David M. Bremner

Effects of diet quality on urea fates in sheep as assessed by refined, non-invasive [15N15N]urea kinetics.

The effect of diet quality on urea production, entry into the gastrointestinal tract (GIT) and subsequent diversion to anabolic or catabolic fates was examined in four sheep (mean live weight 49.5 kg). The animals received, in a crossover design, each of two rations, hay-grass pellets (1:1 HG) and a mixed concentrate-forage (CF). Measurements were made of N balance and urea kinetics based on a 4 d continuous intravascular infusion of [15N15N]urea. Enrichments of [15N15N]- and [14N15N]urea in the urine, and faecal 15N content were determined each day. After 24 h of infusion, urinary [15N15N]urea enrichments reached constant enrichment but a further 24 h was required before [14N15N]urea enrichment was at plateau. The latter is derived from hydrolysis of urea to 15NH3 in the digestive tract with subsequent absorption and reconversion to urea. The diets were not isonitrogenous (14.3 v. 17.1 g N supplied daily for HG and CF respectively) but showed no difference in N balance. Urea-N production was much greater (16.3 v. 11.1 g/d; P = 0.011) for CF compared with HG and more urea-N entered the GIT (9.9 v. 7.7; P = 0.07). A larger proportion of GIT entry was returned to ureagenesis (51 v. 42%; P = 0.047) for the CF diet but a smaller fraction was lost in the faeces (3.3% v. 7.1%; P = 0.013). In consequence, most of the additional urea-N which entered the GIT on the CF diet was returned to the ornithine cycle (probably as NH3) and the absolute amount available for anabolic purposes was similar between the rations (3.9 v. 4.5 g N/d).

Oxidation of essential amino acids by the ovine gastrointestinal tract.

It is not known if the ruminant animal gastrointestinal tract (GIT) can oxidise essential amino acids (AA) other than leucine. Therefore, the oxidation of four essential AA (leucine, lysine, methionine and phenylalanine), supplied systemically as labelled 1-13C forms, was monitored across the mesenteric-drained viscera (MDV; small intestine) and portal-drained viscera (PDV; total GIT), as part of a Latin square design, in four wether sheep (35-45 kg) fed at 1.4 x maintenance. Oxidation was assessed primarily by appearance of 13CO2, corrected for sequestration of [13C]bicarbonate. The GIT contributed 25 % (P<0.001) and 10 % (P<0.05) towards whole-body AA oxidation for leucine and methionine respectively. This reduced net appearance across the PDV by 23 and 11 % respectively. The contribution of MDV metabolism to total PDV oxidation was 40 % for leucine and 60 % for methionine. There was no catabolism of systemic lysine or phenylalanine across the GIT. Production and exchange of secondary metabolites (e.g. 4-methyl-2-oxo-pentanoate, homocysteine, 2-aminoadipate) across the GIT was also limited. Less AA appeared across the PDV than MDV (P<0.001), indicative of use by tissues such as the forestomach, large intestine, spleen and pancreas. The PDV: MDV net appearance ratios varied (P<0.001) between AA, e.g. phenylalanine (0.81), lysine (0.71), methionine (0.67), leucine (0.56), histidine (0.71), threonine (0.63) and tryptophan (0.48). These differences probably reflect incomplete re-absorption of endogenous secretions and, together with the varied oxidative losses measured, will alter the pattern of AA net supply to the rest of the animal.

Effects of a high-protein, low-carbohydrate v . high-protein, moderate-carbohydrate weight-loss diet on antioxidant status, endothelial markers and plasma indices of the cardiometabolic profile

There are concerns that weight-loss (WL) diets based on very low carbohydrate (LC) intake have a negative impact on antioxidant status and biomarkers of cardiovascular and metabolic health. Obese men (n 16) participated in a randomised, cross-over design diet trial, with food provided daily, at approximately 8.3 MJ/d (approximately 70 % of energy maintenance requirements). They were provided with two high-protein diets (30 % of energy), each for a 4-week period, involving a LC (4 % carbohydrate) and a moderate carbohydrate (MC, 35 % carbohydrate) content. Body weight was measured daily, and weekly blood samples were collected. On average, subjects lost 6.75 and 4.32 kg of weight on the LC and MC diets, respectively (P < 0.001, SED 0.350). Although the LC and MC diets were associated with a small reduction in plasma concentrations of retinol, vitamin E (α-tocopherol) and β-cryptoxanthin (P < 0.005), these were still above the values indicative of deficiency. Interestingly, plasma vitamin C concentrations increased on consumption of the LC diet (P < 0.05). Plasma markers of insulin resistance (P < 0.001), lipaemia and inflammation (P < 0.05, TNF-α and IL-10) improved similarly on both diets. There was no change in other cardiovascular markers with WL. The present data suggest that a LC WL diet does not impair plasma indices of cardiometabolic health, at least within 4 weeks, in otherwise healthy obese subjects. In general, improvements in metabolic health associated with WL were similar between the LC and MC diets. Antioxidant supplements may be warranted if LC WL diets are consumed for a prolonged period.

