Synergistic effect of synthetic conjugated linoleic acid & non fat milk on fat deposition & lipid metabolism in mice.

Background & objectives: Conjugated linoleic acid (CLA) reduces fat deposition in the body, but the mechanism of action is not clear. The present study was conducted to investigate the effects of CLA on body fat metabolism. Since milk fat is the best natural source of dietary CLA, intervention of non-fat milk constituents on CLA treatment was also investigated. Methods: Diets containing CLA (1%) with or without skim milk powder (SMP) was fed to male Swiss albino mice for 60 days. Adipose depots weight, faecal fat and the activities of selected enzymes of lipid metabolism were determined. Results: The mice on CLA and CLA+SMP diets gained weight similar to those on control diet, despite higher feed intake in the former two groups. Total fat pad mass was significantly (P<0.05) less in CLA group than in control group, and inclusion of SMP in the diet enhanced the fat reducing effect of CLA. Adiposity index was also less on CLA and CLA+SMP diets than on control diet, and CLA+SMP was more efficacious in reducing adiposity index. The weight of liver and spleen was increased by CLA, and this effect was eliminated by inclusion of SMP in the diet. The fatty acid synthase (FAS) activity in liver and retroperitoneal adipose tissue decreased substantially on CLA and CLA+SMP diets compared to that on control diet. Interpretation & conclusions: Our preliminary data show that dietary CLA reduces body fat mass by decreasing fatty acid biosynthesis, and the effect is enhanced by inclusion of SMP in the diet.

Maharishi Amrit Kalash, an ayurvedic medicinal preparation, enhances cholinergic enzymes in aged guinea pig brain.

The effect of orally fed Maharishi Amrit Kalash was examined on the activities of cholinergic enzymes in the guinea pig brain. The activity of the cholinergic enzymes viz. choline-acetyltransferase and acetylcholinesterase enzymes was found to be reduced significantly (P<0.05) in the various regions of CNS of the aged guinea pigs. Oral administration of MAK(500 mg/kg body weight daily) for 2 months significantly increased (P<0.05) the activity of choline acetyltransferase and acetylcholinesterase in the older animals. The present study indicates that this food supplement can be helpful in alleviating the cholinergic deficits in the old age.

Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs.

The age-related changes in the activities of antioxidant enzymes of mitochondrial and cytosolic fractions were measured in different regions of the central nervous system (CNS) in 10 and 32 months old guinea pigs. In old animals, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced (p < 0.05) in all the regions of CNS studied but catalase (CAT) declined significantly only in the cerebral cortex, hypothalamus and cerebellum. Glutathione reductase (GRd) activity declined in cerebral cortex and hypothalamus in the cytosolic fractions and only in cerebellum in the mitochondrial fraction. It is concluded that age-related decline in the activities of antioxidant enzymes is both region and enzyme specific. The endogenous lipid peroxide was found to be significantly higher (p < 0.05) in the 32 month old animals whereas, lipid peroxidation after incubating the tissue homogenate in air was found to be lower (p < 0.05). The in vitro mitochondrial lipid peroxidation decreased with age. The results indicate that accumulation of lipid peroxides takes place with ageing but the susceptibility of lipid peroxidation decreases in the older animals.

Heterogeneity of transport systems for L-glutamine in mouse mammary gland.

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L-glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+-dependent systems, which we have named BCI(-)-dependent and BCl(-)-independent, are the new systems identified. These are broad specificity systems and were discriminated on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems, L-homoserine inhibited the uptake of L-glutamine via only BCl(-)-dependent system. The uptake of L-glutamine via the BCl(-)-independent system was upregulated by preloading mammary tissue with L-serine, while BCl(-)-dependent system was unaffected. The Na+-independent uptake of L-glutamine was inhibited by 2-aminobicyclo-(2,2,1)heptane carboxylic acid and other neutral amino acids, and identified as the system L.

