Saturated fatty acids inhibit unsaturated fatty acid induced glucose uptake involving GLUT10 and aerobic glycolysis in bovine granulosa cells.

Fatty acids have been shown to modulate glucose metabolism in vitro and in vivo. However, there is still a need for substantial evidence and mechanistic understanding in many cell types whether both saturated and unsaturated fatty acids (SFAs and UFAs) pose a similar effect and, if not, what determines the net effect of fatty acid mixes on glucose metabolism. In the present study, we asked these questions by treating granulosa cells (GCs) with the most abundant non-esterified fatty acid species in bovine follicular fluid. Results revealed that oleic and alpha-linolenic acids (UFAs) significantly increased glucose consumption compared to palmitic and stearic acids (SFAs). A significant increase in lactate production, extracellular acidification rate, and decreased mitochondrial activity indicate glucose channeling through aerobic glycolysis in UFA treated GCs. We show that insulin independent glucose transporter GLUT10 is essential for UFA driven glucose consumption, and the induction of AKT and ERK signaling pathways necessary for GLUT10 expression. To mimic the physiological conditions, we co-treated GCs with mixes of SFAs and UFAs. Interestingly, co-treatments abolished the UFA induced glucose uptake and metabolism by inhibiting AKT and ERK phosphorylation and GLUT10 expression. These data suggest that the net effect of fatty acid induced glucose uptake in GCs is determined by SFAs under physiological conditions.

Heat stress and feeding effects on the mucosa-associated and digesta microbiome and their relationship to plasma and digesta fluid metabolites in the jejunum of dairy cows.

The intestinal microbiota plays a pivotal role in digestive processes and maintains gut health and intestinal homeostasis. These functions may be compromised by increased environmental heat which in turn reduces feed intake and gut integrity, while activating the intestinal immune system. It remains unknown whether high ambient temperatures, causing heat stress (HS) to dairy cows, disturb the eubiosis of the microbial community and if so, to which extent the reduction in feed intake and the impairment of circulating and intestinal metabolites account for the alterations of the jejunal microbiota. To address these questions, jejunal digesta, mucosa, and plasma samples from cows exposed to heat stress (HS: 28°C, temperature-humidity-index (THI) = 76, n = 10), control conditions (CON: 16°C, THI = 60, n = 10), or pair-feeding (PF: 16°C, THI = 60, n = 10) for 7 d were collected. Digesta fluids were examined for pH, acetate, nonesterified fatty acids (NEFA), glucose, and lactate, while plasma samples were analyzed for glucose, lactate, ß-hydroxybutyrate (BHB), triglycerides, NEFA, creatinine and urea. The microbiota of digesta and mucosa samples were analyzed by 16S rRNA sequencing. The α diversity was higher in mucosa than digesta, but not affected by high ambient temperatures. However, the mucosa-associated microbiota appears more responsive to ambient heat than the digesta microbiome. The adaptive responses under HS conditions comprised an increased mucosal abundance of Bifidobacteriaceae, Succinivibrionaceae UCG-001, Clostridia and Lactobacillus. In the digesta, HS has exerted effects on microbial abundance of Colidextribacter and Lachnospiraceae UCG-008. Several correlations between plasma or intestinal metabolites and microbiota were elucidated, including Methanobacteriaceae correlating positively with plasma BHB and digesta glucose concentrations. Moreover, the reduction in feed intake during HS had non-negligible effects on microbial diversity and the abundance of certain taxa, underpinning the importance of nutrient supply on maintaining intestinal homeostasis.

Acute and persistent effects of oral glutamine supplementation on growth, cellular proliferation, and tight junction protein transcript abundance in jejunal tissue of low and normal birthweight pre-weaning piglets.

