Monika Röntgen

Increased anaplerosis, TCA cycling, and oxidative phosphorylation in the liver of dairy cows with intensive body fat mobilization during early lactation.

The onset of milk production lets mammals experience an enormous energy and nutrient demand. To meet these requirements, high-yielding dairy cows mobilize body fat resulting in an augmented hepatic oxidative metabolism, which has been suggested to signal for depressing hunger after calving. To examine how the extent of fat mobilization influences hepatic oxidative metabolism and thus potentially feed intake, blood and liver samples of 19 Holstein cows were taken throughout the periparturient period. Retrospectively grouped according to high (H) and low (L) liver fat content, H cows showed higher fatty acid but lower amino acid plasma concentrations and lower feed intake than L cows. The hepatic phospho-AMPK/total AMP ratio was not different between groups but decreased after parturition. A 2-DE coupled MALDI-TOF-TOF analysis and qRT-PCR studies revealed H cows having lower expressions of major enzymes involved in mitochondrial β-oxidation, urea cycling, and the pentose phosphate pathway but higher expressions of enzymes participating in peroxisomal and endoplasmic fatty acid degradation, pyruvate and TCA cycling, amino acid catabolism, oxidative phosphorylation, and oxidative stress defense. These data indicate that increasing lipolysis leads to augmenting nutrient catabolism for anaplerosis and mitochondrial respiration, providing a molecular link between hepatic oxidative processes and feed intake.

Plasma ghrelin is positively associated with body fat, liver fat and milk fat content but not with feed intake of dairy cows after parturition.

Ghrelin is a gastrointestinal peptide hormone that is present in blood mostly in a non-posttranslationally modified form, with a minor proportion acylated at Ser(3). Both ghrelin forms were initially assigned a role in the control of food intake but there is accumulating evidence for their involvement in fat allocation and utilization. We investigated changes in the ghrelin system in dairy cows, exhibiting differences in body fat mobilization and fatty liver, from late pregnancy to early lactation. Sixteen dairy cows underwent liver biopsy and were retrospectively grouped based on high (H) or low (L) liver fat content post-partum. Both groups had a comparable feed intake in week -6 (before parturition) and week 2 (after parturition). Only before parturition was preprandial total ghrelin concentration higher in L than in H cows and only after parturition was the basal plasma concentration of non-esterified fatty acids higher in H than in L cows. Both before and after parturition, H cows had higher preprandial plasma concentrations of acyl ghrelin, a higher acyl:total ghrelin ratio, lower plasma triacylglyceride concentrations and a lower respiratory quotient compared with L cows. These group differences could not be attributed to an allelic variant of the acyl ghrelin receptor. Rather, the ratio of acyl:total ghrelin correlated with several aspects of fat metabolism and with respiratory quotient but not with feed intake. These results show that endogenous ghrelin forms are associated with fat allocation, fatty liver, and utilization of fat during the periparturient period.

Reduced AgRP activation in the hypothalamus of cows with high extent of fat mobilization after parturition

Agouti-related protein (AgRP), produced by neurons located in the arcuate nucleus of the hypothalamus stimulates feed intake. During early lactation dairy cows increase their feed intake and additionally mobilize their fat reserves leading to increased plasma non-esterified fatty acid (NEFA) concentrations. Since cows with a higher extent of fat mobilization exhibit the lower feed intake, it seems that high NEFA concentrations confine hyperphagia. To test the involvement of AgRP neurons, we investigated 18 cows from parturition until day 40 postpartum (pp) and assigned the cows according to their NEFA concentration on day 40pp to either group H (high NEFA) or L (low NEFA). Both groups had comparable feed intake, body weight, milk yield, energy balance, plasma amino acids and leptin concentrations. Studies in respiratory chambers revealed the higher oxygen consumption and the lower respiratory quotient (RQ) in H compared to L cows. mRNA abundance of neuropeptide Y, peroxisome proliferator-activated receptor-gamma, AMP-activated protein kinase, and leptin receptor in the arcuate nucleus were comparable between groups. Immunohistochemical studies revealed the same number of AgRP neurons in H and L cows. AgRP neurons were co-localized with phosphorylated adenosine monophosphate-activated kinase without any differences between groups. The percentage of cFOS-activated AgRP neurons per total AgRP cells was lower in H cows and correlated negatively with oxygen consumption and NEFA, positively with RQ, but not with feed intake. We conclude that AgRP activation plays a pivotal role in the regulation of substrate utilization and metabolic rate in high NEFA dairy cows during early lactation.