Impact of Short Term Consumption of Diets High in Either Non-Starch Polysaccharides or Resistant Starch in Comparison with Moderate Weight Loss on Indices of Insulin Sensitivity in Subjects with Metabolic Syndrome

This study investigated if additional non-starch polysaccharide (NSP) or resistant starch (RS), above that currently recommended, leads to better improvement in insulin sensitivity (IS) than observed with modest weight loss (WL). Obese male volunteers (n = 14) were given an energy-maintenance (M) diet containing 27 g NSP and 5 g RS daily for one week. They then received, in a cross-over design, energy-maintenance intakes of either an NSP-enriched diet (42 g NSP, 2.5 g RS) or an RS-enriched diet (16 g NSP, 25 g RS), each for three weeks. Finally, a high protein (30% calories) WL diet was provided at 8 MJ/day for three weeks. During each dietary intervention, endogenous glucose production (EGP) and IS were assessed. Fasting glycaemia was unaltered by diet, but plasma insulin and C-peptide both decreased with the WL diet (p < 0.001), as did EGP (−11%, p = 0.006). Homeostatis model assessment of insulin resistance improved following both WL (p < 0.001) and RS (p < 0.05) diets. Peripheral tissue IS improved only with WL (57%–83%, p < 0.005). Inclusion of additional RS or NSP above amounts currently recommended resulted in little or no improvement in glycaemic control, whereas moderate WL (approximately 3 kg fat) improved IS.

Impact of high-protein diets with either moderate or low carbohydrate on weight loss, body composition, blood pressure and glucose tolerance in rats

One approach to achieve weight loss and decrease both obesity and associated morbidities involves high-protein, low-carbohydrate (HPLC) diets. This study compares the impact on metabolic health of HPLC and high-protein, medium-carbohydrate (HPMC) diets offered to diet-induced obese (DIO) rats. Weanling male rats were fed either a 37 % fat diet (n 48) or stock pellets (n 12) for 22 weeks. Rats fed the 37 % fat diet accumulated more body fat (26.6 versus 14.8 % body weight, P < 0.001) compared with those on stock diet. The DIO rats had higher systolic blood pressure (+6.6 mmHg, P = 0.002), fasting insulin (+63 % P = 0.006) and areas under the glucose (+21 %, P < 0.001) and insulin (+81 %, P < 0.001) curves following an oral glucose tolerance test. DIO rats were then separated into four groups and offered for 8 weeks either: (1) the 37 % fat diet; (2) an HPLC or (3) HPMC diet; or (4) fed the 37 % fat diet to the intake of the HPMC group. Rats offered the 37 % fat or HPLC diets gained while those on HPMC lost body fat. Blood pressure was not altered by the dietary switch. Both HPLC and HPMC rats had lowered fasting insulin (P = 0.027) and improved homeostatic assessment (HOMA; P = 0.011) that was not different from those of stock animals. These improvements occurred despite differences in fat gain, and indicate that both weight loss and macronutrient intake can impact favourably on obesity-associated morbidities.

Transfers of N metabolites across the ovine liver in response to short-term infusions of an amino acid mixture into the mesenteric vein

The effect of acute (4.5 h) infusions into the mesenteric vein of an amino acid (AA) mixture, which simulated the composition of rumen microbial protein, on net transfers of NH3, urea and total AA across the portal-drained viscera (PDV) and liver in the ovine has been examined. Four wether sheep were surgically prepared with vascular catheters across the PDV and liver (Lobley et al. 1995) and were offered a basal diet of 1000 g grass pellets/d (approximately 1.4 x energy maintenance). Each animal was infused at weekly intervals with one of four dilutions of the AA mixture. These dilutions provided 0.44, 0.88, 1.32 and 1.84 mmol AA-N/min infused, the lowest of which approximately doubled the net absorption of AA-N from the basal diet. Animals were treated with heparin to allow continuous collection of blood by peristaltic pump for 2 h preceding, and between 0.5-2.5 and 2.5-4.5 h after, the start of the AA infusions. Blood flow in the hepatic artery increased (100 v. 208 g/min; P = 0.002) in response to AA infusion, while hepatic portal venous flow decreased (2090 v. 1854 g/min; P = 0.006). The AA infusion also stimulated O2 uptake by the PDV (P < 0.001) and liver (P = 0.016). Absorption across the PDV and hepatic removal of NH3 were unchanged between basal and amino acid infusion conditions. Urea-N removal across the PDV was unaltered, but hepatic production increased (P < 0.001) with level of AA infusion. During infusions, net appearance of AA across the PDV was below the theoretical level. This may have been due to inhibition of AA uptake from the small intestine, and/or increased removal by the digestive tract of AA from the systemic circulation associated with greater arterial concentrations. Hepatic extraction of AA increased with level of infusion, both for total AA and those included in the infusate. Total hepatic urea-N production tended towards a maximum (estimated as 2 mumol N/g liver wet weight per min). The AA removed by the liver and not used for ureagenesis remained similar (170 mumol AA-N/min) between basal and AA infusions. This was presumed available for anabolic purposes (mainly synthesis of export proteins). The proportion of net AA-N appearance (absorption plus infused) across the PDV removed by the liver declined from 0.71 to 0.53 between basal and AA infusions. In contrast to findings from cattle (Wray-Cahen et al. 1997), increased AA infusion did not alter the net removal of glutamine across the liver. This may reflect differences between the studies in NH3: AA-N absorbed. Further differences between the cattle study and the current findings may relate to the different physiological state (pregnancy v. growth), which may alter the partition of AA between anabolic and catabolic fates.

