Modulation of GCN2/eIF2α/ATF4 Pathway in the Liver and Induction of FGF21 in Young Goats Fed a Protein- and/or Phosphorus-Reduced Diet.

Mammals respond to amino acid (AA) deficiency by initiating an AA response pathway (AAR) that involves the activation of general control nonderepressible 2 (GCN2), phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and activation of transcription factor 4 (ATF4). In this study, the effects of protein (N) and/or phosphorus (P) restriction on the GCN2/eIF2α/ATF4 pathway in the liver and the induction of fibroblast growth factor 21 (FGF21) in young goats were investigated. An N-reduced diet resulted in a decrease in circulating essential AA (EAA) and an increase in non-essential AA (NEAA), as well as an increase in hepatic mRNA expression of GCN2 and ATF4 and protein expression of GCN2. Dietary N restriction robustly increased both hepatic FGF21 mRNA expression and circulating FGF21 levels. Accordingly, numerous significant correlations demonstrated the effects of the AA profile on the AAR pathway and confirmed an association. Furthermore, activation of the AAR pathway depended on the sufficient availability of P. When dietary P was restricted, the GCN2/eIF2α/ATF4 pathway was not initiated, and no increase in FGF21 was observed. These results illustrate how the AAR pathway responds to N- and/or P-reduced diets in ruminants, thus demonstrating the complexity of dietary component changes.

Postnatal development of salivary phosphate, sodium and potassium secretion in calves.

The transition of young lambs and calves from a non-ruminating to a ruminating animal include substantial, developmental changes to alter saliva production. Due to the simultaneous development of the forestomach system, the salivary glands must transfer more and more substances such as bicarbonate and phosphate (Pi), but also sodium (Na), into saliva in order to create and to maintain optimal environmental conditions for microbial metabolism. The objective of the present study was to characterize the effects of different dietary energy levels on the ability of the salivary glands to concentrate minerals in young ruminants in more detail during the first 7 weeks of life. Blood and saliva samples were collected from twelve female calves of the German Holstein breed fed different levels of milk replacer. Plasma and saliva samples were collected over 7 weeks postpartum and Na, Pi and potassium (K) concentrations were measured. Salivary Na and Pi concentrations (p < 0.001) increased as a function of time and were not affected by varying energy intake, while K concentration (p < 0.001) decreased over the developmental period and was also not affected by energy intake. This suggests that the ability to specifically concentrate minerals such as Na and Pi in saliva follows a genetic program in the salivary glands rather than being influenced by dietary factors such as energy intake in young ruminants.

Modulation of Intestinal Phosphate Transport in Young Goats Fed a Low Phosphorus Diet.

The intestinal absorption of phosphate (Pi) takes place transcellularly through the active NaPi-cotransporters type IIb (NaPiIIb) and III (PiT1 and PiT2) and paracellularly by diffusion through tight junction (TJ) proteins. The localisation along the intestines and the regulation of Pi absorption differ between species and are not fully understood. It is known that 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) and phosphorus (P) depletion modulate intestinal Pi absorption in vertebrates in different ways. In addition to the apical uptake into the enterocytes, there are uncertainties regarding the basolateral excretion of Pi. Functional ex vivo experiments in Ussing chambers and molecular studies of small intestinal epithelia were carried out on P-deficient goats in order to elucidate the transepithelial Pi route in the intestine as well as the underlying mechanisms of its regulation and the proteins, which may be involved. The dietary P reduction had no effect on the duodenal and ileal Pi transport rate in growing goats. The ileal PiT1 and PiT2 mRNA expressions increased significantly, while the ileal PiT1 protein expression, the mid jejunal claudin-2 mRNA expression and the serum 1,25-(OH)2D3 levels were significantly reduced. These results advance the state of knowledge concerning the complex mechanisms of the Pi homeostasis in vertebrates.

Dietary phosphorus restriction affects bone metabolism, vitamin D metabolism and rumen fermentation traits in sheep.