Breeding for higher fertility has resulted in a higher number of low birthweight (LBW) piglets. It has been shown that LBW piglets grow slower than normal birthweight (NBW) littermates. Differences in growth performance have been associated with impaired small intestinal development. In suckling and weaning piglets, glutamine (Gln) supplementation has been associated with improved growth and intestinal development. This study was designed to examine the effects of oral Gln supplementation on growth and small intestinal parameters in LBW and NBW suckling piglets. At birth (day 0), a total of 72 LBW (1.10 ± 0.06 kg) and 72 NBW (1.51 ± 0.06) male piglets were selected. At day 1, litters were standardized to 12 piglets, and experimental piglets supplemented daily with either Gln (1 g/kg BW) or isonitrogenous amounts of Alanine (Ala) as control (1.22 g/kg BW) until day 12. Creep feed was offered from day 14 onward. Subgroups of piglets were euthanized at days 5, 12, and 26 for the analyses of jejunal morphometry, cellular proliferation, glutathione concentration and transcript abundance of tight junction proteins. From age day 11 to 21, Gln supplemented LBW (LBW-Gln) piglets were heavier than Ala supplemented LBW (LBW-Ala) littermates (P = 0.034), while NBW piglets were heavier until age day 26 compared to LBW littermates. Villus height was higher in LBW-Gln compared to LBW-Ala on age day 12 (P = 0.031). Sporadic differences among supplementation and birthweight groups were detected for jejunal cellular proliferation, cellular population and glutathione concentration, whereas age was the most dominant factor. These results show that Gln supplementation improved the growth of LBW piglets compared to LBW-Ala beyond the termination of Gln supplementation, but this was not associated with consistent effects on selected parameters of jejunal development.

Upcycling of recycled minerals from sewage sludge through black soldier fly larvae (Hermetia illucens): Impact on growth and mineral accumulation.

Phosphorous (P) resources are finite. Sewage sludge recyclates (SSR) are not only of interest as plant fertilizer but also as potential source of minerals in animal nutrition. However, besides P and calcium (Ca), SSR contain heavy metals. Under EU legislation, the use of SSR derivatives in animal feed is not permitted, but given the need to improve nutrient recycling, it could be an environmentally sound future mineral source. Black soldier fly larvae (BSFL) convert low-grade biomass into valuable proteins and lipids, and accumulate minerals in their body. It was hypothesized that BSFL modify and increase their mineral content in response to feeding on SSR containing substrates. The objective was to evaluate the upcycling of minerals from SSR into agri-food nutrient cycles through BSFL. Growth, nutrient and mineral composition were compared in BSFL reared either on a modified Gainesville fly diet (FD) or on FD supplemented with either 4% of biochar (FD + BCH) or 3.6% of single-superphosphate (FD + SSP) recyclate (n = 6 BSFL rearing units/group). Larval mass, mineral and nutrient concentrations and yields were determined, and the bioaccumulation factor (BAF) was calculated. The FD + SSP substrate decreased specific growth rate and crude fat of BSFL (P < 0.05) compared to FD. The FD + SSP larvae had higher Ca and P contents and yields but the BAF for Ca was lowest. The FD + BCH larvae increased Ca, iron, cadmium and lead contents compared to FD. Larvae produced on FD + SSP showed lower lead and higher arsenic concentration than on FD + BCH. Frass of FD + BCH had higher heavy metal concentration than FD + SSP and FD (P < 0.05). Except for cadmium and manganese, the larval heavy metal concentration was below the legally permitted upper concentrations for feed. In conclusion, the SSR used could enrich BSFL with Ca and P but at the expense of growth. Due to the accumulation of Cd and Mn, BSFL or products thereof can only be a component of farmed animal feed whereas in BSFL frass heavy metal concentrations remained below the upper limit authorized by EU.

Cryptosporidium parvumcompetes with the intestinal epithelial cells for glucose and impairs systemic glucose supply in neonatal calves.

Cryptosporidiosis is one of the main causes of diarrhea in children and young livestock. The interaction of the parasite with the intestinal host cells has not been characterized thoroughly yet but may be affected by the nutritional demand of the parasite. Hence, we aimed to investigate the impact of C. parvum infection on glucose metabolism in neonatal calves. Therefore, N = 5 neonatal calves were infected with C. parvum on the first day of life, whereas a control group was not (N = 5). The calves were monitored clinically for one week, and glucose absorption, turnover and oxidation were assessed using stable isotope labelled glucose. The transepithelial transport of glucose was measured using the Ussing chamber technique. Glucose transporters were quantified on gene and protein expression level using RT-qPCR and Western blot in the jejunum epithelium and brush border membrane preparations. Plasma glucose concentration and oral glucose absorption were decreased despite an increased electrogenic phlorizin sensitive transepithelial transport of glucose in infected calves. No difference in the gene or protein abundance of glucose transporters, but an enrichment of glucose transporter 2 in the brush border was observed in the infected calves. Furthermore, the mRNA for enzymes of the glycolysis pathway was increased indicating enhanced glucose oxidation in the infected gut. In summary, C. parvum infection modulates intestinal epithelial glucose absorption and metabolism. We assume that the metabolic competition of the parasite for glucose causes the host cells to upregulate their uptake mechanisms and metabolic machinery to compensate for the energy losses.

