Effect of a high-protein diet on food intake and liver metabolism during pregnancy, lactation and after weaning in mice.

Major hepatic metabolic pathways are involved in the control of food intake but how dietary proteins affect global metabolism to adjust food intake is incompletely understood, particularly under physiological challenging conditions such as lactation. In order to identify these molecular events, mice were fed a high-protein (HP) diet from pregnancy, during lactation until after weaning and compared with control fed counterparts. Liver specimens were analyzed for regulated proteins using 2-DE and MALDI-TOF-MS and plasma samples for metabolites. Based on the 26 differentially expressed proteins associated with depleted liver glycogen content, elevated urea and citrulline plasma concentrations, we conclude that HP feeding during lactation leads to an activated amino acid, carbohydrate and fatty acid catabolism while it activates gluconeogenesis. From pregnancy to lactation, plasma arginine, tryptophan, serine, glutamine and cysteine decreased, whereas urea concentrations increased in both groups. Concomitantly, hepatic glycogen content decreased while total fat content remained unaltered in both groups. Consideration of 59 proteins differentially expressed between pregnancy and lactation highlights different strategies of HP and control fed mice to meet energy requirements for lactation by adjusting amino acid degradation, carbohydrate and fat metabolism, citrate cycle, but also ATP-turnover, protein folding, secretion of proteins and (de)activation of transcription factors.

Proteome analysis of fatty liver in feed-deprived dairy cows reveals interaction of fuel sensing, calcium, fatty acid, and glycogen metabolism.

The liver of dairy cows is involved in signaling the current hepatic metabolic state to the brain via metabolites and nerval afferents to control and adjust feed intake. Feed deprivation may result in mobilization of body reserves favoring hepatic steatosis. While the overall metabolic changes are well characterized, specific regulatory mechanisms are not readily understood. To identify molecular events associated with metabolic adaptation and the control of energy homeostasis, liver specimens from six ad libitum-fed and six feed-deprived cows were analyzed for selected metabolites, for the activation of AMP kinase, and for regulatory/regulated proteins using two-dimensional gel electrophoresis and MALDI-TOF-MS. Feed deprivation increased total liver fat and the calcium content, as well as augmented AMPK phosphorylation, while it decreased the contents of protein, glucose, glycogen, and cholesterol when expressed as a percentage of dry matter. Among 34 differentially expressed proteins identified, we found downregulation of proteins associated with fatty acid oxidation, glycolysis, electron transfer, protein degradation, and antigen processing, as well as cytoskeletal rearrangement. Proteins upregulated after feed deprivation included enzymes of the urea cycle, fatty acid or cholesterol transport proteins, an inhibitor of glycolysis, and previously unknown changes in calcium signaling network. Direct correlation was found between expression of glycolytic enzymes and glucose/glycogen content, whereas inverse correlation exists between expression of beta-oxidative enzymes and total liver fat content. In conclusion, the regulatory response of identified proteins may help to explain development and consequences of hepatic lipidosis but also offers novel candidates potentially involved in signaling for maintaining energy homeostasis.

Effect of inulin supplementation on selected gastric, duodenal, and caecal microbiota and short chain fatty acid pattern in growing piglets.

We explored whether bifidobacteria and lactobacilli numbers and other selected bacteria in the upper intestine and the caecum of growing pigs were affected by diet and intake of inulin. Starting at two weeks after weaning (28 d) 72 pigs were fed two types of diets (wheat/barley (WB) or maize/gluten (MG)), without or with 3% inulin (WB + I, MG + I) for three and six weeks. Intestinal bacteria were quantified by fluorescence-in-situ-hybridization (n = 8/group). Duration of feeding had no effect on the variables tested, so data for both periods were pooled. Gastric total bacteria amounted to log(10) 7.4/g digesta. Bifidobacteria were detected in stomach and duodenum two weeks after weaning and disappeared thereafter. In jejunum and caecum bifidobacteria were present at a level of log(10) 7.0/g digesta. Inulin did not alter numbers of lactobacilli, bifidobacteria, enterococci, enterobacteria and bacteria of the Clostridium coccoides/Eubacterium rectale-group. Inulin disappearance in stomach plus jejunum was higher with the MG diet (73.7 vs. 60.7%, p = 0.013). Caecal acetate was lower in inulin-supplemented diets (p < 0.05) whereas propionate and butyrate were higher in pigs fed the WB diets (p < 0.05). With the WB diet total caecal short chain fatty acids concentration was higher which resulted in a lower pH value (p < 0.05).

Proteomics analysis of hypothalamic response to energy restriction in dairy cows.