Homeostasis of calcium (Ca) and phosphate (Pi ) is maintained by a concerted interplay of absorption and reabsorption via the gastrointestinal tract and the kidney and by storage and mobilization from the bone regulated mainly by parathyroid hormone (PTH), 1,25-dihydroxycholecalciferol and calcitonin. The present study aimed at characterizing the effects of dietary P restriction on bone, vitamin D metabolism and rumen fermentation traits reflecting the endogenous P cycle maintaining the ruminal P supply for microbial metabolism. The experiments were done in eleven female, non-pregnant, non-lactating four- to nine-year-old Black Headed Mutton sheep allotted to two feeding groups: "P-restricted" (0.11% P/kg DM and 0.88% Ca/kg DM) and "Control" (0.38% P/kg DM and 0.88% Ca/kg DM). Dietary P restriction did not lead to hypophosphataemia, probably due to a compensation by bone mobilization, demonstrated by increased serum concentrations of a resorption marker and altered gene expression in bone tissue. In addition, the RNA expression of fibroblast growth factor 23, a bone-derived factor involved in the regulation of vitamin D metabolism, was significantly reduced with dietary P restriction. Furthermore, several genes related to vitamin D metabolism and plasma concentrations of 1,25-(OH)2 D were associated with serum concentrations of phosphate (Pi ). In the parotid gland, the expression of the Pi transporter NaPi2b was negatively associated with serum Pi and positively with parathyroid PTH expression. Although Pi concentrations in saliva and the gastrointestinal tract were significantly reduced, we found no adverse effects of the P-restricted ration on the production of short chain fatty acids, but slight differences in the production of butyrate as well as its relationship to rumen Pi and ammonia concentrations that might indicate an impact on ruminal fermentation.

Dietary protein and calcium modulate parathyroid vitamin D receptor expression in young ruminants.

For economic reasons and in order to minimize nitrogen excretion and thus pollution, the crude protein content in the diet of livestock animals should be as low as possible without negatively affecting the animals´ health and performance. As ruminants can efficiently use dietary protein because of the ruminohepatic circulation of urea, they are considered to cope more easily with such a feeding regime than monogastric animals. However, despite unaltered daily weight gain, massive changes in mineral homeostasis and vitamin D metabolism were observed with dietary protein reduction (N-) in young, growing goats. Serum concentrations of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) were decreased with a low N intake, even if calcium (Ca) was also restricted (Ca-). Interestingly, concentrations of cyclic adenosine monophosphate (cAMP) measured as an indirect assessment for the parathyroid hormone (PTH) activity were not affected by low protein. Therefore, it was hypothesized that the sensitivity of the parathyroid gland is modulated during these dietary interventions. Four groups of male German colored goats received a control (N+/Ca+), a reduced protein (N-/Ca+), a reduced Ca (N+/Ca-) or a reduced protein and Ca (N-/Ca-) diet. After six weeks we determined the expression of PTH, PTH receptor, Ca sensing receptor (CASR), vitamin D receptor (VDR), retinoid X receptor (RXRα), Klotho, fibroblast growth factor receptor 1c-splicing form, and the sodium-dependent Pi transporter (PiT1) in the parathyroid glands. Concentrations of cAMP were not affected, while those of Ca and 1,25-(OH)2D3were diminished and that of 25-hydroxyvitamin D3 was increased with N- feeding. The expression patterns of the described target genes were not altered. In contrast, animals fed the Ca- rations showed enhanced serum 1,25-(OH)2D3 and cAMP levels with no changes in blood Ca concentrations demonstrating an efficient adaptation. The mRNA expression of expression of VDR and CASR in the parathyroid gland was significantly diminished and RXRα, PTHR and PiT1 expression was elevated. Instead of the assumed desensitization of the parathyroid gland with N-, our results indicate elevated responsiveness to decreased blood Ca with feeding Ca-.

Modulation of growth hormone receptor-insulin-like growth factor 1 axis by dietary protein in young ruminants.