Responses in gut hormones and hunger to diets with either high protein or a mixture of protein plus free amino acids supplied under weight-loss conditions

High-protein diets are an effective means for weight loss (WL), but the mechanisms are unclear. One hypothesis relates to the release of gut hormones by either protein or amino acids (AA). The present study involved overweight and obese male volunteers (n 18, mean BMI 36·8 kg/m2) who consumed a maintenance diet for 7 d followed by fully randomised 10 d treatments with three iso-energetic WL diets, i.e. with either normal protein (NP, 15% of energy) or high protein (HP, 30%) or with a combination of protein and free AA, each 15% of energy (NPAA). Psychometric ratings of appetite were recorded hourly. On day 10, plasma samples were taken at 30 min intervals over two consecutive 5 h periods (covering post-breakfast and post-lunch) and analysed for AA, glucose and hormones (insulin, total glucose-dependent insulinotropic peptide, active ghrelin and total peptide YY (PYY)) plus leucine kinetics (first 5 h only). Composite hunger was 16% lower for the HP diet than for the NP diet (P<0·01) in the 5 h period after both meals. Plasma essential AA concentrations were greatest within 60 min of each meal for the NPAA diet, but remained elevated for 3-5 h after the HP diet. The three WL diets showed no difference for either fasting concentrations or the postprandial net incremental AUC (net AUCi) for insulin, ghrelin or PYY. No strong correlations were observed between composite hunger scores and net AUCi for either AA or gut peptides. Regulation of hunger may involve subtle interactions, and a range of signals may need to be integrated to produce the overall response.

Effect of abomasal glucose infusion on alanine metabolism and urea production in sheep.

The effect of abomasal infusion of glucose (120 kJ/d per kg body weight (BW)0.75, 758 mmol/d) on urea production, plasma alanine-N flux rate and the conversion of alanine-N to urea was studied in sheep offered a low-N diet at limited energy intake (500 kJ/d per kg BW0.75), based on hay and grass pellets. Glucose provision reduced urinary N (P = 0.040) and urea (P = 0.009) elimination but this was offset by poorer N digestibility. Urea-N production was significantly reduced (822 v. 619 mmol/d, P = 0.024) by glucose while plasma alanine-N flux rate was elevated (295 v. 342 mmol/d, P = 0.011). The quantity of urea-N derived from alanine tended to be decreased by glucose (127 v. 95 mmol/d) but the fraction of urea production from alanine was unaltered (15%). Plasma urea and alanine concentrations (plus those of the branched chain amino acids) decreased in response to exogenous glucose, an effect probably related to enhanced anabolic usage of amino acids and lowered urea production.

Glutamine metabolism in ovine splanchnic tissues: effects of infusion of ammonium bicarbonate or amino acids into the abomasum.

This study investigates the effects of increased NH3 or amino acid supply on glutamine utilisation and production by the splanchnic tissues of fed sheep. Six sheep, prepared with vascular catheters in the aorta, mesenteric, portal and hepatic veins, were fed grass pellets to 1.1 x energy maintenance requirements. Each treatment involved a 4 d abomasal infusion, of either ammonium bicarbonate (AMM; 234 micromol/kg(0.75 per min), water (CONT), or a mixture of amino acids that excluded glutamine and glutamate (AA; 46.8 micromol amino acid-N/kg(0.75) per min). The treatments simulated nutritional extremes in terms of the balance of absorbed N. Kinetics across the whole gut and the liver were monitored during an intra-jugular infusion of [5-(15N)]glutamine. Blood flow across the whole gut or liver were unaffected by treatment. Both AMM and AA infusions doubled the hepatic release of urea-N compared with CONT (P<0.02). AA infusion decreased arterial glutamine concentration by 26% (P<0.01) and 23 % (P<0.05) compared with AMM and CONT respectively. Despite this, whole-body glutamine flux was not affected by treatment. In contrast, AMM infusion increased hepatic glutamine production by 40% compared with CONT (P<0.02). This provided a mechanism to ensure NH3 supply to the periphery was maintained within the normal low physiological levels. Hepatic glutamine utilisation tended to increase during AA infusion, probably to ensure equal inflows of N to the ornithine cycle. Between 6 and 10% of NH3 absorbed across the digestive tract was derived from the amido-N of glutamine. Overall, splanchnic glutamine utilisation accounted for 45-70% of whole-body glutamine flux.

Responses in whole-body amino acid kinetics to an acute, sub-clinical endotoxin challenge in lambs.

Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations.