H.M. Hammon

Gluconeogenesis in dairy cows: the secret of making sweet milk from sour dough.

Gluconeogenesis is a crucial process to support glucose homeostasis when nutritional supply with glucose is insufficient. Because ingested carbohydrates are efficiently fermented to short‐chain fatty acids in the rumen, ruminants are required to meet the largest part of their glucose demand by de novo genesis after weaning. The qualitative difference to nonruminant species is that propionate originating from ruminal metabolism is the major substrate for gluconeogenesis. Disposal of propionate into gluconeogenesis via propionyl‐CoA carboxylase, methylmalonyl‐CoA mutase, and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) has a high metabolic priority and continues even if glucose is exogenously supplied. Gluconeogenesis is regulated at the transcriptional and several posttranscriptional levels and is under hormonal control (primarily insulin, glucagon, and growth hormone). Transcriptional regulation is relevant for regulating precursor entry into gluconeogenesis (propionate, alanine and other amino acids, lactate, and glycerol). Promoters of the bovine pyruvate carboxylase (PC) and PEPCK genes are directly controlled by metabolic products. The final steps decisive for glucose release (fructose 1,6‐bisphosphatase and glucose 6‐phosphatase) appear to be highly dependent on posttranscriptional regulation according to actual glucose status. Glucogenic precursor entry, together with hepatic glycogen dynamics, is mostly sufficient to meet the needs for hepatic glucose output except in high‐producing dairy cows during the transition from the dry period to peak lactation. Lactating cows adapt to the increased glucose requirement for lactose production by mobilization of endogenous glucogenic substrates and increased hepatic PC expression. If these adaptations fail, lipid metabolism may be altered leading to fatty liver and ketosis. Increasing feed intake and provision of glucogenic precursors from the diet are important to ameliorate these disturbances. An improved understanding of the complex mechanisms underlying gluconeogenesis may further improve our options to enhance the postpartum health status of dairy cows.

Tumor necrosis factor-α and nitrite/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and of NO(x) (sum of nitrite and nitrate as indicators of endogenous nitric oxide production) in milk and blood plasma were measured in three mastitis models in dairy cows in early lactation. Escherichia coli P4:O37 bacteria or endotoxin O111:B4 were administered into both left quarters of 12 and 6 cows, respectively. Six of the E. coli-infected cows were treated with a bactericidal antibiotic (Enrofloxacin; Bayer AG, Leverkusen, Germany) i.v. at 10 hr and subcutaneously (sc) at 30 hr after infection. NO(x) concentrations transiently increased maximally 10- to 11-fold in milk of E. coli-infected quarters with or without antibiotic treatment at 24 hr and after endotoxin administration. NO(x) concentrations did not change in milk of unchallenged quarters and in blood plasma. Increases of NO(x) were proceeded by a transient (96- to 149-fold) rise of milk TNF-alpha concentrations, which in endotoxin-administered quarters was maximal at 6 hr and in infected quarters without or with Enrofloxacin treatment at 10 and 14 hr. In blood plasma TNF-alpha concentrations only moderately increased to peaks in endotoxin-administered cows at 6 hr and in E. coli-infected cows at 14 hr postchallenge. In one severely sick, nontreated E. coli-infected cow milk, TNF-alpha response at 14 hr was excessive and followed by a spectacular rise of NO(x) concentration in milk between 48 and 72 hr. In conclusion, a possible clinical relevance of nitric oxide production associated with a rise of intramammary and systemic TNF-alpha during acute mastitis by E. coli infection and endotoxin in lactating dairy cows is indicated, but could not be inhibited by antibiotic treatment.

Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows

Fat mobilization to meet energy requirements during early lactation is inevitable because of insufficient feed intake, but differs greatly among high-yielding dairy cows. Therefore, we studied milk production, feed intake, and body condition as well as metabolic and endocrine changes in high-yielding dairy cows to identify variable strategies in metabolic and endocrine adaptation to overcome postpartum metabolic load attributable to milk production. Cows used in this study varied in fat mobilization around calving, as classified by mean total liver fat concentrations (LFC) postpartum. German Holstein cows (n=27) were studied from dry off until d 63 postpartum in their third lactation. All cows were fed the same total mixed rations ad libitum during the dry period and lactation. Plasma concentrations of metabolites and hormones were measured in blood samples taken at d 56, 28, 15, and 5 before expected calving and at d 1 and once weekly up to d 63 postpartum. Liver biopsies were taken on d 56 and 15 before calving, and on d 1, 14, 28, and 49 postpartum to measure LFC and glycogen concentrations. Cows were grouped accordingly to mean total LFC on d 1, 14, and 28 in high, medium, and low fat-mobilizing cows. Mean LFC (±SEM) differed among groups and were 351±14, 250±10, and 159±9 mg/g of dry matter for high, medium, and low fat-mobilizing cows, respectively, whereas hepatic glycogen concentrations postpartum were the highest in low fat-mobilizing cows. Cows in the low group showed the highest dry matter intake and the least negative energy balance postpartum, but energy-corrected milk yield was similar among groups. The decrease in body weight postpartum was greatest in high fat-mobilizing cows, but the decrease in backfat thickness was greatest in medium fat-mobilizing cows. Plasma concentrations of nonesterified fatty acids and β-hydroxybutyrate were highest around calving in high fat-mobilizing cows. Plasma triglycerides were highest in the medium group and plasma cholesterol concentrations were lowest in the high group at calving. During early lactation, the decrease in plasma glucose concentrations was greatest in the high group, and plasma insulin concentrations postpartum were highest in the low group. The revised quantitative insulin sensitivity check index values decreased during the transition period and postpartum, and were highest in the medium group. Plasma cortisol concentrations during the transition period and postpartum period and plasma leptin concentrations were highest in the medium group. In conclusion, cows adapted differently to the metabolic load and used variable strategies for homeorhetic regulation of milk production. Differences in fat mobilization were part of these strategies and contributed to the individual adaptation of energy metabolism to milk production.

Metabolism and lactation performance in dairy cows fed a diet containing rumen-protected fat during the last twelve weeks of gestation.

Effects of dietary fat supplementation prepartum on liver lipids and metabolism in dairy cows are contradictory. Thus, we examined in 18 German Holstein cows (half-sib; first lactation 305-d milk yield >9,000 kg) whether dietary fat:carbohydrate ratio during the last trimester of gestation affects lipid metabolism and milk yield. The diets were formulated to be isoenergetic and isonitrogenous but differed in rumen-protected fat (FD; 28 and 46.5 g/kg of dry matter during far-off and close-up dry period; mainly C16:0 and C18:1) and starch concentration [carbohydrate diet (CD); 2.3 times as much starch as FD]. Diets were given ad libitum starting 12 wk before expected parturition. After parturition all cows were fed a single lactation diet ad libitum for 14 wk. With the FD treatment, dry matter intake was depressed prepartum, milk yield during first 4 wk of lactation was lower (36.9 vs. 41.0 kg/d), and postpartum energy balance during this period was more negative. During the first 4 wk, cows in the FD group had lower lactose percentage and yield but higher milk fat, whereas milk protein and fat yield as well as energy-corrected milk did not differ. Between wk 5 and 14, milk fat and milk protein percentage was lower in CD than in FD. Milk fat C14:0 was lower and C16:1 was higher in the FD group. For FD cows, plasma triacylglycerol, nonesterified fatty acids, and cholesterol concentrations were higher prepartum, whereas plasma beta-hydroxybutyrate and glucose concentrations were lower. During the first 10 d after parturition, plasma triacylglycerol concentration was higher in FD, and prepartum plasma glucose and cholesterol differences persisted during the first 14 wk of lactation. Irrespective of prepartum nutrient composition, concentrations of plasma leptin and subcutaneous fat leptin mRNA decreased between -10 d to +10 d relative to parturition, and liver lipids and glycogen reached maximum and minimal values, respectively, 10 d after parturition. Acetyl-coenzyme A carboxylase alpha mRNA abundance in subcutaneous fat decreased between -10 d to +1 d relative to parturition by 97%, whereas it was generally much lower in the liver and remained at a low level until wk 14 of lactation. In conclusion, feeding a diet containing rumen-protected fat during late lactation and dry period until calving negatively affected dry matter intake, energy balance, and milk yield during subsequent lactation, did not change acetyl-coenzyme A carboxylase alpha mRNA abundance in subcutaneous fat, and was not beneficial for liver lipid accumulation.

Metabolomic profiles indicate distinct physiological pathways affected by two loci with major divergent effect on Bos taurus growth and lipid deposition

