of PKC isoforms in signal transduction in equine skeletal muscle might be dependent on fiber type.     

Immunohistological study on bovine, swine and ovine skeletal muscle fibers for the localization of fatty acid translocase FAT/CD36

The present immunohistological study was conducted to investigate the localization of fatty acid translocase CD36 (FAT/CD36) in the skeletal muscle (Biceps femoris) fibers of bovine, swine and ovine. The results showed that CD36 was mainly localized in type I muscle fiber of these animals. In contrast, FAT/CD36 localization in type II fiber was insignificant in the types of muscle in the present experiments, suggesting that type II fiber of bovine, swine and ovine might lack fatty acid translocase FAT/CD36.     

Comparison of sensitivity of sodium currents to tetrodotoxin in equine muscle specimens with that in murine and human muscle specimens

OBJECTIVE: To determine sensitivity of equine skeletal muscle to tetrodotoxin and compare that with sensitivity of murine and human skeletal muscles. SAMPLE POPULATION: Semimembranosus, vastus lateralis, triceps brachii, and masseter muscle specimens from 22 euthanatized horses, vastus lateralis muscle biopsy specimens from 25 clinically normal humans, and diaphragmatic muscle specimens from 6 mice. PROCEDURE: Electrically elicited twitch responses were measured in muscle specimens incubated in medium alone and with tetrodotoxin (100 nM, 400 nM, 1.6 microM for equine specimens and 100 nM, 200 nM, 400 nM, 800 nM, 1.6 microM for murine and human specimens). Percentages of tetrodotoxin-sensitive and -resistant sodium channels were determined and compared among muscles and species. RESULTS: 2 sodium channels with different sensitivities to tetrodotoxin were identified in equine muscle. One was blocked with 100 nM tetrodotoxin and the other was unaffected by tetrodotoxin at concentrations up to 1.6 microM. The only difference detected among the 4 equine muscles was that masseter muscle specimens had a higher percentage of tetrodotoxin-sensitive channels than triceps brachii muscle specimens. Tetrodotoxin-resistant sodium channels constituted 31 to 66% of the equine muscle twitch response, which was greater than that determined for normal human and murine muscle (< 5%). CONCLUSION AND CLINICAL RELEVANCE: Equine skeletal muscle contains a high percentage of tetrodotoxin-resistant sodium channels. The 4 equine muscles evaluated were more similar to each other than to murine and human muscles. Shifts in expression of sodium channel subtypes may play a role in the manifestation of certain myopathies.     

Effect of exercise on activation of the p38 mitogen-activated protein kinase pathway, c-Jun NH2 terminal kinase, and heat shock protein 27 in equine skeletal muscle

OBJECTIVE: To investigate the effects of exercise on activation of mitogen-activated protein kinase (MAPK) signaling proteins in horses. ANIMALS: 6 young trained Standardbred geldings. PROCEDURE: Horses performed a 20-minute bout of exercise on a treadmill at 80% of maximal heart rate. Muscle biopsy specimens were obtained from the vastus lateralis and pectoralis descendens muscles before and after exercise. Amount of expression and intracellular location of phosphospecific MAPK pathway intermediates were determined by use of western blotting and immunofluorescence staining. RESULTS: Exercise resulted in a significant increase in phosphorylation of p38 pathway intermediates, c-Jun NH2 terminal kinase (JNK), and heat shock protein 27 (HSP27) in the vastus lateralis muscle, whereas no significant changes were found in phosphorylation of extracellular regulated kinase. In the pectoralis descendens muscle, phosphorylation of p38 and HSP27 was significantly increased after exercise. Immunohistochemical analysis revealed fiber-type- specific locations of phosphorylated JNK in type 2a/b intermediate and 2b fibers and phosphorylated p38 in type 1 fibers. Phosphorylated HSP27 was strongly increased after exercise in type 1 and 2a fibers. CONCLUSIONS AND CLINICAL RELEVANCE: The p38 pathway and JNK are activated in the vastus lateralis muscle after a single 20-minute bout of submaximal exercise in trained horses. Phosphorylation of HSP27 as detected in the study reported here is most likely induced through the p38 signaling pathway.     

Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses

Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.     

Age-related changes in percentage of fiber types and mean fiber diameters of the ovine quadriceps muscles.