Insulin signaling of glucose uptake in skeletal muscle of lactating dairy cows

Insulin response in skeletal muscles is thought to be impaired in dairy cows during early lactation to favor nutrient supply, especially glucose, towards the mammary gland. However, the molecular mechanisms of insulin action on glucose metabolism in cows and in other ruminants are still not completely understood, particularly during early lactation. Previous studies provide evidence that peripartal insulin resistance in skeletal muscle of ruminants is caused by post-receptor changes of the insulin signaling pathway. The protein content of glucose transporter 4 (GLUT4) is reduced in skeletal muscle of goats and cows at begin of lactation (Balage et al., 1997; Kuhla et al., 2011). On the other hand, lactation does not have an effect on the number, the affinity or the activity of the tyrosine kinase of the insulin receptor (InsR) in sheep (Wilson et al., 1996) and goats (Balage et al., 1992). Furthermore, Duhlmeier et al. (2005) showed differences in protein amount of GLUT 1 and 4 in oxidative and glycolytic muscles of lactating cows. We have tested the hypothesis that insulin signaling in skeletal muscle of dairy cows is related to the stage of lactation and differs among various muscle types. Furthermore, we have investigated the influence of different post-calving metabolic status of dairy cows as reflected by divergent fat mobilization and liver fat concentration (LFC) on insulin signaling in muscle tissue.

Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile.

During postnatal development, hyperplastic and hypertrophic processes of skeletal muscle growth depend on the activation, proliferation, differentiation, and fusion of satellite cells (SC). Therefore, molecular and functional SC heterogeneity is an important component of muscle plasticity and will greatly affect long-term growth performance and muscle health. However, its regulation by cell intrinsic and extrinsic factors is far from clear. In particular, there is only minor information on the early postnatal period which is critical for muscle maturation and the establishment of adult SC pools. Here, we separated two SC subpopulations (P40/50, P50/70) from muscle of 4-day-old piglets. Our results characterize P40/50 as homogeneous population of committed (high expression of Myf5), fast-proliferating muscle progenitors. P50/70 constituted a slow-proliferating phenotype and contains high numbers of differentiated SC progeny. During culture, P50/70 is transformed to a population with lower differentiation potential that contains 40% Pax7-positive cells. A reversible state of low mitochondrial activity that results from active down-regulation of ATP-synthase is associated with the transition of some of the P50/70 cells to this more primitive fate typical for a reserve cell population. We assume that P40/50 and P50/70 subpopulations contribute unequally in the processes of myofiber growth and maintenance of the SC pool.

A longitudinal proteomic approach to investigate liver metabolism in periparturient dairy cows with different body fat mobilization

In late gestation dairy cows start to mobilize body reserves primarily glycogen, protein and fat depots. The mobilization of fat reserves results in an increased plasma concentration of free fatty acids and in a fat deposition in the liver accompanied by changes of hepatic lipid metabolism after calving. According to the hepatic oxidation theory (HOT) [1], increased hepatic fatty acid oxidation is suggested to depress feed intake during early lactation. Therefore, we investigated the expression of enzymes participating in hepatic beta-oxidation in dairy cows with different body fat mobilization to identify correlations with dry matter intake (DMI). Daily ad libitum DMI was recorded for 19 multiparous German Holstein cows (2nd to 4th lactation, >10,000 kg/305d in at least one previous lactation) housed in a tie stall from week 7 before until 5 weeks after calving. Cows were fed three different total mixed rations according to their physiological state, in far-off dry period (week −7 to −4, 5.87 MJ NEL and 128 g nXP/kg DM), close-up dry period (week −3 till calving, 6.49 MJ NEL and 137 g nXP/kg DM) and lactation (week 1 to 5, 7.06 MJ NEL and 163 g nXP/kg DM). The liver of each cow was biopsied at five time points [days −34, -17, 3, 18, 30 rtp (relative to parturition)] and at slaughter (day 40 rtp) and the obtained samples were immediately frozen in liquid nitrogen. Total liver fat content (LFC) was determined by the CN-method [2] and used as a parameter of body fat mobilization in early lactation. According to the average post partum (days 3, 18, and 30 rtp) LFC, cows were grouped into high-mobilizing (HMC; LFC >24.4% DM; n = 10) and low-mobilizing (LMC; LFC <;24.0% DM; n = 9) cows. Also, samples (50 mg) of frozen liver tissue were crushed to a fine powder and extracted for a proteomic approach as described earlier for feed restriction-induced fatty liver [3]. Briefly, extracts were applied to 2-dimensional gel electrophoresis followed by colloidal Coomassie staining (Figure 1). For spot detection and quantification using the normalized spot volume, 2D gels were processed using Delta 2D software (DECODON). Data were analysed by the Mixed Model of SAS with LFC and time relative to parturition as fixed effects. All spots that were differentially expressed between groups (P < 0.05; Mixed Model of SAS, LFC and time as fixed effects) were picked, tryptic digested and analysed on a 5800 MALDI TOF/TOF analyser.

