Insulin stimulates GLUT4 translocation in the semitendinosus muscle of Shetland ponies.

Glucose homeostasis depends on insulin-regulated glucose uptake in the skeletal muscles and fat tissues via glucose transporter (GLUT) 4 translocation into cellular plasma membranes. The present study sought to elucidate GLUT4 expression, GLUT1 and GLUT4 translocation and glucose uptake in the skeletal muscles of Shetland ponies. Semitendinosus muscle explants were removed by open muscle biopsy from six Shetland pony geldings under general anaesthesia. The expression of GLUT4 was analysed by measuring muscle crude membrane (CM) GLUT4 protein contents. To determine the insulin-stimulated GLUT translocation, GLUT1 and GLUT4 concentrations were measured in partially purified plasma membranes (PM) and cytoplasmic vesicles (CV). GLUT contents were determined semi-quantitatively by Western blotting. Insulin-stimulated glucose uptake was analysed using 3-O-d-methyl[(3)H]glucose uptake. Incubation of semitendinosus muscle strips with 0.1 and 20mIU/mL insulin significantly increased GLUT4 translocation (PM GLUT4 contents), but had no significant effect on GLUT4 expression (CM GLUT4 concentrations) or PM GLUT1. The uptake of myocyte 3-O-Methylglucose was not significantly increased following insulin stimulation. The sub-cellular fractionation technique proved to be an appropriate tool for determining insulin-stimulated GLUT4 translocation in equine skeletal muscle. GLUT4 translocation in equines is insulin-dependent, as has been described in rodents and farm animals, but insulin-stimulated GLUT4 activation in ponies is lower than reported for pigs and cows under the same experimental conditions. Poor insulin-activated GLUT4 translocation may account for insulin resistance in ponies in previous euglycaemic, hyperinsulinaemic clamp tests.

pH gradients and a micro-pore filter at the luminal surface affect fluxes of propionic acid across guinea pig large intestine.

A neutral pH microclimate had been shown at the luminal surface of the large intestine. The aim was to estimate to what extent fluxes of propionic acid/propionate are affected by changes of the luminal pH when this microclimate is present, largely reduced or absent. Fluxes of propionic acid/propionate (J(Pr)) across epithelia from the caecum, the proximal and the distal colon of guinea pigs were measured in Ussing chambers with and without a filter at the luminal surface. With bicarbonate and with a neutral or an acid pH of mucosal solutions (pH 7.4 or 6.4), mucosal-to-serosal fluxes (J(ms)(Pr) ) were 1.5 to 1.9-fold higher at the lower pH, in bicarbonate-free solutions and carbonic anhydrase (CA) inhibition 2.1 to 2.6-fold. With a filter at the mucosal surface and with bicarbonate containing solutions, J (ms) (Pr) was not or only little elevated at the lower pH. Without bicarbonate J(ms)(Pr) was clearly higher. We conclude that the higher J(ms)(Pr) after luminal acidification is due to vigorous mixing in Ussing chambers resulting in a markedly reduced unstirred layer. Therefore, an effective pH microclimate at the epithelial surface is missing. J(ms)(Pr) is not or is little affected by lowering of pH because in the presence of bicarbonate the filter maintains the pH microclimate. However, in bicarbonate-free solutions J(ms)(Pr) was higher at pH 6.4 because a pH microclimate does not develop. Findings confirm that 30-60% of J(ms)(Pr) results from non-ionic diffusion.

Mechanisms of insulin-dependent glucose transport into porcine and bovine skeletal muscle.

Euglycemic, hyperinsulinemic clamp tests have shown that adult ruminants are less insulin-sensitive than monogastric omnivores. The present study was carried out to elucidate possible cellular mechanisms contributing to this impaired insulin sensitivity of ruminants. Western blotting was used to measure glucose transporters 1 and 4 (GLUT1, GLUT4) in oxidative (musculus masseter and diaphragm) and glycolytic (musculus longissimus dorsi and semitendinosus) skeletal muscle in the crude membranes of pigs and cows. Muscles were characterized biochemically. To determine insulin-stimulated 3-O-D-[(3)H]-methylglucose (3-O-MG) uptake and GLUT4 translocation, porcine and bovine musculus semitendinosus strips were removed by open muscle biopsy and incubated without and with 0.1 or 20 mIU insulin/ml. GLUT4 translocation was analyzed using subcellular fractionation techniques to isolate partially purified plasma membranes and cytoplasmic vesicles and using Western blotting. GLUT4 protein contents were significantly higher in oxidative than in glycolytic muscles in pigs and cows. GLUT1 protein contents were significantly higher in glycolytic than in oxidative muscles in bovines but not in porcines. The 3-O-MG uptake into musculus semitendinosus was similar in both species. Maximum insulin-induced GLUT4 translocation into musculus semitendinosus plasma membrane was significantly lower in bovines than in porcines. These results indicate that GLUT1 is the predominant glucose transporter in bovine glycolytic muscles and that a reinforced insulin-independent glucose uptake via GLUT1 may compensate for the impaired insulin-stimulated GLUT4 translocation, resulting in a similar 3-O-MG uptake in bovine and porcine musculus semitendinosus. These findings may explain at least in part the impaired in vivo insulin sensitivity of adult ruminants compared with that of omnivorous monogastric animals.