Mean fiber diameters and percentages of each fiber type of the vastus lateralis, vastus medialis, rectus femoris, and vastus intermedius muscles from 15 sheep, ranging from 1 day to 5 years of age, were determined. Myosin adenosine triphosphatase and nicotinamide adenine dinucleotide-tetrazolium reductase stained sections were used. The vastus lateralis, vastus medialis, and rectus femoris contained 3 fiber types (I, IIA, and IIB). The vastus intermedius was composed almost entirely of type I fibers. From birth to 5 years of age, mean fiber diameters of type I fibers increased from 15.8 to 47.0 micron in the vastus lateralis, 15.6 to 50.7 micron in the vastus medialis, 17.5 to 46.5 micron in the rectus femoris, and 26.7 to 51.8 micron in the vastus intermedius. Means of fiber diameters of type II fibers increased from 16.1 to 44.6 micron in the vastus lateralis, 19.8 to 44.0 micron in the vastus medialis, and 17.0 to 44.5 micron in the rectus femoris. The percentage of type II fibers in the vastus lateralis, vastus medialis, and rectus femoris decreased from 85% to 90% at birth to approximately 72% at 5 years of age. The vastus intermedius consisted of only type I fibers in sheep 2 years and older.     

Biochemical evaluation of mitochondrial respiratory chain enzymes in canine skeletal muscle

OBJECTIVE: To perform respiratory chain enzymatic activity assays on canine skeletal muscle biopsy specimens and establish reference range values of skeletal muscle enzyme activities for dogs. SAMPLE POPULATION: Biopsy specimens from the vastus lateralis muscle were obtained from 24 dogs (8 sexually intact males and 14 sexually intact females) ranging from 15 months to 6 years of age. PROCEDURE: Mean values of citrate synthase, cytochrome-c oxidase, succinate dehydrogenase, succinate dehydrogenase-cytochrome-c reductase, nicotinamide adenine dinucleotide (NADH) dehydrogenase, and NADH dehydrogenase-cytochrome-c reductase activities were established by use of 6 standard spectrophotometric assays for respiratory chain enzyme analysis. RESULTS: Compared with published data for skeletal muscle enzyme activities in humans, skeletal muscle enzyme activities in dogs were 2- to 4-fold higher. Additionally, citrate synthase activity, a marker for mitochondrial volume, was positively correlated with age in dogs, suggesting that mitochondrial volume increases with age, although no apparent change in respiratory chain enzymatic activity with an increase in age was found. CONCLUSIONS AND CLINICAL RELEVANCE: Reference range values for skeletal muscle enzyme activities of dogs are needed to accurately interpret results of respiratory chain enzymatic activity assays. During investigation of metabolic myopathies, if skeletal muscle biopsy specimens are evaluated for respiratory chain enzyme kinetics, they should be performed and evaluated in concert with skeletal muscle biopsy specimens from clinically normal animals of the same species.     

Evaluation of developmental changes in the coexpression of myosin heavy chains and metabolic properties of equine skeletal muscle fibers

OBJECTIVE: To determine the growth-related changes in metabolic and anatomic properties in equine muscle fiber type, including hybrid fibers identified with immunohistochemical analysis. ANIMALS: 24 2-, 6-, 12-, and 24-month-old female Thoroughbreds. PROCEDURE: Samples were obtained from the gluteus medius muscle of all horses. Expression of myosin heavy chain (MHC) isoforms MHC-I, -IIa, -IIb, and -IIx in each muscle fiber was detected by use of 4 primary monoclonal antibodies: BA-D5, SC-71, BF-F3, and BF-35, respectively. Five muscle fiber types (types I, I/IIA, IIA, IIA/IIX, and IIX) were immunohistochemically identified. The area and activity of succinic dehydrogenase (SDH) in each fiber type were determined by use of quantitative histochemical staining and image analysis. RESULTS: Although the proportion of type I and IIX fibers did not change with age, the proportion of type IIA and IIA/IIX fibers significantly increased and decreased, respectively, from 2 months to 24 months of age. The increase in proportion of type IIA fibers with growth may have been attributable to muscle fiber-type transition from type IIA/IIX fibers but not from type IIX fibers. Values for SDH activity and fiber area in hybrid fiber types were intermediate to those for their respective pure phenotypes. CONCLUSIONS AND CLINICAL RELEVANCE: Hybrid fibers have an important role for determining the proportion of muscle fiber type in horses < 24 months old, and the metabolic and anatomic properties of the hybrid fibers are well coordinated, as in mature horses.     

