Elevating serotonin pre-partum alters the Holstein dairy cow hepatic adaptation to lactation

<div><p>Serotonin is known to regulate energy and calcium homeostasis in several mammalian species. The objective of this study was to determine if pre-partum infusions of 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin synthesis, could modulate energy homeostasis at the level of the hepatocyte in post-partum Holstein and Jersey dairy cows. Twelve multiparous Holstein cows and twelve multiparous Jersey cows were intravenously infused daily for approximately 7 d pre-partum with either saline or 1 mg/kg bodyweight of 5-HTP. Blood was collected for 14 d post-partum and on d30 post-partum. Liver biopsies were taken on d1 and d7 post-partum. There were no changes in the circulating concentrations of glucose, insulin, glucagon, non-esterified fatty acids, or urea nitrogen in response to treatment, although there were decreased beta-hydroxybutyrate concentrations with 5-HTP treatment around d6 to d10 post-partum, particularly in Jersey cows. Cows infused with 5-HTP had increased hepatic serotonin content and increased mRNA expression of the serotonin 2B receptor on d1 and d7 post-partum. Minimal changes were seen in the hepatic mRNA expression of various gluconeogenic enzymes. There were no changes in the mRNA expression profile of cell-cycle progression marker cyclin-dependent kinase 4 or apoptotic marker caspase 3, although proliferating cell nuclear antigen expression tended to be increased in Holstein cows infused with 5-HTP on d1 post-partum. Immunofluorescence assays showed an increased number of CASP3- and Ki67-positive cells in Holstein cows infused with 5-HTP on d1 post-partum. Given the elevated hepatic serotonin content and increased mRNA abundance of <i>5HTR2B</i>, 5-HTP infusions may be stimulating an autocrine-paracrine adaptation to lactation in the Holstein cow liver.</p></div

Hepatic serotonin content and <i>TPH1</i> and <i>SERT</i> mRNA abundance of multiparous Holstein cows and multiparous Jersey cows administered pre-partum daily I.V. infusions of 1 liter of saline or 1 liter of 1.0 mg/kg bodyweight of 5-hyroxy-L-tryptophan (5-HTP) reconstituted in saline.

<p>Final treatment groups were saline-infused Holsteins (<i>n</i> = 6), 5-HTP infused Holsteins (<i>n</i> = 6), saline-infused Jerseys (<i>n</i> = 6), and 5-HTP infused Jerseys (<i>n</i> = 6). (A) Liver serotonin content corrected to ug of protein (B) mRNA abundance of tryptophan hydroxylase 1 (<i>TPH1</i>) and (C) mRNA abundance of serotonin reuptake transporter (<i>SERT</i>). Stars indicate statistical difference between group means (* = 0.05<<i>P</i><0.01). All values are reported as LS means ± SEM.</p

Immunofluorescent images and counts of cells positive for Ki67 and CASP3 in multiparous Holstein cows and multiparous Jersey cows administered pre-partum daily I.V. infusions of 1 liter of saline or 1 liter of 1.0 mg/kg bodyweight of 5-hyroxy-L-tryptophan (5-HTP) reconstituted in saline.

<p>Final treatment groups were saline-infused Holsteins (<i>n</i> = 6), 5-HTP infused Holsteins (<i>n</i> = 6), saline-infused Jerseys (<i>n</i> = 6), and 5-HTP infused Jerseys (<i>n</i> = 6). (A) Percent of cells positive for Ki67 stain of all counted cells in representative images (<i>n</i> = 3) from each treatment group. (B) Immunofluorescent images of Ki67-positive cells. The scale bar indicates 15 microns. The arrow indicates an example of a cell considered positive for Ki67. d1 and d7 are days 1 and 7 post-partum, respectively. CON and 5-HTP represent saline-infused and 5-HTP infused groups, respectively. (C) Immunofluorescent images of CASP3-positive cells. The scale bar indicates 15 microns. The arrow indicates an example of a cell considered positive for CASP3. (D) Count of cells positive for CASP3 in representative images (<i>n</i> = 3) from each treatment group. Stars indicate statistical difference between group means (* = 0.05<<i>P</i><0.01; *** = 0.001<<i>P</i><0.0001). All values are reported as LS means ± SEM.</p

Primer sequences for genes quantified by real-time PCR.

<p>Primer sequences for genes quantified by real-time PCR.</p

The Goat (<i>Capra hircus</i>) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis

<div><p>Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.</p></div

Hepatic mRNA abundance of <i>CCND1</i>, <i>CDK4</i>, <i>PCNA</i>, and <i>CASP3</i> in multiparous Holstein cows and multiparous Jersey cows administered pre-partum daily I.V. infusions of 1 liter of saline or 1 liter of 1.0 mg/kg bodyweight of 5-hyroxy-L-tryptophan (5-HTP) reconstituted in saline.

<p>Final treatment groups were saline-infused Holsteins (<i>n</i> = 6), 5-HTP infused Holsteins (<i>n</i> = 6), saline-infused Jerseys (<i>n</i> = 6), and 5-HTP infused Jerseys (<i>n</i> = 6). mRNA abundance of (A) cyclin D1 (<i>CCND1</i>) (B) cyclin-dependent kinase 4 (<i>CDK4</i>) (C) proliferating cell nuclear antigen (<i>PCNA</i>) and (D) caspase-3 (<i>CASP3</i>). All values are reported as LS means ± SEM.</p

Second dimension separation of the subunits of mitochondrial protein complexes from Majorera breed, under denaturing conditions, BN-PAGE/SDS-PAGE.

<p>Identified proteins are referred in the figure by the gene identifier and reference number. Proteins were stained with CCB and identified with MALDI-TOF/TOF. Detailed information of protein identification is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151599#pone.0151599.s002" target="_blank">S2 Table</a>.</p

Protein complexes, and respective subunits, identified by nano-LC-MS/MS.

<p>Detailed information of the identification of proteins is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151599#pone.0151599.s001" target="_blank">S1 Table</a>.</p

Protein spots showing differential abundance in Majorera and Palmera individuals subjected to food restriction.

<p>Values are reported as log normalized volumes. Statistical differences in comparison to control with P<0.05 (*).</p

Separation of mitochondrial protein complexes from Majorera breed by BN-PAGE.

<p>Proteins were solubilized with the detergent DDM at a protein ratio of 1:10; 1:7.5 and 1:5 (protein: detergent, w/w). Gel stained with Coomassie Blue Colloidal. Gel bands were excised and analysed by nano-LC-MS/MS.</p