Ketogenesis is the conversion of acetyl-CoA to the ketone bodies acetoacetate and beta-hydroxybutyrate (BHBA). In hepatic ketogenesis, which occurs during fasting in both nonruminant and ruminant animals, the source of acetyl-CoA is the mitochondrial oxidation of predominantly long-chain fatty acids. In the mature, fed ruminant animal, the ruminal epithelium is also capable of producing ketone bodies. In this case, the source of acetyl-CoA is the mitochondrial oxidation of butyrate produced by the microbial fermentation of feed. The purposes of this study were to determine ontogenic and dietary effects on ketogenic enzyme gene expression in developing lamb ruminal epithelium. Twenty-seven conventionally reared lambs and twenty-seven milk-fed lambs were slaughtered between 1 and 84 d of age. Six additional milk-fed lambs were weaned (the fed group) or maintained on milk replacer with a volatile fatty acid gavage (the VFA group) until 84 d of age. At slaughter, total RNA was extracted from samples of ruminal epithelium. The expression of the genes encoding acetoacetyl-CoA thiolase, the first enzyme in the ketogenic pathway, and 3-hydroxy-3-methylglutaryl-CoA synthase, the rate-limiting enzyme in the ketogenic pathway in nonruminant liver, were examined. Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase mRNA concentrations increased with age independent of diet. 3-Hydroxy-3-methylglutaryl-CoA synthase mRNA levels in ruminal epithelium obtained from milk-fed lambs were low before 42 d of age, but a marked increase occurred by 42 d of age. At 84 d of age, there were no differences in acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase expression due to diet. The pattern of the expression of these genes, in particular, 3-hydroxy-3-methylglutaryl-CoA synthase, parallels the rate of production of BHBA by rumen epithelial cells isolated from the same lambs, which increased to conventionally reared adult levels at 42 d of age and did not differ with diet. In conclusion, development of the ketogenic capacity of the ruminal epithelium occurs as the animal ages, regardless of dietary treatment. Thus, the expression of the genes encoding the ketogenic enzymes are not affected by the presence of VFA in the ruminal lumen.

This study examined the time course of rumen metabolic development in the absence of solid feed consumption and the effect of delayed solid feed consumption on sheep rumen development. Twenty-seven lambs consumed milk replacer until slaughter at nine ages from 1 to 84 d (milk group). Three additional lambs consumed milk replacer from 1 to 48 d. From 49 d until slaughter at 84 d, these lambs were weaned onto solid feed (fed group). At slaughter, rumen contents were removed for VFA analysis and rumen epithelium was preserved for morphological examination. Rumen epithelial cells were isolated and incubated in media containing 2.5 mM U-[14C]-glucose or 10 mM 1-[14C]-butyrate. Rumen VFA concentrations did not change with age in lambs given milk replacer. At 84 d of age, intraruminal VFA concentrations were elevated in lambs consuming solid feed compared to 84-d-old lambs given milk replacer (P < .05). The number of ruminal papillae per square centimeter decreased (P < .05) while papillae length and width did not change significantly with age in rumen epithelium from lambs given milk replacer. At 84 d of age, rumen epithelium from lambs in the fed group had fewer and larger papillae/per square centimeter than rumen epithelium from lambs given milk replacer (P < .05). Rates of glucose and butyrate oxidation and acetoacetate and lactate production by rumen cells isolated from lambs given milk replacer did not change with age. Beta-hydroxybutyrate (BHBA) production was undetectable before 42 d of age in lambs given milk replacer and increased to levels found in conventionally raised adults by 84 d. At 84 d there were no differences in rates of glucose and butyrate oxidation or acetoacetate and lactate production by rumen cells between the two treatment groups. Thus, the change in substrate oxidation from glucose to butyrate, indicative of rumen metabolic maturation, does not occur in the absence of solid feed consumption. However, the development of rumen ketogenesis, as evidenced by increased BHBA production, does occur in the absence of solid feed consumption. Delaying the initiation of solid feed consumption results in rumen morphological development but does not stimulate rumen metabolic development. Increased intraruminal VFA concentrations, earlier exposure to VFA, or a longer period of exposure to VFA may be required to induce the genes responsible for rumen metabolic development.