Overexpression of Na + /Mg 2+ exchanger SLC41A1 attenuates pro-survival signaling

The Na+/Mg2+ exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg2+ efflux system, and its overexpression decreases [Mg2+]intracellular. IMH plays an important role in the regulation of many cellular processes, including cellular signaling. However, whether the overexpression of A1 and the consequent drop of [Mg2+]i impact on intracellular signaling is unknown. To examine the latter, we utilized dynamic mass redistribution (DMR) assay, PathScan® RTK signaling antibody (PRSA) array, confirmatory Western blot (WB) analyses of phosphorylation of kinases selected by PRSA, and mag-fura 2-assisted fast filter spectrometry (FFS). We demonstrate here that the overexpression of A1 quantitatively and qualitatively changes the DMR signal evoked by the application of PAR-1-selective activating peptide and/or by changing [Mg2+]extracellular in HEK293 cells. PRSA profiling of the phosphorylation of important signaling nodes followed by confirmatory WB has revealed that, in HEK293 cells, A1 overexpression significantly attenuates the phosphorylation of Akt/PKB on Thr308 and/or Ser473 and of Erk1/2 on Thr202/Tyr204 in the presence of 0 or 1 mM (physiological) Mg2+ in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg2+]i in the presence of [Mg2+]e = 0 or 1 mM. This correlates with the observed attenuation of prosurvival Akt/PKB – Erk1/2 signaling in these cells. Thus, A1 expression status and [Mg2+]e (and consequently also [Mg2+]i) modulate the complex physiological fingerprint of the cell and influence the activity of kinases involved in anti-apoptotic and, hence, pro-survival events in cells.

Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles

Muscle stem cells, termed satellite cells (SC), and SC-derived myogenic progenitor cells (MPC) are involved in postnatal muscle growth, regeneration, and muscle adaptability. They can be released from their natural environment by mechanical disruption and tissue digestion. The literature contains several isolation protocols for porcine SC/MPC including various digestion procedures, but comparative studies are missing. In this report, classic trypsinization and a more complex trypsin, collagenase, and DNase (TCD) digestion were performed with skeletal muscle tissue from 4- to 5-d-old piglets. The two digestion procedures were compared regarding cell yield, viability, myogenic purity, and in vitro cell function. The TCD digestion tended to result in higher cell yields than digestion with solely trypsin (statistical trend p = 0.096), whereas cell size and viability did not differ. Isolated myogenic cells from both digestion procedures showed comparable proliferation rates, expressed the myogenic marker Desmin, and initiated myogenic differentiation in vitro at similar levels. Thus, TCD digestion tended to liberate slightly more cells without changes in the tested in vitro properties of the isolated cells. Both procedures are adequate for the isolation of SC/MPC from juvenile porcine muscles but the developmental state of the animal should always be considered.

Correlations between plasma ghrelin and parameters of fat metabolism in early lactating dairy cows

The peptide hormone ghrelin is produced in the ruminal and proximal duodenal wall with a small portion of ghrelin being post-translationally modified by fatty acids at Ser3. Both forms (desacyl and acyl ghrelin) are released into the blood stream and have been initially assigned a role in the control of feed intake. While acyl ghrelin increases feed intake, desacyl ghrelin may exert opposing effects. However, there is accumulating evidence for the involvement of ghrelin in fat allocation and utilization involving liver, muscle and adipose tissue (Barazzoni et al., 2005; Jennings et al., 2011; Rodriguez et al., 2009). Therefore, it seems possible that ghrelin may be involved in body fat metabolism of dairy cows during the transition period. Here we examined the hypothesis that ghrelin could play a role in the extent of body fat mobilization during early lactation in dairy cows.

Skeletal muscle fatty acid oxidation in lactating dairy cows during early lactation

The mobilization of adipose tissue of dairy cows during early lactation is reflected by increased fatty acid (FA) plasma concentrations. The capacity of the liver for complete oxidation of non-esterified FA (NEFA) is limited leading to an increased formation of ketone bodies, re-esterification, and accumulation of triacylglycerides in the liver. This is accompanied by a lower feed intake and a high incidence of metabolic disorders (Drackley et al., 2005). The skeletal muscle may also oxidize FA and thus relieve the liver from FA load. Here we hypothesized that skeletal muscle FA degradation adapts to the extent of fat mobilization during early lactation.