Ketotic dairy cows exhibit a state of negative energy balance (NEB) characterized by elevated circulating levels of β-hydroxybutyrate (BHB) and fatty acids. Impaired hepatic insulin signaling in dairy cows occurs frequently during the transition into lactation, but its role on liver function during this period is not well known. In nonruminants, endoplasmic reticulum (ER) stress is a causal factor contributing to impaired insulin signaling in the liver. Thus, the aim of this study was to investigate the status of hepatic insulin and ER stress signaling and whether ER stress contributes to impaired insulin signaling in dairy cows with ketosis. Healthy (control cows, n = 10, BHB ≤0.6 mM) and ketotic (ketotic cows, n = 10, BHB ≥1.2 mM) cows at 3 to 10 d in milk were selected for liver biopsy and blood sampling before feeding. In vitro experiments were conducted with isolated hepatocytes from 5 healthy calves (1 d old, fasted female, 30-40 kg of body weight). Treatments included BHB (0, 0.9, 1.8, 3.6 mM), tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress), and different incubation times (0.5, 1, 2, 3, 5, 7, 9, or 12 h). Ketotic cows had lower daily milk yield (median: 29.50 vs. 23.00 kg), higher plasma nonesterified fatty acid (NEFA) (median: 0.33 vs. 1.17 mM), BHB (median: 0.43 vs. 3.22 mM), aspartate aminotransferase (median: 70.58 vs. 155.70 U/L), alanine aminotransferase (median: 18.31 vs. 37.90 U/L), lower plasma glucose (median: 4.32 vs. 2.37 mg/dL), and revised quantitative insulin sensitivity check index (median: 0.39 vs. 0.37) compared with healthy cows. Increased abundance of phosphorylated insulin receptor substrate-1 (IRS1) and decreased abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in ketotic cows indicated a state of insulin resistance. In addition, abundance of phosphorylated protein kinase RNA-like ER kinase (PERK) and inositol requiring protein-1α (IRE1α), and cleavage of activating transcription factor-6 (ATF6) were greater in the liver of ketotic cows. In vitro, at the early stages of incubation, treatment with BHB upregulated abundance of phosphorylated of IRE1α, PERK, and the cleavage of ATF6, as well as several unfolded protein response genes [X-box-binding protein-1 (XBP1), 78 kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP)]. Furthermore, in response to increasing doses of BHB, the phosphorylation level of PERK, IRE1α, and the cleavage of ATF6, and the abundance of XBP1, GRP78, and CHOP increased. In addition, BHB treatment increased phosphorylation of IRS1 and decreased phosphorylation of AKT and GSK3β, and upregulated abundance of gluconeogenic genes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase). Importantly, these changes were reversed by inhibiting ER stress with TUDCA treatment. Overall, the present study indicated that reversing ER stress during ketosis might help alleviate hepatic insulin resistance. Targeting ER stress may represent a potential therapeutic target for controlling the negative aspects of ketosis on liver function.

We investigated effects of rumen-protected Met (RPM) during a heat stress (HS) challenge on (1) hepatic abundance of mTOR, insulin, and antioxidant signaling proteins, (2) enzymes in 1-carbon metabolism, and (3) innate immunity. Holstein cows (n = 32; mean ± standard deviation, 184 ± 59 d in milk) were randomly assigned to 1 of 2 environmental groups, and 1 of 2 diets [total mixed ration (TMR) with RPM (Smartamine M; 0.105% dry matter as top-dress) or TMR without (CON); n = 16/diet] in a split-plot crossover design. There were 2 periods with 2 phases. During phase 1 (9 d), all cows were in thermoneutral conditions (TN; temperature-humidity index = 60 ± 3) and fed ad libitum. During phase 2 (9 d), half the cows (n = 8/diet) were exposed to HS using electric heat blankets. The other half (n = 8/diet) remained in TN, but was pair-fed to HS counterparts. After a 14-d washout and 7-d adaptation period, the study was repeated (period 2) and environmental treatments were inverted relative to phase 2, but dietary treatments were the same. Blood was collected on d 6 of each phase 2 to measure immune function and isolate whole-blood RNA. Liver biopsies were performed at the end of each period for cystathione β-synthase (CBS) and methionine adenosyltransferase activity, glutathione concentration, and protein abundance. Data were analyzed using PROC MIXED in SAS. Abundance of CUL3, inhibitor of antioxidant responses, tended to be downregulated by HS suggesting increased oxidative stress. Heat-shock protein 70 abundance was upregulated by HS. Phosphorylated mTOR abundance was greater overall with RPM, suggesting an increase in pathway activity. An environment × diet (E × D) effect was observed for protein kinase B (AKT), whereas there was a tendency for an interaction for phosphorylated AKT. Abundance of AKT was upregulated in CON cows during HS versus TN, this was not observed in RPM cows. For phosphorylated AKT, tissue from HS cows fed CON had greater abundance compared with all other treatments. The same effect was observed for EIF2A (translation initiation) and SLC2A4 (insulin-induced glucose uptake). An E × D effect was observed for INSR due to upregulation in CON cows during HS versus TN cows fed CON or RPM. There was an E × D effect for CBS, with lower activity in RPM versus CON cows during HS. The CON cows tended to have greater CBS during HS versus TN. An E × D effect was observed for methionine adenosyltransferase, with lower activity in RPM versus CON during HS. Although activity increased in CON during HS versus TN, RPM cows tended to have greater activity during TN. Neutrophil and monocyte oxidative burst and monocyte phagocytosis decreased with HS. An (E × D) effect was observed for whole-blood mRNA abundance of CBS, SOD1 and CSAD; RPM led to upregulation during TN versus HS. Regardless of diet, CDO1, CTH, and SOD1 decreased with HS. Although HS increased hepatic HSP70 and seemed to alter antioxidant signaling, feeding RPM may help cows maintain homeostasis in mTOR, insulin signaling, and 1-carbon metabolism. Feeding RPM also may help maintain whole-blood antioxidant response during HS, which is an important aspect of innate immune function.