Effects of oral glutamine supplementation on jejunal morphology, development, and amino acid profiles in male low birth weight suckling piglets.

BACKGROUND: It has been shown that small intestine development in low birth weight (LBW) piglets is impaired. Glutamine (Gln) has been reported to improve piglet health and intestinal function in weaned piglets, but data is scarce in suckling piglets. This study was conducted to investigate the effects of oral Gln supplementation compared to Alanine (Ala) on jejunal development and function in 5 and 12 d old male LBW and normal birth weight (NBW) suckling piglets. RESULTS: Gln had no effect on the jejunal morphology, development, tissue and digesta amino acid profiles and mRNA abundance of genes involved in amino acid transport, metabolism, glutathione synthesis in LBW piglets when compared to Ala supplementation and birth weight controls at 5 and 12 d. Only the concentration of Gln in jejunal tissue was higher in NBW piglets supplemented with Gln compared to Ala at 5 d (P < 0.05). A comparison of the birth weight groups showed no differences between LBW and NBW piglets at 5 and 12 d in any parameter. Jejunal crypt depth, villus height / width, tunica muscularis thickness, number of goblet and IgA positive cells, the ratio of jejunal RNA to DNA and the concentration of DNA, protein and RNA changed (P < 0.05) from 5 compared to 12 d. The concentrations of several free, and protein bound amino acids as well as amino metabolites differed between age groups in jejunal tissue but the digesta concentrations were affected to a lesser extent. CONCLUSIONS: Oral Gln supplementation to suckling male piglets over the first 12 d of life was not associated with changes in jejunal parameters measured in this study. The absence of effects may indicate that Gln is absorbed as well as metabolized in the upper intestinal tract and thus could benefit intestinal development at a more proximal location.

Glutamine supplementation moderately affects growth, plasma metabolite and free amino acid patterns in neonatal low birth weight piglets.

Low birth weight (LBW) neonates show impaired growth compared with normal birth weight (NBW) neonates. Glutamine (Gln) supplementation benefits growth of weaning piglets, while the effect on neonates is not sufficiently clear. We examined the effect of neonatal Gln supplementation on piglet growth, milk intake and metabolic parameters. Sow-reared pairs of newborn LBW (0·8-1·2 kg) and NBW (1·4-1·8 kg) male piglets received Gln (1 g/kg body mass (BM)/d; Gln-LBW, Gln-NBW; n 24/group) or isonitrogenous alanine (1·22 g/kg BM/d; Ala-LBW; Ala-NBW; n 24/group) supplementation at 1-5 or 1-12 d of age (daily in three equal portions at 07:00, 12:00 and 17:00 by syringe feeding). We measured piglet BM, milk intake (1, 11-12 d), plasma metabolite, insulin, amino acid (AA) and liver TAG concentrations (5, 12 d). The Gln-LBW group had higher BM (+7·5%, 10 d, P = 0·066; 11-12 d, P < 0·05) and milk intake (+14·7%, P = 0·015) than Ala-LBW. At 5 d, Ala-LBW group had higher plasma TAG (+34·7%, P < 0·1) and lower carnosine (-22·5%, P < 0·05) than Ala-NBW and Gln-LBW, and higher liver TAG (+66·9%, P = 0·029) than Ala-NBW. At 12 d, plasma urea was higher (+37·5%, P < 0·05) with Gln than Ala supplementation. Several proteinogenic AA in plasma were lower (P < 0·05) in Ala-NBW v. Gln-NBW. Plasma arginine was higher (P < 0·05) in Gln-NBW v Ala-NBW piglets (5, 12 d). Supplemental Gln moderately improved growth and milk intake and affected lipid metabolism in LBW piglets and AA metabolism in NBW piglets, suggesting effects on intestinal and liver function.

The Effect of Dietary Protein Imbalance during Pregnancy on the Growth, Metabolism and Circulatory Metabolome of Neonatal and Weaned Juvenile Porcine Offspring.

