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.

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.

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.

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.

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.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

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.