Voluntary wheel running reduces voluntary consumption of ethanol in mice: identification of candidate genes through striatal gene expression profiling.

Hedonic substitution, where wheel running reduces voluntary ethanol consumption, has been observed in prior studies. Here, we replicate and expand on previous work showing that mice decrease voluntary ethanol consumption and preference when given access to a running wheel. While earlier work has been limited mainly to behavioral studies, here we assess the underlying molecular mechanisms that may account for this interaction. From four groups of female C57BL/6J mice (control, access to two-bottle choice ethanol, access to a running wheel, and access to both two-bottle choice ethanol and a running wheel), mRNA-sequencing of the striatum identified differential gene expression. Many genes in ethanol preference quantitative trait loci were differentially expressed due to running. Furthermore, we conducted Weighted Gene Co-expression Network Analysis and identified gene networks corresponding to each effect behavioral group. Candidate genes for mediating the behavioral interaction between ethanol consumption and wheel running include multiple potassium channel genes, Oprm1, Prkcg, Stxbp1, Crhr1, Gabra3, Slc6a13, Stx1b, Pomc, Rassf5 and Camta2. After observing an overlap of many genes and functional groups previously identified in studies of initial sensitivity to ethanol, we hypothesized that wheel running may induce a change in sensitivity, thereby affecting ethanol consumption. A behavioral study examining Loss of Righting Reflex to ethanol following exercise trended toward supporting this hypothesis. These data provide a rich resource for future studies that may better characterize the observed transcriptional changes in gene networks in response to ethanol consumption and wheel running.

Effect of skim milk supplementation on blood cholesterol concentration, blood pressure, and triglycerides in a free-living human population.

In 82 subjects, aged 21-73, we studied the effect of skim milk supplementation on serum cholesterol concentration, blood pressure, and serum triglyceride level. The study involved a 1-week pretreatment baseline period followed by 8 weeks of milk supplementation. Sixty-four people were designated to a test group and 18 people were placed in a seasonal index group. The study was designed as a free-living trial, i.e., participants were requested to maintain their normal lifestyles, including dietary pattern, except for the supplementation of one quart of 2% solids-not-fat fortified skim milk to the daily diet in the test group. Supplemental milk treatment was associated with a 6.6% reduction (p = 0.0004) of serum cholesterol in the high cholesterol (greater than or equal to 190 mg/dl) test subgroup within the first 4 weeks. No change was noted in serum cholesterol in the low-cholesterol (less than 190 mg/dl) subgroup throughout the study. Body weight and seasonal variation of blood cholesterol did not significantly influence serum cholesterol levels. Reduction (p = 0.0140) in percentage of calories from fat in the high-cholesterol subgroup was not correlated with the decrease in serum cholesterol in this test subgroup. Reductions in systolic and diastolic blood pressure occurred in the test subgroups; the low-cholesterol subgroup had a greater reduction (p = 0.0002) in diastolic blood pressure than the high-cholesterol group (p = 0.0049). Milk supplementation was associated with reduction (p = 0.0370) in serum triglycerides in the high-cholesterol subgroup.

Effects of source and amount of protein on ruminal fermentation and passage of nutrients to the small intestine of lactating cows.

Four Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square to investigate the effects of source (corn gluten meal or soybean meal) and amount (14.5 or 11.0%) of CP on ruminal fermentation, passage of nutrients to the small intestine, and animal performance. Cows wee fed for ad libitum intake a diet of 60% corn silage and 40% concentrate on a DM basis. The treatments, arranged in a 2 x 2 (source x amount of CP) factorial, were 1) 14.5% CP, soybean meal; 2) 11.0% CP, soybean meal; 3) 14.5% CP, corn gluten meal; and 4) 11.0% CP, corn gluten meal. Digestion in the rumen of OM, starch, ADF, and NDF was not affected by source or amount of CP in the diet. Total VFA and NH3 concentrations in ruminal fluid were increased by feeding diets that contained 14.5% CP or soybean meal. FLows of non-NH3 N and amino acids to the duodenum were greater in cows fed the 14.5% CP diets because of a greater flow of non-NH3 nonmicrobial N to the duodenum. Larger amounts of lysine passed to the duodenum when cows were fed soybean meal compared with corn gluten meal. Microbial N flow to the duodenum and efficiency of microbial growth were not affected by treatments, suggesting that ruminal NH3 concentration was not limiting for maximal microbial protein synthesis. Feeding 14.5% CP diets increased the production of milk (29.5 vs. 26.8 kg/d) and milk protein compared with 11.0% CP diets, possibly because of greater passage of amino acids to the small intestine. Feeding soybean meal to cows increased production of milk protein compared with feeding corn gluten meal, possibly because more lysine passed to the small intestine.