Myosin heavy chain composition in normal and atrophic equine laryngeal muscle

The myosin heavy chain (MHC) composition of a given muscle determines the contractile properties and, therefore, the fiber type distribution of the muscle. MHC isoform expression in the laryngeal muscle is modulated by neural input and function, and it represents the cellular level changes that occur with denervation and reinnervation of skeletal muscle. The objective of this study was to evaluate the pattern of MHC isoform expression in laryngeal muscle harvested from normal cadavers and cadavers with naturally occurring left laryngeal hemiplegia secondary to recurrent laryngeal neuropathy. Left and right thyroarytenoideus (TA) and cricoarytenoideus dorsalis (CAD) were obtained from 7 horses affected with left-sided intrinsic laryngeal muscle atrophy and from 2 normal horses. Frozen sections were evaluated histologically for degree of atrophy and fiber type composition. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of muscle protein. Histologic atrophy was seen in all atrophic muscles and some right-sided muscles of 3 affected horses, as well as the left TA of 1 normal horse. Fiber type grouping or loss of type I muscle fibers was observed in the left-sided laryngeal muscles in all but 1 affected horse, as well as in the right muscles of 2 affected horses, and the left TA of 1 normal horse. SDS-PAGE showed 2 bands corresponding to the type I and type IIB myosin isoforms in the CAD and TA of the 2 normal horses. Affected horses demonstrated a trend toward increased expression of the type IIB isoform and decreased expression of the type I isoform in atrophic muscles. This study confirmed the presence of histologic abnormalities in grossly normal equine laryngeal muscle, and it demonstrated an increased expression of type IIB MHC with a concurrent decreased expression of type I MHC in affected muscles. Evaluation of muscle fiber changes at the cellular level under denervated and reinnervated conditions may aid in assessing future strategies for reinnervation or regeneration of atrophic laryngeal muscle.     

Correlation between histochemically assessed fiber type distribution and isomyosin and myosin heavy chain content in porcine skeletal muscles.

Highly sensitive enzyme assays developed to differentiate skeletal muscle fibers allow the recognition of three main fiber types: slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG). Myosin, the predominant contractile protein in mammalian skeletal muscle, can be separated based on the electrophoretic mobility under nondissociating conditions into SM2, SM1, IM, FM3, and FM2 isoforms, or under dissociating conditions into myosin heavy chain (MHC) I, IIb, IIx/d, and IIa. The purpose of the present study was to determine whether the histochemical method of differentiation of fiber types is consistent with the electrophoretically identified isomyosin and MHC isoforms. These comparisons were made using serratus ventralis (SV), gluteus medius (GM), and longissimus muscles (LM) from 13 pigs. Two calculation methods for the histochemical assessed fiber type distribution were adopted. The first method incorporated the number of fibers counted for each fiber type and calculated a percentage of the total fiber number (fiber number percentage: FNP). The second method expressed the cross-sectional area of each fiber type as a percentage of the total fiber area measured per muscle (fiber area percentage: FAP). Independent of the calculation methods, correlation analyses revealed in all muscles a strong relation between SO fibers, the slow isomyosin (SM1 and SM2), and MHCI, as well as between the FG fibers, the fast isomyosin (FM3 and FM2), and MHCIIx/b content (P<.05). There were no correlations between FOG fiber population assessed by histochemical analysis and intermediate isoform (IM) or MHCIIa content. The present results did not provide conclusive evidence as to which of the calculation methods (FNP or FAP) was more closely related to myosin composition of skeletal muscles. Despite some incompatibility between the methods, the present study shows that histochemical as well as electrophoretic analyses yielded important information about the composition of porcine skeletal muscle. The combination of the two methods may be essential to accurately characterize porcine skeletal muscles.     

胎儿期与成年期蒙古马骨骼肌肌纤维类型转化研究

为探索影响蒙古马胎儿期和成年期肌纤维类型差异机理.本研究选取3匹4月龄胎儿(两母一公)与3匹5岁健康成年母马身体4块分布全身、具有代表性的肌肉组织(长臂三头肌、夹肌、背最长肌、臀中肌)作为一个整体.胎儿期蒙古马肌纤维和成年期蒙古马肌纤维因存在差异各做为一组,试验进行3个生物学重复.首先对蒙古马骨骼肌肌肉样品进行免疫组化...     