A reduced protein intake causes a decrease in insulin-like growth factor 1 (IGF1) concentrations and modulates Ca homoeostasis in young goats. IGF1 is synthesised by the liver in response to stimulation by growth hormone (GH). Due to rumino-hepatic circulation of urea, ruminants are suitable for investigating the effects of protein reduction despite sufficient energy intake. The present study aimed to investigate the impact of a protein-reduced diet on the expression of components of the somatotropic axis. Male young goats were divided into two feeding groups receiving either a control diet (20 % crude protein (CP)) or a reduced-protein diet (9 % CP). Blood concentrations of IGF1 and GH were measured, and a 24-h GH secretion profile was compiled. Moreover, ionised Ca and insulin concentrations as well as mRNA and protein expression levels of hepatic proteins involved in GH signalling were quantified. Due to the protein-reduced diet, concentrations of ionised Ca, insulin and IGF1 decreased significantly, whereas GH concentrations remained unchanged. Expression levels of the hepatic GH receptor (GHR) decreased during protein reduction. GHR expression was down-regulated due to diminished insulin concentrations as both parameters were positively correlated. Insulin itself might be reduced due to reduced blood Ca levels that are involved in insulin release. The protein-reduced diet had an impact on the expression of components of the somatotropic axis as a disruption of the GH-IGF1 axis brought about by diminished GHR expression was shown in response to a protein-reduced diet.

Mechanisms and regulation of epithelial phosphate transport in ruminants: approaches in comparative physiology.

Ruminants have a unique utilization of phosphate (Pi) based on the so-called endogenous Pi recycling to guarantee adequate Pi supply for ruminal microbial growth and for buffering short-chain fatty acids. Large amounts of Pi enter the gastrointestinal tract by salivary secretion. The high saliva Pi concentrations are generated by active secretion of Pi from blood into primary saliva via basolateral sodium (Na+)-dependent Pi transporter type II. The following subsequent intestinal absorption of Pi is mainly carried out in the jejunum by the apical located secondary active Na+-dependent Pi transporters NaPi IIb (SLC34A2) and PiT1 (SLC20A1). A reduction in dietary Pi intake stimulates the intestinal Pi absorption by increasing the expression of NaPi IIb despite unchanged plasma 1,25-dihydroxyvitamin D3 concentrations, which modulate Pi homeostasis in monogastric species. Reabsorption of glomerular filtrated plasma Pi is mainly mediated by the Pi transporters NaPi IIa (SLC34A1) and NaPi IIc (SLC34A3) in proximal tubule apical cells. The expression of NaPi IIa and the corresponding renal Na+-dependent Pi capacity were modulated by high dietary phosphorus (P) intake in a parathyroid-dependent manner. In response to reduced dietary Pi intake, the expression of NaPi IIa was not adapted indicating that renal Pi reabsorption in ruminants runs at a high level allowing no further increase when P intake is diminished. In bones and in the mammary glands, Na+-dependent Pi transporters are able to contribute to maintaining Pi homeostasis. Overall, the regulation of Pi transporter activity and expression by hormonal modulators confirms substantial differences between ruminant and non-ruminant species.

A reduced protein diet modulates enzymes of vitamin D and cholesterol metabolism in young ruminants.

Besides other adverse effects, a low protein diet has been shown to modulate cholesterol and vitamin D metabolism in monogastric species like rats and humans. As ruminants can increase the efficiency of the rumino-hepatic circulation of urea, it is assumed that goats should be able to compensate for a low dietary protein intake better. After a dietary protein restriction (9% vs. 20%) for six weeks, plasma concentrations of urea, albumin, 1,25-dihydroxyvitamin D3 and calcium were decreased, while plasma 25-hydroxyvitamin D3 (25-OHD3), and total cholesterol were significantly increased in young goats. Because this was not accompanied by any decrease in expression of CYP24A1 mRNA, we investigated mRNA expression of additional enzymes with known 24- and/or 25-hydroxylase activities (CYP2R1, CYP2J2, CYP3 A24, CYP27A1), receptors involved in their regulation (VDR, PXR, RXRα) and vitamin D binding protein (VDBP). CYP2R1expression was stimulated with the low dietary protein intake, negatively correlated with plasma urea and positively associated with serum 25-OHD3. The greater plasma concentrations of total cholesterol could be explained with the reduction of CYP2J2 and CYP27A1 expression. None of the receptors investigated were affected by the dietary protein restriction but mRNA expression of VDBP was slightly reduced. Taken together our results show that dietary protein restriction has an impact on vitamin D and cholesterol metabolism in ruminants, too. Therefore, further investigations are needed before dietary interventions aiming at diminishing nitrogen excretion can be implemented.

Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet.

Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.

Dietary nitrogen and calcium modulate bone metabolism in young goats.