Effects of source of protein and carbohydrate on ruminal fermentation and passage of nutrients to the small intestine of lactating cows.

Four early lactation multiparous Holstein cows were used in a 4 x 4 Latin square to investigate the effects of source of protein (fish meal or soybean meal) and carbohydrate (corn or barley) on ruminal fermentation, flow of nutrients to the small intestine, and animal performance. The treatments, arranged in a 2 x 2 (protein x carbohydrate) factorial were: 1) corn plus soybean meal; 2) corn plus fish meal; 3) barley plus soybean meal; and 4) barley plus fish meal. Dry matter and starch intakes were greater when corn was fed than when barley was fed. Barley-based diets were more extensively degraded in the rumen than corn-based diets and therefore provided more energy for microbial growth. However, passage of amino acids and starch to the duodenum was greater for corn-based diets than barley-based diets, because of the greater intake and lower ruminal degradability of the corn-based diets. Microbial protein constituted a larger portion of the total N and had a greater influence on the pattern and quantity of amino acids that passed to the duodenum than did protein from fish meal or soybean meal, which escaped ruminal degradation. Feeding corn-based diets increased production of milk and milk protein compared with feeding barley-based diets.

The modulating effect of an inhibitor of cholesterolgenesis present in bovine milk upon the synthesis of cholesterol, dolichol and ubiquinone.

Bovine milk contains two inhibitors of hepatic cholesterol genesis. One of these, identified as orotic acid, influences the early segment of the cholesterol biosynthetic pathway and suppresses the conversion of acetate to mevalonate. In this study the other inhibitor was shown to curtail the formation of compounds past farnesyl pyrophosphate on the squalene-cholesterol branch of the pathway. Thus cholesterol synthesis may be suppressed while the production of two other products of the branched pathway, dolichol and ubiquinone, is allowed to continue. The possible role of these ingested regulators in the metabolism of the young until they achieve sufficient development is discussed.

Isolation of an inhibitor of hepatic cholesterolgenesis from human milk.

Two preparations active in reducing hepatic cholesterolgenesis in vitro were demonstrated in human milk. These appear to affect the cholesterol synthetic pathway at different loci. One inhibits the synthesis before the formation of mevalonic acid and has been isolated and subsequently identified as uric acid. The other inhibitor has yet to be identified. The apparent paradox of the active component being uric acid, of which high levels are known to cause gout, is discussed.

Origins of the cholesterol in milk.

Studies were conducted to investigate the origin of milk cholesterol in the ruminant. In the first experiment, [1(-14)C]sodium acetate was infused into one side of the udder of a lactating goat via the test canal whereas in the second, (1,2-3H]cholesterol was injected intravenously and concurrently with a [14C]acetate intramammary infusion. In both experiments, blood and milk samples were collected at intervals for 6 days postinjection. Maximum unesterified cholesterol specific activity (sp act) in whole milk appeared at 78 hr after intravenous injections of 3H cholesterol and within 3-7 hr after infusion of [14C]acetate. Virtually all the tritium in milk was associated with unesterified cholesterol. The sp act of 14C-labeled cholesterol was only 20% of gland-synthesized decanoic acid. Decanoic acid is known to be completely synthesized in the mammary gland, and, like cholesterol, acetate is its precursor. The results indicate that, although some milk cholesterol is synthesized in the mammary gland, it is derived principally from serum cholesterol. The data show also that serum cholesterol equilibrates with membrane cholesterol of the lactating cell prior to its secretion in milk.

Multi-origins of milk serum albumin in the lactating goat.

L-[U-14C]Leucine was infused into the right-hand mammary glands of lactating goats. Milk from both glands of the animals was sampled at intervals for 36 h. After 3 h the specific activity of milk serum albumin from the infused glands was more than six times that from the non-infused glands. The specific activity of milk serum albumin was considerably lower than that of alpha-lactalbumin or beta-lactoglobulin which are exclusively synthesized by mammary secretory cells. Following the intravenous injection of 125I-labeled serum albumin, maximum specific activity of this protein appeared in milk in 12 h. The specific activity of serum albumin in milk attained no more than 45% of the specific activity of the serum albumin in blood. It is concluded that milk serum albumin has multiple origins and that a portion of it, at least (10-20%), is made in the mammary gland.

Effect of milk constituents on hepatic cholesterol-genesis.

