Adipose tissue proteomic analysis in ketotic or healthy Holstein cows in early lactation1

Abstract

Ketosis is a major metabolic disorder of high-yielding dairy cows during the transition period. Although metabolic adaptations of the adipose tissue are critical for a successful transition, beyond lipolysis, alterations within adipose tissue during ketosis are not well known. The objective of this study was to investigate the adipose tissue proteome of healthy or ketotic postpartum cows to gain insights into biological adaptations that may contribute to disease outcomes. Adipose tissue biopsy was collected on 5 healthy and 5 ketotic cows at 17 (±4) d postpartum and ketosis was defined according to the clinical symptoms and serum β-hydroxybutyrate concentration. Morphology micrographs stained by hematoxylin–eosin showed that adipocytes were smaller in ketotic cows than in healthy cows. The isobaric tag for relative and absolute quantification was applied to quantitatively identify differentially expressed proteins (DEP) in the adipose tissue. We identified a total of 924 proteins, 81 of which were differentially expressed between ketotic and healthy cows (P < 0.05 and fold changes >1.5 or <0.67). These DEP included enzymes and proteins associated with various carbohydrate, lipid, and amino acid metabolism processes. The top pathways differing between ketosis and control cows were glycolysis/gluconeogenesis, glucagon signaling pathway, cysteine and methionine metabolism, biosynthesis of amino acids, and the cGMP–PKG signaling pathway. The identified DEP were further validated by western blot and co-immunoprecipitation assay. Key enzymes associated with carbohydrate metabolism such as pyruvate kinase 2, pyruvate dehydrogenase E1 component subunit α), lactate dehydrogenase A , phosphoglucomutase 1, and 6-phosphofructokinase 1 were upregulated in ketotic cows. The expression and phosphorylation state of critical regulators of lipolysis such as perilipin-1 and hormone-sensitive lipase were also upregulated in ketotic cows. Furthermore, key proteins involved in maintaining innate immune response such as lipopolysaccharide binding protein and regakine-1 were downregulated in ketotic cows. Overall, data indicate that ketotic cows during the transition period have altered carbohydrate, lipid metabolism, and impaired immune function in the adipose tissue. This proteomics analysis in adipose tissue of ketotic cows identified several pathways and proteins that are components of the adaptation to ketosis.

Adenosine 5′-monophosphate-activated protein kinase ameliorates bovine adipocyte oxidative stress by inducing antioxidant responses and autophagy

“…The intense lipid mobilization caused by severe negative energy balance (NEB) in transition cows leads to adipose tissue metabolic stress and increased susceptibility to ketosis (Kabara et al, 2014;Wankhade et al, 2017;Xu et al, 2019b). The regulating effect of AMPK, NFE2L2, and autophagy under oxidizing conditions are well characterized in human and animal models of oxidative stress (Kiffin et al, 2006;Ma, 2013;Wu et al, 2014).…”

“…Subcutaneous adipose tissue was collected at 17 (±4) d postpartum from the tail-head depot of all cows on the same day through the methods described previously (Xu et al, 2019b). In brief, the pelage on the tail-head region and one side of the tail-head were thoroughly scrubbed with surgical soap before tissue collection.…”

All-trans retinoic acid controls differentiation, proliferation, and lipolysis in isolated subcutaneous adipocytes from peripartal Holstein cows

“…All cows were examined by the attending veterinarian to ensure there were no other complications such as hypocalcemia or mastitis. Subcutaneous adipose tissue (SAT) was collected from the tail-head of 5 healthy Holstein cows at 17 (±4) d postpartum (Xu et al, 2019b) and processed as described previously (Xu et al, 2019a). In brief, SAT (1 g) was digested with collagenase type I digestion solution (1 mg/mL; Sigma-Aldrich, St. Louis, MO) and then filtered through a 40-μm cell filter (Solarbio, Beijing, China).…”

“…no. 26149; Thermo Fisher Scientific) as previously described (Xu et al, 2019b). A portion of the same adipocyte samples used for western blot analysis was washed with ice-cold PBS and lysed in Co-IP lysis buffer.…”

Metabolomic and Proteomic Profiles Associated With Ketosis in Dairy Cows

“…Using the same method, Fan et, al (Fan et al, 2017) identified differentially expressed proteins related to a metabolic disorder of subclinical hypocalcemia. Moreover, proteomic analysis using liver tissues (Xu and Wang, 2008) and adipose tissues (Xu et al, 2019) has also been used in cow ketosis studies, which would provide novel opportunities to unravel the complex biology of the disease.…”