Differential response of cull cow muscles to the hypertrophic actions of ractopamine-hydrogen chloride

Ractopamine-HCl (RAC) is a beta-adrenergic agonist with variable effects on cattle performance and carcass variables. Cull cows fed RAC (200 mg . head(-1) . d(-1)) demonstrate an increased size of type I and II muscle fibers that does not translate into a larger ribeye area. The objective of this study was to examine the dose-dependent effects of RAC on cull cow muscle morphometrics. Eighty-eight cull beef cows representing 2 breed types (n = 44 each) were fed 0, 100, 200, and 300 mg . head(-1) . d(-1) of RAC for the last 28 d of a 54-d feeding period. On d 54, cows were slaughtered, and samples of the LM and semimembranosus muscle (SM) from 16 randomly selected carcasses (n = 4 per treatment) were taken for measurement of beta (2)-adrenergic receptors and type I, IIA, and IIX myosin heavy chain (MyHC) gene expression. Twenty-four hours postmortem, LM, SM, infraspinatus (INF), and vastus lateralis samples from 40 randomly selected carcasses (n = 10 per treatment) were obtained and frozen for immunohistochemical analysis. Muscle fiber cross-sectional area and diameter, MyHC isoform expression, and fiber-associated nuclei numbers were measured. Ractopamine dosage exhibited differential effects on muscle morphometrics and MyHC gene expression. Muscle fiber cross-sectional area and diameter were increased (P < 0.05) by RAC in INF type I and IIA fibers and SM type IIA fibers. Ractopamine increased (P < 0.05) MyHC type IIX mRNA and tended to increase (P < 0.10) beta(2)-adrenergic receptors in the SM; a change in mRNA abundance was not detected for either gene in the LM. Treatment with RAC decreased (P < 0.05) fiber-associated nuclei numbers in the INF, vastus lateralis, and LM but did not affect (P > 0.05) MyHC or beta-adrenergic receptor expression. These results indicate that cull cow feeding programs may consider supplementing RAC as a means of adding value to cuts within the chuck, such as the INF.     

Age-related changes in metabolic properties of equine skeletal muscle associated with muscle plasticity

The purpose of the present study was to determine the age-related changes in myosin heavy chain (MHC) composition and muscle oxidative and glycolytic capacity in 18 horses ranging in age from two to 30 years. Muscle samples were collected by excisional biopsy of the semimebranosus muscle. MHC expression and the key enzymatic activities were measured. There was no significant correlation between horse age and the proportions of type-IIA and type-IIX MHC isoforms. The percentage of type-I MHC isoforms decreased with advancing age. Muscle citrate synthase activity decreased, whereas lactate dehydrogenase activity increased with increasing age. Muscle 3-OH acyl CoA dehydrogenase activity did not change with ageing. The results suggest that, similar to humans, the oxidative capacity of equine skeletal muscle decreases with age. The age-related changes in muscle metabolic properties appear to be consistent with an age-related transition in MHC isoforms of equine skeletal muscle that shifts toward more glycolytic isoforms with age.     

Analysis of proglycogen and macroglycogen content in muscle biopsy specimens obtained from horses

OBJECTIVE: To determine proglycogen (PG) and macroglycogen (MG) content in equine skeletal muscle and to compare 2 analytical methods (acid hydrolysis [AC] and PG plus MG determination) for measurement of total muscle glycogen content (Gly(tot)) in biopsy specimens. SAMPLE POPULATION: Muscle biopsy specimens obtained from 41 clinically normal horses. PROCEDURE: Forty-five muscle biopsy specimens obtained from the middle gluteal (n = 31) or triceps (14) muscle were analyzed, using AC and MG plus PG determination for Gly(tot). Variability within muscle biopsy specimens for each method was calculated from duplicate analyses of muscle specimens. In a second experiment, variation in MG and PG content between muscle biopsy specimens and the effect of sample collection depth on the concentration of MG and PG in the middle gluteal muscle was evaluated. RESULTS: There was a strong correlation (r = 0.99) between Gly(tot) values obtained by use of AC and MG plus PG determination. Coefficients of variation for within- and between-specimen variability of Gly(tot) were approximately 4% for each method. The PG fraction was always in excess of the MG fraction. Biopsy specimens obtained from the superficial part of the middle gluteal muscle contained significantly more Gly(tot) and PG than specimens obtained from deeper parts. CONCLUSIONS AND CLINICAL RELEVANCE: This study confirms that MG and PG exist in equine skeletal muscle and can be measured reliably in biopsy samples. This technique could be applied in future studies to investigate glycogen metabolism in exercising horses and horses with glycogen-storage diseases.     

