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Preadipocyte proliferation and differentiation are critical for normal adipose tissue development, including achieving a mature phenotype, characterized by its ability to accumulate triacylglycerol and release fatty acids. In nonruminants, it is well known that all-trans retinoic acid (ATRA), the most-active form of vitamin A, helps regulate proliferation, differentiation, and apoptosis in several types of cells including adipocytes. The purpose of this study was to evaluate the effect of ATRA on proliferation, apoptosis, differentiation, and lipolysis of primary bovine adipocytes isolated from subcutaneous adipose tissue of 5 healthy Holstein cows at 17 (±4 standard deviations) d postpartum. Cells were stimulated with increasing concentrations of ATRA (0.2, 2, and 20 nM) at the preconfluent (2 d) and postconfluent (8 d) preadipocyte stage or at the mature adipocyte stage (2 d). All concentrations of ATRA inhibited preconfluent preadipocyte proliferation with decreased proportion of S-phase cells and reduced protein abundance of cyclins (CCND1, CCND2, CCND3, CCNE1) and cyclin-dependent kinases (CDK2, CDK4, CDK6). Compared with vehicle, ATRA treatment induced apoptosis in preconfluent preadipocytes. Additionally, ATRA (0.2, 2, and 20 nM) supplementation also inhibited differentiation of postconfluent preadipocytes through downregulation of protein abundance of PPARγ and C/EBPα. After induction of differentiation, basal lipolysis in mature adipocytes increased upon treatment with all concentrations of ATRA. However, data on phosphorylated hormone-sensitive lipase or PLIN1 indicated that ATRA had no effect on epinephrine-stimulated lipolysis in mature adipocytes. Overall, these results demonstrate that ATRA might inhibit lipid accumulation by suppressing preadipocyte proliferation and differentiation, subsequently leading to apoptosis in postconfluent preadipocytes and promoting basal lipolysis in mature adipocytes. Overall, these in vitro responses provide some insights into the potential for nutritional management to modulate adipose tissue lipolysis, particularly in overconditioned cows during the dry period, which are more susceptible to suffer metabolic disorders due to excessive fat mobilization postpartum., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Dairy cows with ketosis display excessive lipolysis in adipose tissue. Heat-shock protein B7 (HSPB7), a small heat-shock protein, plays important roles in mediating cytoprotective responses to oxidative stress in rodent adipose tissue. Accordingly, it is assumed that HSPB7 may also play important roles in the antioxidant response in adipose tissue of ketotic cows. Therefore, the aim of this study is to investigate (1) the redox state of adipose tissue in ketotic cows and (2) the role and mechanism of HSPB7 on the regulation of oxidative stress in adipocytes from preruminant calves. An in vivo study consisting of 15 healthy and 15 clinically ketotic cows was performed to harvest subcutaneous adipose tissue and blood samples. In addition, adipocytes isolated from calves were treated with different concentrations of H 2 O 2 (0, 12.5, 25, 50, 100, or 200 μM) for 2 h, transfected with adenovirus-mediated overexpression of HSPB7 for 48 h, or transfected with small interfering RNA of HSPB7 for 48 h followed by exposure to H 2 O 2 (200 μM) for 2 h. Serum concentrations of nonesterified fatty acids and β-hydroxybutyrate were greater in cows with clinical ketosis, whereas serum concentration of glucose was lower. Compared with healthy cows, the malondialdehyde content was greater but the activity of glutathione peroxidase and superoxide dismutase was lower in adipose tissue of clinically ketotic cows. The abundance of HSPB7 and nuclear factor, erythroid 2 like 2 (NFE2L2) was greater in adipose tissue of clinically ketotic cows. In vitro, H 2 O 2 treatment induced the overproduction of reactive oxygen species and malondialdehyde, and inhibited the activity of antioxidant enzymes glutathione peroxidase and superoxide dismutase in adipocytes from preruminant calves. The low concentration of H 2 O 2 (12.5, 25, and 50 μM) increased the abundance of HSPB7 and NFE2L2, but high concentrations of H 2 O 2 (100 or 200 μM) reduced the abundance of HSPB7 and NFE2L2. The overexpression of HSPB7 improved the H 2 O 2 -induced oxidative stress in adipocytes via increasing the abundance of NFE2L2 and its downstream target genes heme oxygenase-1 (HMOX1) and NADH quinone oxidoreductase 1 (NQO1). Knockdown of HSPB7 markedly inhibited the expression of NFE2L2, HMOX1, and NQO1 and further exacerbated H 2 O 2 -induced oxidative stress. Overall, these results indicate that activation of the HSPB7-NFE2L2 pathway increases cellular antioxidant capacity, thereby alleviating oxidative stress in bovine adipocytes., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Adipose tissue concentration of reactive oxygen species (ROS) increases in dairy cows with ketosis, suggesting that the tissue experiences oxidative stress. Autophagy, an adaptive response to cellular stress, has been shown to promote survival and plays a critical role in antioxidant responses. Dysregulation of adenosine 5′-monophosphate-activated protein kinase (AMPK) is closely related to antioxidant responses and autophagy of adipocytes in animal models of metabolic disorders, but its role in bovine adipose tissue during periods of stress is unknown. We hypothesized that AMPK may play important roles in the regulation of oxidative stress in adipose tissue of ketotic cows. Specific objectives were to evaluate autophagy status and AMPK activity in adipose tissue of ketotic cows, and their link with oxidative stress in isolated bovine adipocytes. Selection of 15 healthy and 15 clinically ketotic Holstein cows at 17 (±4) d postpartum was performed after a thorough veterinary evaluation for clinical symptoms and also based on serum β-hydroxybutyrate concentrations before collection of subcutaneous adipose tissue samples. Primary cultures of bovine adipocytes isolated from the harvested adipose tissue were stimulated with varying concentrations of H 2 O 2 (0, 50, 100, 200, or 400 μ M) for 2 h. In another experiment, adipocytes were cultured with the AMPK activator A769662 or adenovirus-containing small interfering RNA (ad-AMPKα-siRNA) for 3 or 48 h, respectively, followed by H 2 O 2 exposure (200 μ M) for 2 h. Compared with healthy cows, clinical ketosis led to increased abundance of AMPK and nuclear factor erythroid-derived 2-like 2 (NFE2L2), but lower abundance of Kelch-like ECH-associated protein 1 (KEAP1) in adipose tissue. Abundance of the key proautophagy proteins Beclin1, sequestosome 1 (SQSTM1), autophagy-related gene 7 (ATG7), ATG5, and ratio of microtubule-associated protein light chain 3 (LC3) II to LC3I were greater in adipose tissue of ketotic cows. In bovine adipocytes, treatment with H 2 O 2 induced accumulation of ROS and malondialdehyde (MDA), whereas H 2 O 2 stimulation inhibited activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Addition of AMPK activator A769662 increased antioxidant response via activating NFE2L2 and its downstream targets heme oxygenase 1 (HMOX1), superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione-S-transferase (GST) to improve H 2 O 2 -induced oxidative stress in adipocytes. Simultaneously, activation of AMPK increased abundance of Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I. In contrast, inhibition of AMPK downregulated abundance of NFE2L2, HMOX1, SOD1, CAT, Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I, and further aggravated H 2 O 2 -induced oxidative stress. Overall, these data indicate that activation of AMPK, as an adaptive mechanism for acute metabolic regulation of adipose tissue homeostasis, can induce antioxidant responses and autophagy, and further reduce oxidative stress in bovine adipocytes. [ABSTRACT FROM AUTHOR]