Identifying trait-associated genetic variation offers new prospects to reveal novel physiological pathways modulating complex traits. Taking advantage of a unique animal model, we identified the I442M mutation in the non-SMC condensin I complex, subunit G (NCAPG) gene and the Q204X mutation in the growth differentiation factor 8 (GDF8) gene as substantial modulators of pre- and/or postnatal growth in cattle. In a combined metabolomic and genotype association approach, which is the first respective study in livestock, we surveyed the specific physiological background of the effects of both loci on body-mass gain and lipid deposition. Our data provided confirming evidence from two historically and geographically distant cattle populations that the onset of puberty is the key interval of divergent growth. The locus-specific metabolic patterns obtained from monitoring 201 plasma metabolites at puberty mirror the particular NCAPG I442M and GDF8 Q204X effects and represent biosignatures of divergent physiological pathways potentially modulating effects on proportional and disproportional growth, respectively. While the NCAPG I442M mutation affected the arginine metabolism, the 204X allele in the GDF8 gene predominantly raised the carnitine level and had concordant effects on glycerophosphatidylcholines and sphingomyelins. Our study provides a conclusive link between the well-described growth-regulating functions of arginine metabolism and the previously unknown specific physiological role of the NCAPG protein in mammalian metabolism. Owing to the confirmed effect of the NCAPG/LCORL locus on human height in genome-wide association studies, the results obtained for bovine NCAPG might add valuable, comparative information on the physiological background of genetically determined divergent mammalian growth.

mRNA of insulin-like growth factor (IGF) quantification and presence of IGF binding proteins, and receptors for growth hormone, IGF-I and insulin, determined by reverse transcribed polymerase chain reaction, in the liver of growing and mature male cattle

Plasma insulin-like growth factor-I (IGF-I) concentrations were related to hepatic levels of IGF-I mRNA measured by competitive reverse transcription polymerase chain reaction (PCR) (RT-PCR) in neonatal (8 d old) calves, veal calves, fattened castrated bulls and mature intact bulls. Furthermore, the presence of mRNAs of IGF-II and of receptors for IGF-I (IGF-IR), growth hormone (GHR) and insulin (IR), as well as mRNAs of IGF binding proteins (IGFBP-1, -2 and -3) were assessed by RT-PCR. Hepatic IGF-I mRNA levels and plasma IGF-I concentrations in veal calves, fattened castrated bulls and in intact bulls were 4 to 8 times higher than in 8-d old calves and were 2 to 3 times higher in calves fed colostrum than in calves fed only milk replacer. Hepatic IGF-I mRNA concentrations were closely correlated (r = 0.92) with plasma IGF-I concentrations, suggesting that hepatic IGF-I production largely determines plasma IGF-I levels. The presence of IGF II, IGF-IR, GHR, IR and IGFBP-1, -2 and -3 mRNA was confirmed in the liver of 8-d old calves and older cattle as well, and among newborn calves their presence was independent of differences in nutrition. In conclusion, the major hepatic components of the GH-IGF axis were present in neonatal calves, but the IGF-I expression and therefore also plasma IGF-I levels were relatively low.

Energy metabolism in the newborn farm animal with emphasis on the calf: endocrine changes and responses to milk-born and systemic hormones

Neonatal mammals need adaption to changes in nutrient supply because energy intake shifts from continuous parenteral supply of nutrients (mainly glucose, lactate, and amino acids) via the placenta to discontinuous colostrum and milk intake with lactose and fat as main energy sources. Besides ingested lactose, endogenous glucose production is essential in the neonate to assure sufficient glucose availability. Fetal endogenous glucose production is low, but endocrine changes (especially the prenatal rise of glucocorticoid production) promote maturation of metabolic pathways that enable marked glycogen synthesis before and enhanced gluconeogenesis after birth to establish an adequate glucose status during postnatal maturation. In preterm born farm animals gluconeogenic activity is low, mainly because of a low glucocorticoid and thyroid status. In full-term neonates, endogenous glucose production increases with age. Colostral bioactive components (such as growth factors, hormones, bioactive peptides, and cytokines) do not have a direct effect on endogenous glucose production. However, colostrum feeding stimulates intestinal growth and development, an effect at least in part mediated by bioactive substances. Increased nutrient and glucose absorption thus allows increased glucose supply and hepatic glycogen storage, which improves the glucose status. The improved energetic status of colostrum-fed neonates is reflected by an accelerated maturation of the somatotropic axis, leading especially to enhanced production of IGF-I in the neonate. Secretion and production of hormones involved in the regulation of glucose and fat metabolism in neonates depend on the developmental stage and the response to feeding. In addition, many such hormones have actions in the neonate that differ from adult animals. Endocrine action to support endogenous energy supply in neonates is probably not fully established, and therefore, needs postnatal maturation. Therefore, our knowledge on energy metabolism in the neonate needs to be extended to better understand the function and the failure and to assess endocrine responses during the neonatal period.

Involvement of skeletal muscle protein, glycogen, and fat metabolism in the adaptation on early lactation of dairy cows.