Protein imbalance during pregnancy affects women in underdeveloped and developing countries and is associated with compromised offspring growth and an increased risk of metabolic diseases in later life. We studied in a porcine model the glucose and urea metabolism, and circulatory hormone and metabolite profile of offspring exposed during gestation, to maternal isoenergetic low-high (LP-HC), high-low (HP-LC) or adequate (AP) protein-carbohydrate ratio diets. At birth, LP-HC were lighter and the plasma acetylcarnitine to free carnitine ratios at 1 day of life was lower compared to AP offspring. Plasma urea concentrations were lower in 1 day old LP-HC offspring than HP-LC. In the juvenile period, increased insulin concentrations were observed in LP-HC and HP-LC offspring compared to AP, as was body weight from HP-LC compared to LP-HC. Plasma triglyceride concentrations were lower in 80 than 1 day old HP-LC offspring, and glucagon concentrations lower in 80 than 1 day old AP and HP-LC offspring. Plasma urea and the ratio of glucagon to insulin were lower in all 80 than 1 day old offspring. Aminoacyl-tRNA, arginine and phenylalanine, tyrosine and tryptophan metabolism, histidine and beta-alanine metabolism differed between 1 and 80 day old AP and HP-LC offspring. Maternal protein imbalance throughout pregnancy did not result in significant consequences in offspring metabolism compared to AP, indicating enormous plasticity by the placenta and developing offspring.

Jejunal mucosa proteomics unravel metabolic adaptive processes to mild chronic heat stress in dairy cows.

Climate change affects the duration and intensity of heat waves during summer months and jeopardizes animal health and welfare. High ambient temperatures cause heat stress in dairy cows resulting in a reduction of milk yield, feed intake, and alterations in gut barrier function. The objectives of this study were to investigate the mucosal amino acid, glucose and lactate metabolism, as well as the proteomic response of the small intestine in heat stressed (HS) Holstein dairy cows. Cows of the HS group (n = 5) were exposed for 4 days to 28 °C (THI = 76) in a climate chamber. Percentage decrease in daily ad libitum intake of HS cows was calculated to provide isocaloric energy intake to pair-fed control cows kept at 15 °C (THI = 60) for 4 days. The metabolite, mRNA and proteomic analyses revealed that HS induced incorrect protein folding, cellular destabilization, increased proteolytic degradation and protein kinase inhibitor activity, reduced glycolysis, and activation of NF-κB signaling, uronate cycling, pentose phosphate pathway, fatty acid and amino acid catabolism, mitochondrial respiration, ATPase activity and the antioxidative defence system. Our results highlight adaptive metabolic and immune mechanisms attempting to maintain the biological function in the small intestine of heat-stressed dairy cows.

Differences between Holstein dairy cows in renal clearance rate of urea affect milk urea concentration and the relationship between milk urea and urinary nitrogen excretion.

Urine and fecal excretions from cattle contribute to global nitrogen (N) emissions. The milk urea nitrogen (MUN) concentration in dairy cows is positively correlated with urinary urea N (UUN) emissions, and both decline with the reduction in crude protein intake. However, MUN concentration may differ between individual cows despite feeding the same ration. Thus, we hypothesized that due to differences in endogenous N utilization cows with high MUN concentration excrete more UUN than cows with a low MUN concentration. The objective of the present study was to elucidate N partitioning and urea metabolism in dairy cows with divergent MUN concentrations fed two planes of crude protein. Twenty Holstein dairy cows with high (HMU; n = 10) and low (LMU; n = 10) milk urea concentrations were fed two isocaloric diets with a low (LP) and normal (NP) crude protein level. Methane and ammonia emissions were recorded in respiration chambers. Feed intake, feces and urine excretions and milk yield were recorded for four days and subsamples were analyzed for total N and N-metabolites. A carbon-13 labeled urea bolus was administered intravenously followed by a series of plasma samplings. Total N and UUN excretions and ammonia emissions from excreta were lower on the LP diet, however, methane emissions, urinary N excretions and ammonia emissions were comparable between groups. Although plasma and salivary urea concentrations, urea pool size and urea turnover were higher, HMU cows had lower renal urea clearance rates. Additionally, HMU cows had lower renal clearance rates for creatinine, uric acid and creatine and excreted less uric acid (on the LP diet only) and creatine with urine. In conclusion, contrary to our hypothesis, HMU cows did not excrete more UUN than LMU cows. The lower urinary creatine excretion of HMU cows suggests that these animals have a lower environmental nitrogen footprint.

Effects of Oral Glutamine Supplementation on Early Postnatal Muscle Morphology in Low and Normal Birth Weight Piglets.

Adapted nutrition can improve the growth of low birth weight (LBW) piglets. Since maternal milk is thought to provide insufficient glutamine (Gln) for LBW piglets, the current study investigated the influence of Gln supplementation during the early suckling period on development and lipid deposition in skeletal muscle. The weight differences between LBW and normal birth weight (NBW) littermates persisted from birth to slaughter (p < 0.001). However, intramuscular Gln and Ala concentrations were altered in piglets according to the supplementation (p < 0.01). There were larger muscle fibers (p = 0.048) in Gln-supplemented piglets. Capillarization or nuclei number per muscle fiber was not influenced by birth weight (BiW) or Gln supplementation. Abundance of myosin heavy chain (MYH) isoforms was slightly altered by Gln supplementation. LBW piglets had more lipid droplets than NBW piglets at day 5 of life in both muscles (p < 0.01). The differences decreased with age. Adipocyte development increased with age, but was not influenced by BiW or supplementation. The results indicate that BiW differences were accompanied by differences in lipid deposition and muscle fiber structure, suggesting a delayed development in LBW piglets. Supplementation with Gln may support piglets to overcome those disadvantages.

The relationship between methane emission and daytime-dependent fecal archaeol concentration in lactating dairy cows fed two different diets.

Archaeol is a cell membrane lipid of methanogenic archaea excreted in feces and is therefore a potential biomarker for individual methane emission (MEM). The aims of this study were to examine the potential of the fecal archaeol concentration (fArch) to be a proxy for MEM prediction in cows fed different diets and determine if the time of fecal collection affected the archaeol concentration. Thus, we investigated (i) the variation of the fArch concentration in spot samples of feces taken thrice within 8 h during respiration chamber measurements and (ii) the effect of two diets differing in nutrient composition and net energy content on the relationship between fArch and MEM in lactating cows. Two consecutive respiration trials with four primiparous and six multiparous lactating Holstein cows were performed. In the first trial (T1) at 100 ± 3  d in milk (IM), a diet moderate in starch and fat content was fed for ad libitum intake, whereas in the second trial (T2) at 135 ± 3  d IM, cows received a diet lower in starch and fat. Individual MEM (g d - 1 ) was measured for 24 h. Fecal samples were taken at 06:30, 10:00, and 14:30 LT and analyzed for fArch using Soxhlet lipid extraction and GC-MS. Cows produced less methane (364 g  CH 4  d - 1 ) during T1 and had significantly lower fArch concentrations (37.1  µ g g - 1 dry matter; DM) compared to T2 (392 g  CH 4  d - 1 and 47.6  µ g g - 1  DM). A significant positive relationship between fArch ( µ g g - 1 fecal DM) and MEM, expressed on a dry matter intake (DMI) basis (g kg - 1  DMI), was found ( R 2 = 0.53 , n = 20 ). Among samples collected over the day, those collected at 10:00 LT provided the best coefficient of determination for MEM ( R 2 = 0.23 ). In conclusion, fArch offers some potential in serving as a proxy for innovative breeding schemes to lower enteric methane when fecal samples are taken at a certain time of the day, but more data on the sources of variation of the MEM  :  fArch ratios are required.

Oral exposure of pigs to the mycotoxin deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction.

The sensitivity of pigs to deoxynivalenol (DON) might be influenced by systemic inflammation (SI) which impacts liver. Besides following acute-phase proteins, our aim was to investigate both the hepatic fractional albumin (ALB) synthesis rate (FSR) and the ALB concentration as indicators of ALB metabolism in presence and absence of SI induced by LPS via pre- or post-hepatic venous route. Each infusion group was pre-conditioned either with a control diet (CON, 0.12 mg DON/kg diet) or with a DON-contaminated diet (DON, 4.59 mg DON/kg diet) for 4 wk. A depression of ALB FSR was observed 195 min after LPS challenge, independent of feeding group or LPS application route, which was not paralleled by a down-regulated ALB mRNA expression but by a reduced availability of free cysteine. The drop in ALB FSR only partly explained the plasma ALB concentrations which were more depressed in the DON-pre-exposed groups, suggesting that ALB levels are influenced by further mechanisms. The abundances of haptoglobin, C-reactive protein, serum amyloid A, pig major acute-phase protein, fibrinogen and LPS-binding protein mRNA were up-regulated upon LPS stimulation but not accompanied by increases in the plasma concentrations of these proteins, pointing at an imbalance between synthesis and consumption.

Substitution of Dietary Sulfur Amino Acids by dl-2-Hydroxy-4-Methylthiobutyric Acid Reduces Fractional Glutathione Synthesis in Weaned Piglets.

BACKGROUND: Cys is limiting for reduced glutathione (GSH) synthesis and can be synthesized from Met. We hypothesized that the dietary Met hydroxyl analogue dl-2-hydroxy-4-methylthiobutyric acid (dl-HMTBA) affects Cys and GSH metabolism and oxidative stress defense differently than Met. OBJECTIVE: The objective was to elucidate whether dl-HMTBA supplementation of a Met-deficient diet affects Cys flux, GSH fractional synthetic rate (FSR), and the basal oxidative stress level relative to Met supplementation in pigs. METHODS: Twenty-nine male German Landrace piglets aged 28 d were allocated to 3 dietary groups: a basal diet limiting in Met (69% of Met plus Cys requirement) supplemented with either 0.15% l-Met (LMET; n = 9), 0.15% dl-Met (DLMET; n = 11), or 0.17% dl-HMTBA (DLHMTBA; n = 9) on an equimolar basis. At age 54 d the pigs received a continuous infusion of [1-13C]-Cys to calculate Cys flux and Cys oxidation. After 3 d, GSH FSR was determined by [2,2-2H2]-glycine infusion, and RBC GSH and oxidized GSH concentrations were measured. At age 62 d the animals were killed to determine hepatic mRNA abundances of enzymes involved in GSH metabolism, GSH concentrations, and plasma oxidative stress defense markers. RESULTS: The Cys oxidation was 21-39% and Cys flux 5-15% higher in the fed relative to the feed-deprived state (P < 0.001). On average, GSH FSR was 49% lower (P < 0.01), and RBC GSH and total GSH concentrations were 12% and 9% lower, respectively, in DLHMTBA and DLMET relative to LMET pigs (P < 0.05). In the feed-deprived state, Gly flux, the GSH:oxidized glutathione (GSSG) ratio, RBC GSSG concentrations, plasma oxidative stress markers, and the hepatic GSH content did not differ between groups. CONCLUSIONS: Although GSH FSR was higher in LMET compared with DLMET or DLHMTBA feed-deprived pigs, these differences were not reflected by lower oxidative stress markers and antioxidant defense enzymes in LMET pigs.

Substitution of Dietary Sulfur Amino Acids by DL-2-hydroxy-4-Methylthiobutyric Acid Increases Remethylation and Decreases Transsulfuration in Weaned Piglets.

BACKGROUND: DL-2-hydroxy-4-methylthiobutyric acid (DL-HMTBA), an L-methionine (L-Met) hydroxyl analogue, has been suggested to be a dietary L-Met source. How dietary DL-HMTBA compared with L-Met affects whole-body L-Met kinetics in growing individuals is unknown. OBJECTIVES: We determined to what extent DL-HMTBA supplementation of an L-Met-deficient diet affects whole-body L-Met and L-cysteine (L-Cys) kinetics, protein synthesis (PS), and the L-Met incorporation rate in liver protein (L-MetInc) compared with L-Met and DL-Met supplementation in a piglet model. METHODS: Forty-five, 28-d-old weaned piglets (male, German Landrace) were allocated to 4 dietary groups: L-Met-deficient diet [Control: 69% of recommended L-Met plus L-Cys supply; 0.22% standardized ileal digestible (SID) L-Met; 0.27% SID L-Cys; n = 12] and Control diet supplemented equimolarly to 100% of recommended intake with either L-Met (n = 12; LMET), DL-Met (n = 11; DLMET), or DL-HMTBA (n = 10; DLHMTBA). At 47 d of age, the piglets were infused with L-[1-13C; methyl-2H3]-Met and [3,3-2H2]-Cys to determine the kinetics and PS rates. Plasma amino acid (AA) concentrations, hepatic mRNA abundances of L-Met cycle and transsulfuration (TS) enzymes, and L-MetInc were measured. RESULTS: During feed deprivation, L-Met kinetics did not differ between groups, and were ≤3 times higher in the fed state (P < 0.01). Remethylation (RM) was 31% and 45% higher in DLHMTBA than in DLMET and Control pigs, respectively, and the RM:transmethylation (TM) ratio was 50% higher in DLHMTBA than in LMET (P < 0.05). Furthermore, TS and the TS:TM ratio were 32% lower in DLHMTBA than in LMET (P < 0.05). L-MetInc was 42% lower in DLMET and DLHMTBA than in L-Met-deficient Control pigs, whereas plasma AA and hepatic mRNA abundances were similar among DL-HMTBA-, L-Met-, and DL-Met-supplemented pigs. CONCLUSIONS: In piglets, DL-HMTBA compared with L-Met and DL-Met supplementation increases RM and reduces the TS rate to conserve L-Met, but all 3 Met isomers support growth at a comparable rate.

Maternal high-fat diet during suckling programs visceral adiposity and epigenetic regulation of adipose tissue stearoyl-CoA desaturase-1 in offspring.

OBJECTIVE: The lactation-suckling period is critical for white adipose tissue (WAT) development. Early postnatal nutrition influences later obesity risk but underlying mechanisms remain elusive. Here, we tested whether altered postnatal nutrition specifically during suckling impacts epigenetic regulation of key metabolic genes in WAT and alter long-term adiposity set point. METHODS: We analyzed the effects of maternal high-fat (HF) feeding in rats exclusively during lactation-suckling on breast milk composition and its impact on male offspring visceral epidydimal (eWAT) and subcutaneous inguinal (iWAT) depots during suckling and in adulthood. RESULTS: Maternal HF feeding during lactation had no effect on mothers' body weight (BW) or global breast milk composition, but induced qualitative changes in breast milk fatty acid (FA) composition (high n-6/n-3 polyunsaturated FA ratio and low medium-chain FA content). During suckling, HF neonates showed increased BW and mass of both eWAT and iWAT depot but only eWAT displayed an enhanced adipogenic transcriptional signature. In adulthood, HF offspring were predisposed to weight gain and showed increased hyperplastic growth only in eWAT. This specific eWAT expansion was associated with increased expression and activity of stearoyl-CoA desaturase-1 (SCD1), a key enzyme of FA metabolism. SCD1 converts saturated FAs, e.g. palmitate and stearate, to monounsaturated FAs, palmitoleate and oleate, which are the predominant substrates for triglyceride synthesis. Scd1 upregulation in eWAT was associated with reduced DNA methylation in Scd1 promoter surrounding a PPARγ-binding region. Conversely, changes in SCD1 levels and methylation were not observed in iWAT, coherent with a depot-specific programming. CONCLUSIONS: Our data reveal that maternal HF feeding during suckling programs long-term eWAT expansion in part by SCD1 epigenetic reprogramming. This programming events occurred with drastic changes in breast milk FA composition, suggesting that dietary FAs are key metabolic programming factors in the early postnatal period.

Breath water-based doubly labelled water method for the noninvasive determination of CO2 production and energy expenditure in mice.

We explored a novel doubly labelled water (DLW) method based on breath water (BW-DLW) in mice to determine whole body CO2 production and energy expenditure noninvasively. The BW-DLW method was compared to the DLW based on blood plasma. Mice (n = 11, 43.5 ± 4.6 g body mass (BM)) were administered orally a single bolus of doubly labelled water (1.2 g H218O kg BM-1 and 0.4 g 2H2O kg BM-1, 99 atom% (AP) 18O or 2H). To sample breath water, the mice were placed into a respiration vessel. The exhaled water vapour was condensed in a cold-trap. The isotope enrichments of breath water were compared with plasma samples. The 2H/1H and 18O/16O isotope ratios were measured by means of isotope ratio mass spectrometry. The CO2 production (RCO2) was calculated from the 2H and 18O enrichments in breath water and plasma over 5 days. The isotope enrichments of breath water vs. plasma were correlated (R2 = 0.89 for 2H and 0.95 for 18O) linearly. The RCO2 determined based on breath water and plasma was not different (113.2 ± 12.7 vs. 111.4 ± 11.0 mmol d-1), respectively. In conclusion, the novel BW-DLW method is appropriate to obtain reliable estimates of RCO2 avoiding blood sampling.

Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine.

Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin (CGP), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase (CGPase) and results in the release of ß-aspartic acid (Asp)-Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB coding region from Thermosynechococcus elongatus BP-1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant-expressed CGP fed in combination with plant-made CGPase was hydrolysed in the intestine, and high levels of ß-Asp-Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGPase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes.

Early postnatal feed restriction reduces liver connective tissue levels and affects H3K9 acetylation state of regulated genes associated with protein metabolism in low birth weight pigs.

Intrauterine growth retardation is associated with metabolic consequences in adulthood. Since our previous data indicate birth weight-dependent effects of feed restriction (R) on protein degradation processes in the liver, it should be investigated whether effects on connective tissue turnover are obvious and could be explained by global changes of histone H3K9me3 and H3K9ac states in regulated genes. For this purpose, female littermate pigs with low (U) or normal (N) birth weight were subjected to 3-week R (60% of ad libitum fed controls) with subsequent refeeding (REF) for further 5 weeks. The 3-week R-period induced a significant reduction of connective tissue area by 43% in the liver of U animals at 98 d of age, which was not found in age-matched N animals. Of note, after REF at 131 d of age, in previously feed-restricted U animals (UR), the percentage of mean connective tissue was only 53% of ad libitum fed controls (UK), indicating a persistent effect. In U animals, R induced H3K9 acetylation of regulated genes (e.g. XBP1, ERLEC1, GALNT2, PTRH2), which were inter alia associated with protein metabolism. In contrast, REF was mostly accompanied by deacetylation in U and N animals. Thus, our epigenetic data may give a first explanation for the observed birth weight-dependent differences in this connective tissue phenotype.

The Effects of Oral Quercetin Supplementation on Splanchnic Glucose Metabolism in 1-Week-Old Calves Depend on Diet after Birth.

BACKGROUND: Inadequate colostrum supply results in insufficient intake of macronutrients and bioactive factors, thereby impairing gastrointestinal development and the maturation of glucose metabolism in neonatal calves. The flavonoid quercetin has been shown to have health-promoting properties, including effects in diabetic animals. However, quercetin interacts with intestinal glucose absorption and might therefore exert negative effects in neonates. OBJECTIVE: We evaluated the interaction between neonatal diet and quercetin feeding on splanchnic glucose metabolism in neonatal calves. METHODS: Calves (n = 28) were assigned to 4 groups and fed either colostrum or a milk-based formula on days 1 and 2 and supplemented daily with 148 µmol quercetin aglycone/kg body weight [colostrum with quercetin (CQ+)/formula with quercetin (FQ+)] or without this substance [colostrum without quercetin (CQ-)/formula with quercetin (FQ-)] from days 2-8. From day 3 onward, all calves received milk replacer. A xylose absorption test was performed on day 3, and on day 7, blood samples were collected to study glucose first-pass uptake after [(13)C6]-glucose feeding and intravenous [6,6-(2)H2]-glucose bolus injection. Plasma concentrations of metabolites and hormones were measured by taking additional blood samples. A biopsy specimen of the liver was harvested on day 8 to measure the mRNA expression of gluconeogenic enzymes. RESULTS: Higher postprandial plasma concentrations of glucose, lactate, urea, adrenaline, noradrenaline, insulin, and glucagon on day 7 in colostrum-fed calves indicate that metabolic processes were stimulated. Postabsorptive xylose and glucose plasma concentrations each increased by an additional 26%, and splanchnic glucose turnover decreased by 35% in colostrum-fed calves, suggesting improved glucose absorption and lower splanchnic glucose utilization in colostrum-fed calves. Quercetin supplementation resulted in higher noradrenaline concentrations and enhanced peak absorption and oxidation of [(13)C6]-glucose by 10%. Liver mitochondrial phosphoenolpyruvate carboxykinase mRNA abundance was reduced by 34% in colostrum-deprived calves. CONCLUSIONS: Feeding colostrum during the first 2 d of life is crucial for maturation of splanchnic glucose metabolism in calves. Supplementing quercetin improves gastrointestinal absorption capacity, particularly in colostrum-deprived calves.