Effect of Maharishi Amrit Kalash an ayurvedic herbal mixture on lipid peroxidation and neuronal lipofuscin accumulation in ageing guinea pig brain.

The effects of ayurvedic herbal mixture Maharishi Amrit Kalash(MAK) were studied on brain lipid peroxidation, oxygen consumption, and lipofuscin accumulation in 10 months and 32 months old guinea pigs. Brain regions studied were cerebral cortex, hypothalamus, cerebellum and spinal cord. Parameters assessed were lipid peroxidation, oxygen consumption, and lipofuscin accumulation. The endogenous lipid peroxide was found to be increased significantly (P < 0.05) in the 32-month-old animals. Neuronal lipofuscin accumulation in the neurons of cerebral motor cortex, cerebellum and cervical spinal cord was increased (P < 0.05) in the older animals. Oxygen consumption was found to be decreased significantly(P < 0.05) in the 32-month old guinea pigs. Treatment with MAK at a dose of 500 mg/kg body weight daily for two months reduced the lipid peroxidation and lipofuscin pigment accumulation significantly in brain regions and it also helped in restoring the normal oxygen consumption in the older animals. This indicates antioxidant properties of MAK.

Characterization of anionic amino acid transport systems in mouse mammary gland.

L-glutamate was transported into mammary tissue via Na(+)-dependent system XAG- that strongly interacted with both D- and L-isomers of aspartate but only with L-isomer of glutamate. Replacement of Cl- by gluconate from the extracellular medium did not affect the uptake of L-glutamate. Although neutral amino acids weakly inhibited the uptake of L-glutamate, there was no evidence for the heterogeneity of anionic amino acid transport system. The XAG- system was inhibited by sulfhydryl group blocking reagent N-ethylmalemide. Low pH (6) partially inhibited the uptake by L-glutamate by mammary tissue. Prior loading of mammary tissue with L-glutamate slightly down regulated its uptake. Culturing pregnant mouse mammary tissue explants in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin) did not affect appreciably the uptake of L-glutamate.

Discrimination of transport systems of L-tyrosine in mouse mammary gland: characterization of system T.

On the basis of inhibition analysis, tyrosine uptake in mouse mammary gland was found to be mediated by Na(+)-dependent and Na(+)-independent systems. Na(+)-dependent system was insensitive to 2-(methylamino) isobutyric acid (MeAIB), with an apparent Km of 1.67 mM and maximal velocity 74.5 nmol.g-1 cell. min-1. Competition experiments showed the presence of two distinct Na(+)-independent components of tyrosine uptake. One component was sensitive to 2-aminobicyclo (2,2,1) heptane-2-carboxylic acid (BCH) and was similar to the system L, with an apparent Km of 0.23 mM and maximum velocity of 31 nmol.g-1 cell.min-1. Second component was BCH-insensitive but tryptophan-sensitive, with an apparent Km of 15.75 mM and Vmax of 157.5 nmol.g-1cell.min-1. BCH-insensitive, tryptophan-sensitive system was a low affinity system. It approached steady state slowly and was more sensitive, relative to the system L, to n-ethylmaleimide inhibition. Tyrosine uptake through this system did not respond to trans-stimulation, whereas system L mediated uptake responded considerably. BCH-insensitive, tryptophan-sensitive component of Na(+)-independent tyrosine uptake is attributed to the system T, previously described only in human red blood cells and rat liver cells.

Lack of functional correlation between gamma-glutamyl transpeptidase and amino acid transport in the lactating mouse mammary gland.

Gamma-glutamyl transpeptidase (EC 2.3.2.2) in lactating mouse mammary gland was inhibited by affinity labelling of the tissue with 6-diazo-5-oxo-L-norleucine. Amino acid (L-alanine, L-methionine) uptake by the affinity labelled mammary gland tissue and the control tissue was measured in vitro. Uptake of amino acids by the affinity-labelled tissue was comparable to that of control tissue. These findings suggest that gamma-glutamyl cycle is not involved in amino acid uptake by the mammary gland.