The hypothalamus is the central regulatory unit that balances a number of body functions including metabolic rate, hunger, and satiety signals. Hypothalamic neurons monitor and respond to alterations of circulating nutrients and hormones that reflect the peripheral energy status. These extracellular signals are integrated within the cell at the ATP:AMP ratio and at the level of ROS, triggering gene expression associated with glucose and lipid metabolism. In order to identify new molecular factors potentially associated with the control of energy homeostasis, metabolic adaptation, and regulation of feed intake, hypothalami from ad libitum fed and energy restricted cows were characterized using 2-DE and MALDI-TOF-MS. Among 189 different protein spots identified, nine proteins were found to be differentially expressed between groups. Beside the 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase, stress-induced phosphoprotein-1, heat shock protein 70 kDa-protein-5, dihydropyrimidinase-related protein-2, [Cu-Zn]-superoxide dismutase, ubiquitin carboxy-terminal hydrolase-L1, and inorganic pyrophosphatase were found to be up-regulated, whereas glyceraldehyde 3-phosphate dehydrogenase and aconitase-2 were down-regulated in the restricted group. In conclusion, differentially expressed proteins are related to energy and nucleotide metabolism and cellular stress under conditions of dietary energy deficiency. These proteins may be new candidate molecules that are potentially involved in signaling for maintaining energy homeostasis.

Effects of dietary energy intake during gestation and lactation on milk yield and composition of first, second and fourth parity sows.

In order to determine the effects of a varied level of dietary energy intake during pregnancy and lactation on milk yield and composition, first, second and fourth parity sows (Large White x German Landrace) were provided with energy at a level of either: (i) 100% of ME requirement (MEreq) during pregnancy and lactation, (ii) 120% MEreq during pregnancy and 80% during lactation, and (iii) 80% MEreq during pregnancy and 120% during lactation. In spite of equal target levels feed analysis revealed that gestating first parity sows with 120/80 treatment combination and lactating sows of 80/120 treatment combination received 25, and 11-17% more digestible N than in the respective 100/100 treatment combination. Irrespective of this 120/80 sows responded with the highest milk DM, fat, and energy contents, and the lowest lactose concentrations whereas protein levels where not affected, irrespective of parity (p < 0.05). Milk yield of sows in 1st and 4th lactation was 85 and 106% of that in 2nd lactation, respectively. Average milk composition was 18.1% DM, 4.9% protein, 6.8% fat, 5.6% lactose, and 0.8% ash. Milk composition changes ceased at day 7 of lactation with a reduction of milk GE and protein, and an increase of lactose content. Concentrations of threonine, arginine, valine, leucine, tyrosine, phenylalanine, cystine, and tryptophan, as well as stearic, oleic, and linoleic acid were higher in colostrum than in milk at later lactation stages. In contrast, laurine, myristic, palmitic, and palmitoleic acids were lower concentrated in colostrum. In conclusion, these results illustrate the importance of body reserve mobilization for milk production in sows and indicate that low energy supply during gestation cannot be compensated by higher energy supply during lactation.

Inulin alters the intestinal microbiota and short-chain fatty acid concentrations in growing pigs regardless of their basal diet.

Inulin stimulates intestinal bifidobacteria in humans and rodents but its effect in pigs is inconsistent. We assessed the effect of inulin on the intestinal microbiota by fluorescent in situ hybridization in growing pigs (age 9-12 wk). Pigs (n = 64) were assigned to 2 types of basal diets [wheat and barley (WB) or corn and wheat gluten (CG)] with or without 3% inulin (WBI or CGI) for 3 and 6 wk (n = 8/group) to test whether naturally occurring dietary fibers influence the inulin effect. Intestinal organic acids, pH values, and residual inulin were determined. The composition of the microbiota was highly individual. The duration of feeding did not affect any of the variables tested; therefore, data for the 2 periods were pooled. Bifidobacteria were detected in less than half of the pigs. Inulin did not stimulate lactobacilli and bifidobacteria numbers irrespective of the basal diet, although 20-50% of inulin was degraded in the jejunum. The number of pigs with colonic bifidobacteria was higher in those fed diets containing inulin (40 vs. 13%; P < 0.05). Total colonic short-chain fatty acid (SCFA) concentrations were lower in both inulin-fed groups due to reduced acetate (P < 0.05). Proportions of colonic butyrate were higher in pigs fed inulin-supplemented diets (P < 0.05). Colonic pH tended to be lower in the WB groups (WB; 6.6 +/- 0.6), and was higher due to inulin (CGI, 7.1 +/- 0.1; P < 0.05). In conclusion, inulin affected intestinal SCFA and the number of pigs harboring bifidobacteria; this effect was independent of the basal diet.

Effect of partial dehulling of two- and six-row barley varieties on precaecal digestibility of amino acids in pigs.