The purpose of this study was to determine whether the continuous intraruminal infusion of calculated physiological concentrations of volatile fatty acids (VFA) stimulated the metabolic development of the neonatal rumen. Eight 1-wk-old lambs were assigned to one of three treatments: saline infusion (three lambs), VFA infusion (three lambs), or no infusion (two lambs). Rumen catheters were surgically implanted into lambs in the infusion groups. The amount of VFA infused, beginning at 2 wk of age, increased weekly in equal increments of 12.5% of the estimated net energy requirement until, at 6 wk of age, lambs received 50% of their estimated net energy requirement from the infused VFA. All lambs consumed milk replacer for ad libitum intake and had free access to water. The lambs that were infused with VFA tended to have longer rumen papillae. There were no differences in width or number of papillae per square centimeter across treatments. Rumen epithelial cells isolated from lambs that were infused with VFA tended to oxidize less glucose and produce more acetoacetate than did cells from lambs that were infused with saline or from uninfused lambs. beta-Hydroxybutyrate production by isolated rumen epithelial cells and concentrations of blood glucose, acetoacetate, and beta-hydroxybutyrate were not different among the three treatments. Thus, infusion of physiological concentrations of VFA appears to stimulate some aspects of rumen metabolic development.

The ontogeny of glucose and butyrate metabolism in developing sheep ruminal epithelium was determined using an isolated ruminal cell system. Ruminal cells were isolated from 21 lambs at seven ages before weaning. Rumen weight increased in proportion to increases in body weight, except between 28 and 42 d, when rumen weight increased threefold, whereas body weight increased only 33%. Glucose oxidation rates [expressed as nmol/(1 x 10(6) ruminal cells.120 min)] by ruminal cells were low at birth (14.2 +/- 5.08), increased sharply by 14 d (71.38 +/- 16.71), and remained elevated until 42 d. Following 42 d, glucose oxidation declined to rates lower than those observed at birth (6.11 +/- 0.83). Butyrate oxidation to CO2 increased from birth (20.03 +/- 3.41) to a peak at 4 d (134.0 +/- 31.71) and decreased throughout the remainder of the preweaning period (56 d; 36.32 +/- 7.48). Butyrate inhibited glucose oxidation by ruminal cells isolated at 14, 28 and 42 d. Similarly, glucose inhibited butyrate oxidation by ruminal cells isolated from 4 d to 28 d following birth. beta-Hydroxybutyrate production [nmol/(1 x 10(6) ruminal cells.120 min)] from butyrate by ruminal cells was undetectable at birth, but measurable by 4 d (3.28 +/- 2.15). Rates of beta-hydroxybutyrate production remained unchanged through 42 d; however, by 56 d, production had increased 10-fold (36.71 +/- 0.67). The metabolic adaptations of the ruminal epithelium are intimately associated with the physical development of the tissue, and major shifts in the fate of glucose and butyrate carbon occur prior to weaning.

ABSTRACTcDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blothybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.The a-ketoacid dehydrogenase complexes-the pyruvate dehydrogenase multienzyme complex, the a-ketoglutarate dehydrogenase multienzyme complex, and the branchedchain a-ketoacid dehydrogenase multienzyme complexcatalyze the oxidative decarboxylation of pyruvate, aketoglutarate, and the branched-chain a-ketoacids, respectively (1-4). These three complexes occupy key positions in energy metabolism and are involved (i) in connecting glycolysis with the Krebs cycle, (ii) in the Krebs cycle itself, and (iii) in the regulation of the oxidation of branched-chain amino acids, respectively. Each complex consists of at least three catalytic components. The decarboxylase component, which has been designated E1, is specific for each complex, and it most likely represents the rate-limiting step in the overall reaction. The dihydrolipoamide acyltransferase component, designated E2, also substrate specific, is involved in the transfer of the acyl group to CoA. The third component of each complex, dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4), designated E3, is considered to be common to all three complexes. E3, a homodimer (subunit Mr = 55,000), contains a noncovalently bound molecule of FAD per subunit and catalyzes the reoxidation of the disulfhydryl form of the lipoyl residue bound to the E2 molecules of the complexes: E3 E2-Lip-(SH)2 + NAD+ <--E2-LiP(S)2 + NADH + H+ Three lines of evidence indicate that the E3 present in these three complexes is identical; they are reconstitution experiments (5), immunological cross-reactivity (6-9), and genetic disorders of E3, which cause simultaneous increases in the blood levels of pyruvate, a-ketoglutarate, and branched-chain ketoacids (10). The glycine cleavage system also contains a dihydrolipoamide dehydrogenase component known as L protein (11). However, this system has not been purified as an intact complex. We have recently suggested th...