During early lactation, high-yielding dairy cows cannot consume enough feed to meet nutrient requirements. As a consequence, animals drop into negative energy balance and mobilize body reserves including muscle protein and glycogen for milk production, direct oxidation, and hepatic gluconeogenesis. To examine which muscle metabolic processes contribute to the adaptation during early lactation, six German Holstein cows were blood sampled and muscle biopsied throughout the periparturient period. From pregnancy to lactation, the free plasma amino acid pattern imbalanced and plasma glucose decreased. Several muscle amino acids, as well as total muscle protein, fat, and glycogen, and the expression of glucose transporter-4 were reduced within the first 4 weeks of lactation. The 2-DE and MALDI-TOF-MS analysis identified 43 differentially expressed muscle protein spots throughout the periparturient period. In early lactation, expression of cytoskeletal proteins and enzymes involved in glycogen synthesis and in the TCA cycle was decreased, whereas proteins related to glycolysis, fatty acid degradation, lactate, and ATP production were increased. On the basis of these results, we propose a model in which the muscle breakdown in early lactation provides substrates for milk production by a decoupled Cori cycle favoring hepatic gluconeogenesis and by interfering with feed intake signaling.

Intestinal Glucose Absorption but Not Endogenous Glucose Production Differs between Colostrum- and Formula-Fed Neonatal Calves

Glucose supply markedly changes during the transition to extrauterine life. In this study, we investigated diet effects on glucose metabolism in neonatal calves. Calves were fed colostrum (C; n = 7) or milk-based formula (F; n = 7) with similar nutrient content up to d 4 of life. Blood plasma samples were taken daily before feeding and 2 h after feeding on d 4 to measure glucose, lactate, nonesterified fatty acids, protein, urea, insulin, glucagon, and cortisol concentrations. On d 2, additional blood samples were taken to measure glucose first-pass uptake (FPU) and turnover by oral [U-(13)C]-glucose and i.v. [6,6-(2)H(2)]-glucose infusion. On d 3, endogenous glucose production and gluconeogenesis were determined by i.v. [U-(13)C]-glucose and oral deuterated water administration after overnight feed deprivation. Liver tissue was obtained 2 h after feeding on d 4 and glycogen concentration and activities and mRNA abundance of gluconeogenic enzymes were measured. Plasma glucose and protein concentrations and hepatic glycogen concentration were higher (P < 0.05), whereas plasma urea, glucagon, and cortisol (d 2) concentrations as well as hepatic pyruvate carboxylase mRNA level and activity were lower (P < 0.05) in group C than in group F. Orally administered [U-(13)C]-glucose in blood was higher (P < 0.05) but FPU tended to be lower (P < 0.1) in group C than in group F. The improved glucose status in group C resulted from enhanced oral glucose absorption. Metabolic and endocrine changes pointed to elevated amino acid degradation in group F, presumably to provide substrates to meet energy requirements and to compensate for impaired oral glucose uptake.

A systems biology approach using metabolomic data reveals genes and pathways interacting to modulate divergent growth in cattle

BackgroundSystems biology enables the identification of gene networks that modulate complex traits. Comprehensive metabolomic analyses provide innovative phenotypes that are intermediate between the initiator of genetic variability, the genome, and raw phenotypes that are influenced by a large number of environmental effects. The present study combines two concepts, systems biology and metabolic analyses, in an approach without prior functional hypothesis in order to dissect genes and molecular pathways that modulate differential growth at the onset of puberty in male cattle. Furthermore, this integrative strategy was applied to specifically explore distinctive gene interactions of non-SMC condensin I complex, subunit G (NCAPG) and myostatin (GDF8), known modulators of pre- and postnatal growth that are only partially understood for their molecular pathways affecting differential body weight.ResultsOur study successfully established gene networks and interacting partners affecting growth at the onset of puberty in cattle. We demonstrated the biological relevance of the created networks by comparison to randomly created networks. Our data showed that GnRH (Gonadotropin-releasing hormone) signaling is associated with divergent growth at the onset of puberty and revealed two highly connected hubs, BTC and DGKH, within the network. Both genes are known to directly interact with the GnRH signaling pathway. Furthermore, a gene interaction network for NCAPG containing 14 densely connected genes revealed novel information concerning the functional role of NCAPG in divergent growth.ConclusionsMerging both concepts, systems biology and metabolomic analyses, successfully yielded new insights into gene networks and interacting partners affecting growth at the onset of puberty in cattle. Genetic modulation in GnRH signaling was identified as key modifier of differential cattle growth at the onset of puberty. In addition, the benefit of our innovative concept without prior functional hypothesis was demonstrated by data suggesting that NCAPG might contribute to vascular smooth muscle contraction by indirect effects on the NO pathway via modulation of arginine metabolism. Our study shows for the first time in cattle that integration of genetic, physiological and metabolomics data in a systems biology approach will enable (or contribute to) an improved understanding of metabolic and gene networks and genotype-phenotype relationships.