Five barrows (German Landrace; initial BW 58 kg, final BW 80 kg) fitted with an ileo-rectal anastomosis were used to determine the effect of partial dehulling and addition of barley hulls of two- and six-row barley varieties on the precaecal digestibility (pD) of CP and amino acids. The following diets were provided according to a standardized diet formulation and tested in seven consecutive periods (repeated group-period design): two-row barley (TRB) + casein (C), dehulled TRB + C, TRB + C + 10% hulls, six-row barley (SRB) + C, dehulled SRB + C, SRB + C + 1% hulls, and wheat starch + C. The diets were supplied at daily rates of 79-86 g DMI x kg BW(-0.75) in barley containing diets and at 49 g DMI x kg BW(-0.75) in the casein diet. The digestibility of amino acids in barley varieties was determined by the difference method (casein as basal diet) using quantitative digesta collection. In both varieties of barley the pD of CP and amino acids did not differ. The pD of CP was unchanged in regard to the treatments in both barley varieties. Due to dehulling in TRB the pD was improved significantly for most indispensable amino acids and in SRB for Met and Cys. Addition of 10% hulls to TRB led to equivalent pD of Arg, His, Leu, Tyr, and Trp compared to TRB, but the pD of Lys, Phe, Thr and Val was significantly decreased below the levels of TRB. Addition of even 1% hulls to SRB impaired the pD of Lys below the level in SRB. In conclusion, addition of barley hulls to pig diets impairs amino acids absorption in the small intestine. The pD values, measured under standardized experimental conditions (without a correction using basal endogenous amino acids), are similar to the values of true digestibility published by NRC (1998).

Intraruminal infusion of n-butyric acid induces an increase of ruminal papillae size independent of IGF-1 system in castrated bulls.

The objective of this study was to explore morphological alterations of rumen papillae induced by n-butyric acid in relation to the insulin-like growth factor (IGF) system in adult castrated bulls. Three animals fitted with rumen cannula were fed twice daily at a low and high nutritional level (LL and HL), i.e., at 1.1 x maintenance (M) and 1.6 x M, respectively. Diets contained artificial dried grass and concentrate (74:26 and 52:48). Bulls received no (B0) or daily intraruminal infusions of 500 g n-butyric acid (B500) over 14 d. The infusion started 1 h after the morning feeding (9:00) and lasted for 3.5 h. Thus, four treatments (BOLL, B500LL, BOHL, and B500HL) were compared. Blood and rumen mucosa samples from the atrium ruminis were taken at the last day of each period. Length, width and surface of rumen papillae were greater (p < 0.001) in BOHL than in BOLL. Treatment with n-butyric acid resulted in an increase of the papillae surface of 20-40% (p = 0.047) for both nutritional levels as compared to periods without n-butyric acid treatments. The higher nutritional level and intraruminal n-butyric acid infusion induced epithelial cell death. The percentage of proliferative cells was doubled by n-butyric acid treatment. The mRNA of IGF-1 and IGF type 1 receptor (IGF-1R), as well as IGF-1R binding capacity were unaffected by butyric acid treatments. The abundance of IGF-1 mRNA tended to be lower (p = 0.1) and IGF-1R abundance was lower (p = 0.03) in response to the HL. The plasma IGF-1 concentration was lower with butyric acid treatment (p < 0.01), but was unaffected by the nutritional level. In conclusion, under described experimental preconditions of daily short-time intraruminal n-butyric acid infusion alterations of rumen papillae morphology is not mediated by ruminal IGF type 1 receptor and by local IGF-1 expression in papillae in castrated bulls.

An energy-rich diet causes rumen papillae proliferation associated with more IGF type 1 receptors and increased plasma IGF-1 concentrations in young goats.

We tested the hypothesis that the dietary energy-dependent alterations of the rumen papillae size are accompanied by corresponding changes in systemic insulin-like growth factor (IGF)-1 concentration and in rumen papillary IGF type 1 receptors (IGF-1R). Young male goats (n=24) were randomly allocated to two groups (n=12) and fed a high level (HL) metabolizable energy [1200 kJ/(kg(0.75).d)] or a low level (LL) [500 kJ/(kg(0.75).d)] diet for 42 d. The concentration of ruminal total SCFA did not differ between the groups, but the molar proportion of butyric acid was enhanced by 70% in the HL group (P<0.05). Both the length and width of the papillae were greater (P<0.05) in the HL group, and the surface was 50-100% larger (P<0.05) in the tissue sampled from the artrium ruminis, the ventral ruminal sac and the ventral blind sac. Transport of Na+ across the rumen epithelium, which is amiloride sensitive, was higher (P<0.05) in the HL than in the LL group. Furthermore, the plasma IGF-1 concentration was about twofold higher in the HL group (P<0.05), and the maximal rumen epithelial IGF-1R binding was also higher in the HL (P<0.05) than in the LL group. IGF-1R mRNA and IGF-1 mRNA were detected in rumen papillae; however, they were unaffected by dietary treatments. DNA synthesis and cell proliferation of cultured rumen epithelial cells were higher (P<0.05) after IGF-1 treatment (25 or 50 microg/L) compared with those in the medium without IGF-1. Thus dietary energy-dependent alterations of rumen morphology and function are accompanied by corresponding changes in systemic IGF-1 and ruminal IGF-1R.