Characterisation and starvation induced regulation of methionine uptake sites in mouse mammary gland.

The sites of methionine uptake by 10 day lactating mouse mammary gland were determined in vitro. Four modes of methionine entry characterised were: (i) A sodium-dependent, N-(methylamino) isobutyric acid (MeAIB)--sensitive system with a Vmax of 18.8 nmol/g cells/min (this mode of entry was similar to the A site in other tissues); (ii) A sodium-dependent, MeAIB--insensitive uptake system with a Vmax of 12.4 nmol/g cells/min); this mode of entry was inhibited by substrates preferred by ASC system); (iii) A sodium-independent, 2-amino-bicyclo heptane 2-carboxylic acid (BCH)-sensitive system L with a Vmax of 30 nmol/g cells/min; and (iv) A sodium-independent entry which was not inhibited by high concentrations of MeAIB or BCH. The Km value of each of the former three carrier mediated transport systems was 0.46 mM. Starvation of animals brought about important increase in the Vmax of the A system by 97% and that of ASC system by 1003% which was accompanied by similar increases in the Km values of these systems. These results show an adaptive regulation of these two sodium-dependent sites as a result of starvation.

Characterisation of the routes of methionine transport in mouse mammary glands.

The sites of methionine uptake by mammary glands from late pregnant and lactating mice were studied in vitro. Using the specific A system inhibitor, N-(methylamino) isobutyric acid (MeAIB) and the specific L system inhibitor, 2-amino-bicyclo (2.2.1) heptane 2-carboxylic acid (BCH), we have defined four modes of methionine entry into these tissues. (i) A sodium-dependent A system with a Vmax of 13.4 and 18.8 n mol/g cells/min in pregnant and lactating mice, respectively. This mode of entry was completely inhibited by MeAIB and its Km value was similar (0.45 mM) in both groups. (ii) A sodium-dependent mode with a Vmax of 6.7 and 12.4 n mol/g cells/min and a Km of 0.24 and 0.46 mM in pregnant and lactating mice, respectively. This mode of entry was insensitive to inhibition by MeAIB, and was similar to the ASC (alanine, serine, cysteine) system in other tissues. (iii) A sodium-independent L system with a Vmax of 13.8 and 30.0 n mol/g cells/min and a Km of 0.27 and 0.46 mM in pregnant and lactating mice, respectively. This mode of entry was completely inhibited by BCH. (iv) A sodium-independent non-specific entry amounting to 25 per cent of the total entry at 0.1 mM external methionine which was not inhibited by high concentration of BCH. The results of our studies show an increase in the number of active carriers of the A, ASC and L systems of methionine uptake in mammary glands of mouse during lactation.

Effect of skim milk on progression of atherosclerosis in cholesterol-fed rabbits.

Effect of skim milk on progression of atherosclerosis was studied in cholesterol-fed rabbits. Rabbits were given a high cholesterol food (0.5%) with skim milk powder (16%) or no milk (control group). At 12 wk, the plasma cholesterol level was significantly higher in the control group (1605 mg/d1) than in the milk-fed group (1146 mg/d1). The contents of esterified cholesterol and elastin in the aorta were higher in the control group than in the milk-fed group by 28 an 94 per cent, respectively. The differences between the two groups in the contents of aortic triacylglycerols, mucopolysaccharides, collagen and unesterified cholesterol were not significant. The difference in sudanophilic area in the aorta between the control (35%) and the milk-fed groups (31%) was not significant. However, intimal proliferation and medial involvement in the aortic lesions were more severe in the control group. These findings suggest that skim milk can slow down the process of cholesterol induced atherogenesis.

Effect of skim milk on cholesterol induced blood platelet hyperaggregability in rabbit.