Characterisation of glucose transporter (GLUT) gene expression in broiler chickens

1. Glucose transporter (GLUT) proteins, one of which is the major insulin-responsive transporter GLUT4, play a crucial role in cellular glucose uptake and glucose homeostasis in mammals. The aim of this study was to identify the extent of mRNA expression of GLUT1, GLUT2, GLUT3 and GLUT8 in chickens intrinsically lacking GLUT4. 2. GLUT1 mRNA was detected in most tissues of 3-week-old broiler chickens, with the highest expression measured in brain and adipose tissue. GLUT2 was expressed only in the liver and kidney. GLUT3 was highly expressed in the brain. GLUT8 was expressed ubiquitously, with expression in kidney and adipose tissue relatively higher than that of other tissues. 3. Expression levels of GLUT isoforms 1, 3 and 8 in skeletal muscle tissue were very low compared to the other tissues tested. 4. [3H]Cytochalasin B binding assays on tissue from 3-week-old chickens showed that the number of cytochalasin B binding sites in skeletal muscle plasma membranes was higher than in liver plasma membranes. These results suggest that GLUT proteins and/or GLUT-like proteins that bind cytochalasin B are expressed in chicken skeletal muscles. 5. It is proposed that GLUT expression and glucose transport in chicken tissues are regulated in a manner different from that in mammals.     

Morphologic and morphometric studies of the intrinsic laryngeal muscles in clinically normal adult dogs

To establish normal histologic and histochemical data and to determine reference values for fiber type proportions (percentages, mean fiber diameters, atrophy and hypertrophy factors, and variability coefficients), a histochemical study was carried out on intrinsic muscles of the larynx (cricothyroid, cricoarytenoid lateralis, cricoarytenoid dorsalis, and thyroarytenoid muscles) of clinically normal dogs. Using myosin adenosine triphosphatase stain under acidic preincubation (pH 4.3) conditions, 3 histochemical fiber types--1, 2A, and 2C--were recognized. The percentage of type-2C fibers varied from 1 to 2% in thyroarytenoid muscles to approximately 10% in cricoarytenoid lateralis muscles. There was no significant difference in mean diameter between left- and right-side specimens of each muscle for type 1 vs type 2. The largest fibers (mean +/- SD) of both types were observed in the cricothyroid muscles (type 1, 38.19 +/- 7.76 microns; type 2, 43.25 +/- 8.66 microns), and the smallest fibers were found in the thyroarytenoid muscles (type 1, 29.38 +/- 5.12 microns; type 2, 33.84 +/- 6.20 microns). Respective mean diameters of fiber types from cricoarytenoid dorsalis (type 1, 32.05 +/- 5.69 microns; type 2, 38.95 +/- 7.75 microns) and cricoarytenoid lateralis (type 1, 33.75 +/- 5.98 microns; type 2, 37.09 +/- 7.01 microns) muscles were similar. The histographic distribution of fiber type diameters was unimodal in all muscles. In each muscle, the mean fiber diameter of type-2 fibers was greater than that of type-1 fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin sensitivity in Belgian horses with polysaccharide storage myopathy

OBJECTIVE: To determine insulin sensitivity, proportions of muscle fiber types, and activities of glycogenolytic and glycolytic enzymes in Belgians with and without polysaccharide storage myopathy (PSSM). ANIMALS: 10 Quarter Horses (QHs) and 103 Belgians in which PSSM status had been determined. PROCEDURES: To determine insulin sensitivity, a hyperinsulinemic euglycemic clamp (HEC) technique was used in 5 Belgians with PSSM and 5 Belgians without PSSM. Insulin was infused i.v. at 3 mU/min/kg for 3 hours, and concentrations of blood glucose and plasma insulin were determined throughout. An i.v. infusion of glucose was administered to maintain blood glucose concentration at 100 mg/dL. Activities of glycogenolytic and glycolytic enzymes were assessed in snap-frozen biopsy specimens of gluteus medius muscle obtained from 4 Belgians with PSSM and 5 Belgians without PSSM. Percentages of type 1, 2a, and 2b muscle fibers were determined via evaluation of >or= 250 muscle fibers in biopsy specimens obtained from each Belgian used in the aforementioned studies and from 10 QHs (5 with PSSM and 5 without PSSM). RESULTS: Belgians with and without PSSM were not significantly different with respect to whole-body insulin sensitivity, muscle activities of glycogenolytic and glycolytic enzymes, or proportions of muscle fiber types. However, Belgians had an increased proportion of type 2a and decreased proportion of type 2b muscle fibers, compared with proportions in QHs, regardless of PSSM status. CONCLUSIONS AND CLINICAL RELEVANCE: PSSM in Belgians may be attributable to excessive glycogen synthesis rather than decreased glycogen utilization or enhanced glucose uptake into muscle cells.     

Glucose transport in the equine hoof

Reasons for performing study: Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin‐responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. Objectives: To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. Methods: Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2‐deoxy‐D‐[2,6‐³H]glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin‐induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushing's syndrome. Results: Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin‐independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. Conclusions: The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1‐mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. Potential relevance: Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.