Two preparations active in reducing hepatic cholesterol biosynthesis were isolated from bovine skim milk. One of the inhibitors was in the dialysate and was identified as orotic acid (OA). The other inhibitor, present in the retentate, was not identified. Orotic acid appears to act by inhibiting cholesterol biosynthesis before the formation of mevalonate, whereas the retentate inhibitor exerts its effect beyond the formation of mevalonate in the biosynthetic pathway. Human milk also inhibited the incorporation of both labeled acetate and mevalonate into cholesterol by rat liver. Orotic acid was not detectable in human milk samples employed in this study. Administration of [6-14C]orotate to rats revealed its conversion to uracil in the liver. Subsequent work demonstrated that uracil had inhibitory activity on hepatic cholesterol biosynthesis similar to that of orotate when incubated with rat liver slices.

Protein and methionine hydroxy analog for lactating cows.

A 2 X 2 factorial experiment (protein 12.5 and 15.5; methionine hydroxy analog 0 and .125% dry matter) included 144 cows for one complete lactation, distributed over seven locations. Rations were formulated to the desired protein, methionine analog, and constant amounts of fiber 17%, sulfur .225%, calcium .6%, phosphorus .4%, and salt .5%. Treatment effects were not apparent for dry matter intake, daily milk and fat-corrected milk production, conversion of energy, and body weight changes. Conversion of dietary crude protein into milk protein was 34.5% for the low and 25.8% for the high protein ration. Methionine analon (0% = 2.54; .125% = 1.90). Effect of methionine analog was most apparent at low protein as 0 analog cows produced 247 kg fat, required 2.9 services/contraception, and had 156 days open whereas cows on other treatments (.125% analog and/or high protein) produced 264 kg fat, required 1.8 to 2.2 services/conception, and had 124 to 134 days open. Methionine analog response is discussed in relation to tuminal and postruminal effects as well as the interrelation with protein and energy.

Bovine milk intake and xanthine oxidase activity in blood serum.

Xanthine oxidase activity in blood serum was measured by a sensitive radio-enzymatic assay. Pigs receiving 7.6 liters of milk daily for 100 days did not show any detectable enzymatic activity in their blood Xanthine oxidase activity in blood serum of 25 human volunteers had an average of 6.7 milliunits per liter with a range of 0 to 34.6 milliunits per liter. Neither a causal nor statistically significant relationship existed between xanthine oxidase activity in blood and average daily milk consumption, age, or sex.

Transport of dietary cholesterol into blood and milk of the goat.

Position 4 labeled carbon-14 cholesterol was placed in abomasums (stomachs) of two lactating goats. Blood and milk samples were collected from the animals for 5 to 13 days. Specific activities of cholesterol and cholesteryl esters in various fractions of blood serum and milk were determined to reveal pathways by which dietary cholesterol enters milk. Results with the two animals showed similar trends. Within 24 h both cholesterol and cholesteryl esters of the three principal serum lipoproteins of the goat were labeled, and this labeling persisted in substantial degree for the 13-day experiment. Specific acities for cholesteryl esters in milk fat globules exhibited several remarkable attributes: they fluctuated in intensity with a 3-to 4-day cycle reaching a maximum at 7 to 8 days after tracer injection; they exceeded cholesteryl ester specific activity in the skim milk by an order of magnitude; and at their maximum they exceeded all specific activities for serum components. The results of this investigation exemplify the ease with which dietary cholesterol enters and crosses membranes in the animal body.

Structure and synthesis of milk fat XI. Effects of heparin on paths of incorporation of glucose and palmitic acid into milk fat.

Intravenous injection of heparin increases lipoprotein lipase activity of circulating serum presumably by removing the enzyme from its location on the capillary endothelium. The incorporation of carbon-14 uniformly labeled glucose and carbon-14 1-labeled palmitic acid into fractionated milk fat triglycerides was studied in both normal and heparin treated lactating goats. The objective was to remove lipoprotein lipase from the mammary gland capillaries and to contrast normal milk fat synthesis with a situation presumed to cause the gland to be solely dependent on the phosphatidic acid pathway. The studies with labeled glucose indicated that under normal conditions there are two sources of milk glyceride glycerol; while following heparin injections, there is a single glycerol pool providing most of the glyceride glycerol. The investigations with labeled palmitic acid indicated that under normal conditions there are two sources of palmitic acid coming from the blood which enter nonequilibrating cellular pools. Palmitic acid from both pools is available for triglyceride synthesis. Following heparin injections there appears to be a common intracellular pool of pre-formed palmitic acid derived from the blood. The data indicate that lipoprotein lipase operating on blood triglycerides yields a 2-mono-glyceride which subsequently enters the gland and is utilized for milk fat synthesis.