Ruminants, possessing the rumino-hepatic circulation, are thought to cope easily with reduced dietary nitrogen (N) supply which is of economic and environmental interest to diminish N output. Nevertheless, feeding an N reduced diet to young goats resulted in a decrease in calcitriol and calcium (Ca) plasma concentrations. Although a dietary Ca reduction alone stimulated calcitriol synthesis and plasma Ca concentrations were restored, in combination with a reduced N supply this stimulating effect was abolished. Based on the important role bone tissue plays in maintaining Ca homeostasis, aim of the present study was to determine effects of an N reduced diet with or without a concomitant Ca reduction on bone metabolism in young goats. A dietary N reduction alone resulted in a significant rise in plasma concentrations of bone resorption marker C-terminal telopeptide of type I collagen (CTX) and bone formation marker osteocalcin (OC), while reduced intake of Ca as well as the combination of both dietary interventions increased bone markers only slightly. Bone mineral content and bone mineral density of metatarsi were decreased by reduced N intake, while Ca and phosphorus (P) content of dried bones remained unaffected. In contrast, a dietary Ca reduction alone led to decreased Ca and P content of dried bones. From these data it can be concluded that a dietary N reduction alone or in combination with a reduced dietary Ca supply modulated bone metabolism in young goats.

Modulation of intestinal calcium and phosphate transport in young goats fed a nitrogen- and/or calcium-reduced diet.

Feeding ruminants a reduced N diet is a common approach to reduce N output based on rumino-hepatic circulation. However, a reduction in N intake caused massive changes in Ca and inorganic phosphate (Pi) homoeostasis in goats. Although a single dietary Ca reduction stimulated intestinal Ca absorption in a calcitriol-dependent manner, a concomitant reduction of Ca and N supply led to a decrease in calcitriol, and therefore a modulation of intestinal Ca and Pi absorption. The aim of this study was to examine the potential effects of dietary N or Ca reduction separately on intestinal Ca and Pi transport in young goats. Animals were allocated to a control, N-reduced, Ca-reduced or combined N- and Ca-reduced diet for about 6-8 weeks, whereby N content was reduced by 25 % compared with recommendations. In Ussing chamber experiments, intestinal Ca flux rates significantly decreased in goats fed a reduced N diet, whereas Pi flux rates were unaffected. In contrast, a dietary Ca reduction stimulated Ca flux rates and decreased Pi flux rates. The combined dietary N and Ca reduction withdrew the stimulating effect of dietary Ca reduction on Ca flux rates. The expression of Ca-transporting proteins decreased with a reduced N diet too, whereas Pi-transporting proteins were unaffected. In conclusion, a dietary N reduction decreased intestinal Ca transport by diminishing Ca-transporting proteins, which became clear during simultaneous N and Ca reduction. Therefore, N supply in young ruminant nutrition is of special concern for intestinal Ca transport.

Modulation of aquaporin 2 expression in the kidney of young goats by changes in nitrogen intake.

In ruminants, a decrease of dietary nitrogen (N) is an appropriate feeding concept to reduce environmental pollution and costs. In our previous study, when goats were kept on an N-reduced diet, a decrease of plasma urea concentration and an increase of renal urea transporters were demonstrated. Renal urea absorption plays a crucial role for renal water absorption and urine concentration. Renal collecting duct water absorption is mainly mediated by the water channel aquaporin 1 and 2 (AQP1 and AQP2). Therefore, the aim of the present study was to investigate the effects of a dietary N reduction on expression of renal AQP1 and AQP2 in young goats. Twenty male White Saanen goats, 3 months old, were divided equally into two feeding groups, receiving either a diet with an adequate or a reduced-N supply. Goats fed a reduced-N diet showed significantly higher amounts of AQP1 mRNA in cortical tissue, and the expression of AQP2 mRNA and protein were highly elevated in renal outer medulla. An increase of vasopressin concentrations in plasma were detected for the N-reduced fed goats. Therefore, a stimulation of renal water absorption can be assumed. This might be an advantage for ruminants in times of N reduction due to higher urea concentrations in the tubular fluid and which might result in higher absorption of urea by renal urea transporters. Therefore, interplay of aquaporin water channels and urea transporters in the kidney may occur to maintain urea metabolism in times of N scarcity in young goats.