Excessive apoptosis of guinea pig colonocytes may lead to an imbalance between phagocytosis and degradation in vivo.

The success or failure of the clearance of apoptotic cell remains depends on the ability of phagocytic cells to recognize, phagocytoze, and digest these remains prior to their lysis, which would cause tissue inflammation. We have recently shown that, after mass-induced apoptosis of guinea pig colonocytes in vivo, phagocytosis by resident macrophages, although efficient, does not prevent a pre-inflammatory response of the mucosa. The present study has investigated the cause(s) of this clearance failure. Immunohistochemistry and transmission electron microscopy were applied. Antibodies directed against the epithelial plasma membrane protein E-cadherin, the lysosomal membrane protein LAMP-1, and the lysosomal matrix protease cathepsin-D were used. The results revealed that: (1) anti-E-cadherin labeled the membrane of epithelial apoptotic bodies internalized in macrophages, (2) double and triple labeling demonstrated that the anti-LAMP-1 and anti-cathepsin-D antibodies recognized and were co-localized in lysosomes and/or phagolysosomes in macrophages but left E-cadherin-positive structures unlabeled, (3) the more numerous were the E-cadherin-positive inclusions in macrophages, the smaller was the number of those that stained positive for lysosomal markers. In parallel with electron microscopy, these findings showed that not all apoptotic bodies phagocytozed by macrophages were subsequently digested, suggesting that the phagocytotic ability of these cells was not matched by their digestive capability.

Intracellular pH regulation in guinea-pig caecal and colonic enterocytes during and after loading with short-chain fatty acids and ammonia.

Absorption of short-chain fatty acids (SCFA) and ammonia implies considerable fluxes of protons across the epithelium of the large intestine. Efficient regulation of intracellular pH (pH(i)) is therefore essential in these cells. The aim of the present study was to examine the effects of SCFA and of ammonia on pH(i), on pH(i) regulation and to characterize the mechanisms involved in pH(i) regulation in surface enterocytes of the guinea-pig caecal and colonic mucosa. Intact epithelia from the caecum and the distal colon were mounted in a microperfusion chamber. pH(i) was measured by fluorescence microscopy using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Addition of SCFA or ammonia to the serosal side changed the enterocyte pH(i) markedly, whereby ammonia caused larger changes in pH(i) than SCFA. In contrast, addition of SCFA to the mucosal solution had no effect on pH(i) and ammonia increased pH(i) only slightly. Basolaterally located pH(i) regulation mechanisms, Na(+)-H(+) exchange and Cl(-)-HCO(3)(-) exchange, are involved mainly in returning pH(i) to normal values. It is concluded that, due to apparently lower permeability of the apical membranes, the caecal and colonic epithelium is protected against pH(i) disturbances caused by the naturally high luminal SCFA and NH(3) concentrations. The major regulation mechanisms of pH(i) are located in the basolateral membrane of the enterocytes.

Permeability properties of apical and basolateral membranes of the guinea pig caecal and colonic epithelia for short-chain fatty acids.

Unidirectional fluxes of short-chain fatty acids (SCFA) indicated marked segmental differences in the permeability of apical and basolateral membranes. The aim of our study was to prove these differences in membrane permeability for a lipid-soluble substance and to understand the factors affecting these differences. Apical and basolateral membrane fractions from guinea pig caecal and colonic epithelia were isolated. Membrane compositions were determined and the permeability of membrane vesicles for the protonated SCFA was measured in a stopped-flow device. Native vesicles from apical membranes of the caecum and proximal colon have a much lower permeability than the corresponding vesicles from the basolateral membranes. For the distal colon, membrane permeabilities of native apical and basolateral vesicles are similar. In vesicles prepared from lipid extracts, the permeabilities for the protonated SCFA are negatively correlated to cholesterol content, whereas no such correlation was observed in native vesicles. Our findings confirm that the apical membrane in the caecum and proximal colon of guinea pig is an effective barrier against a rapid diffusion of small lipid-soluble substances such as SCFAH. Besides cholesterol and membrane proteins, there are further factors that contribute to this barrier property.

Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes.

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.