Effect of high cholesterol diet on blood platelet aggregation, and the influence of skim milk on cholesterol induced platelet hyperaggregability were studied in rabbits. The level of aggregated platelets (AP) increased to 19900/mm3 and that of plasma cholesterol to 1605mg/dl, while platelet aggregate ratio (PAR) decreased to 0.32 in rabbits fed high cholesterol diet (0.5% cholesterol) for 12 wk. The levels of AP (12500/mm3) and plasma cholesterol (1196mg/dl) were lower, and the PAR (0.46) was higher in rabbits which received the cholesterol diet supplemented with 16 per cent skim milk powder. In the second experiment, hypercholesterolaemia and platelet aggregation were induced in rabbits by high cholesterol diet given for 6 wk. The animals then received cholesterol-free diets for 8 wk, one group receiving the diet supplemented with 16 per cent skim milk powder and the other without milk served as the control group. The mean PAR in the milk powder supplemented group was restored to normal at 6 wk, while in the control group the level continued to be lower even up to 8 wk. The results thus demonstrate that the milk can prevent the process of cholesterol induced platelet aggregation.

Effect of vitamin A deficiency on rat intestinal digestive & absorptive functions.

The effect of vitamin A deficiency on the intestinal absorption of nutrients and the activities of brush border enzymes were studied in albino rats. Intestinal uptakes of D-glucose, L-methionine, L-tryptophan and L-histidine were significantly greater in vitamin A-deficient animals than in controls. The specific activities of total adenosine triphosphatase (ATPase), ouabain-sensitive ATPase, maltase and sucrase in the intestinal mucosa of vitamin A-deprived rats were 121, 124, 131 and 134 per cent respectively, of the corresponding values in control animals. The DNA content of the small intestine in vitamin A-deficient rats was 36.5 per cent lower than in control rats. The stimulation in digestive and absorptive capacity appears to be an adaptive change in vitamin A-deficiency which decreases the intestinal cell population.

The activity of gamma-glutamyl transpeptidase in the small intestine of some species of animals at different stages of growth.

The activity of gamma-glutamyl transpeptidase (GTP), the key enzyme of gamma-glutamyl cycle, was studied in the intestine of new born, suckling and adult animals (rats, mice, guinea pigs and rabbits). In all the four species, the activity of GTP was greater in new borns than in adults. The activity was highest in rats and almost negligible in guinea pigs.

Effect of protein deprivation and subsequent rehabilitation on the intestinal transport of L-methionine in vivo.

The effect of protein deprivation and subsequent rehabilitation on the intestinal transport of L-methionine was studied in albino rats of both sexes. The rats given diet containing no protein or 3 per cent maize protein for 28 days, lost their intestinal cell population by 50 and 20 per cent respectively. The net absorption rate of L-methionine was little affected, while absorptive capacity of intestinal cells was considerably enhanced in protein-deficient rats. The increase in absorptive capacity of intestinal cells was much higher in rats given protein-free diet than in those given maize diet. The augmentation is absorptive capacity of intestinal cells of protein-deprived rats was a temporary adaptation to the conditions that prevent the formation of new cells. Rehabilitation of malnourished rat on diet containing 17 per cent casein, resulted in a rapid increase in intestinal cell population, return of the absorptive capacity of intestinal cells to normal, and augmentation in net absorption rates.

Studies on the action of hormones on the intestinal transport of L-histidine.

Intestinal absorption of L-histidine as affected by insulin, diabetes, hydrocortisone, adrenocorticotrophic hormone (ACTH) and thyroxine has been studied. Intestinal absorption of L-histidine is not significantly increased following insulin administration. Similarly, addition of insulin in vitro did not change the transport activity significantly. When rats are made diabetic on administration of alloxan monohydrate, the transport activity is increased. Hydrocortisone, adrenocorticotrophic hormone and thyroxine treatment increased the absorption of L-histidine by small intestine. However, addition in vitro of ACTH and hydrocortisone did not change the absorption of L-histidines by small intestine. It appears that the facilitative action of these hormones is not due to their direct action of the membrane.