Lipolysis is increased in adipose tissue of cows with fatty liver during the transition period. Autophagy, a major cellular degradation process, plays a critical role in adipose tissue homeostasis. The objective of this study was to explore the relationship between lipolysis and autophagy in adipose tissue of cows with fatty liver. Using a nested case-control design, we compared blood and adipose tissue samples from 10 control cows [parity: median = 3, range = 2-4; days in milk: median = 8 d, range = 5-10 d; hepatic triacylglycerol content: median = 0.55% liver wt, range = 0.48-0.61% liver wt] and 10 lactation stage-matched cows with fatty liver (parity: median = 3, range = 2-4; days in milk: median = 9 d, range = 5-11 d; hepatic triacylglycerol content: median = 6.28% liver wt, range = 2.86-7.75% liver wt). Data were analyzed using paired t-tests. Serum concentrations of free fatty acids and β-hydroxybutyrate were greater and glucose concentration was lower in cows with fatty liver, which we determined by using commercially-available kits. Furthermore, western blotting showed that increased protein abundance of ATGL (adipose triglyceride lipase), ATG5 (autophagy-related gene 5), and ATG7; ratio of phosphorylated (p)-HSL (hormone-sensitive lipase) to HSL and MAP1LC3 (microtubule-associated protein 1 light chain 3, also called LC3-II) to LC3-I along with decreased abundance of PLIN1 (perilipin 1), SQSTM1 (sequestosome-1, also called p62), and the ratio of p-mTOR (phosphorylated mechanistic target of rapamycin) to mTOR in cows with fatty liver. Quantitative reverse-transcription PCR revealed an increase in abundance of MAP1LC3 mRNA and a decrease in SQSTM1 mRNA in cows with fatty liver. These findings were replicated using an adipocyte model. Primary cultures of calf adipocytes isolated from the adipose tissue of the peritoneal omentum and mesentery were treated with 10 mM 3-methyladenine (3-MA), 5 nM rapamycin, 1 µM isoproterenol (ISO), and 1 µM ISO + 10 mM 3-MA. Comparisons among groups were analyzed using one-way ANOVA. Compared with the control, the 1 µM ISO treatment upregulated the abundance of ATGL, the ratio of p-HSL to HSL and LC3-II to LC3-I, and the glycerol content, whereas it downregulated the abundance of PLIN1 and p62 in calf adipocytes. Compared with the 1 μM ISO treatment group, 1 µM ISO + 10 mM 3-MA downregulated the abundance of ATGL, the ratio of p-HSL to HSL and LC3-II to LC3-I, and the glycerol content, whereas it upregulated the abundance of PLIN1 and p62. Compared with the control, the 5 nM rapamycin treatment upregulated the abundance of ATGL, the ratio of p-HSL to HSL and LC3-II to LC3-I, and the glycerol content, whereas it downregulated the abundance of PLIN1 and p62 in calf adipocytes. Overall, these data indicated that increased lipolysis in adipose tissue of cows with fatty liver was associated with enhanced autophagy. However, the specific molecular mechanisms that link lipolysis and autophagy need to be further investigated.

Disruption of endoplasmic reticulum (ER) homeostasis, a condition termed "ER stress," contributes to the development of liver injury in nonruminants. Because liver injury is a prominent pathological feature associated with overproduction of ketone bodies in dairy cows with ketosis, understanding the ER stress state and its functional consequences on liver injury is of particular interest. Here, 30 multiparous cows (within 3 wk postpartum) classified based on blood β-hydroxybutyrate (BHB) as healthy (n = 15, BHB 3.0 mM) were used. Compared with healthy cows, ketotic cows had greater levels of serum fatty acids and activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, and glutamate dehydrogenase but lower serum glucose. Furthermore, dairy cows with ketosis had greater protein abundance of ER stress markers in liver tissue, including protein kinase RNA-like ER kinase (PERK), inositol-requiring protein-1α (IRE1α), and cleaved activating transcription factor-6 (ATF6). Cows with ketosis also had higher mRNA levels of hepatic 78-kDa glucose-regulated protein (GRP78) and spliced X-box binding protein 1 (sXBP1). These data confirmed an enhanced ER stress state in clinically ketotic cows. To explore whether enhanced hepatic ER stress was induced by elevated ketone bodies and the possible contribution of ER stress to liver injury, in vitro experiments were then performed using isolated primary calf hepatocytes treated with incremental concentrations of BHB (0, 0.6, 1.2, 3.0, and 4.8 mM) for 12 h with or without overexpression of GRP78 (the master regulator of unfolded protein response). Phosphorylation levels of PERK and IRE1α proteins, level of cleaved ATF6 protein, and mRNA abundance of GRP78 and sXBP1 in hepatocytes increased after treatment with high (3.0 and 4.8 mM) BHB, indicating a mechanistic link between excessive BHB and enhanced hepatic ER stress. Furthermore, treatment with 3.0 and 4.8 mM BHB markedly elevated activities of aspartate aminotransferase and alanine aminotransferase in cell supernatant, indicating exacerbated hepatocyte damage after ER stress was enhanced. Overexpression of GRP78 attenuated both BHB-induced ER stress and the ensuing cellular damage, suggesting that hepatocyte damage caused by excessive BHB can be mediated via enhanced ER stress. Overall, the present study revealed that ER stress may exacerbate liver injury development in clinically ketotic cows, underscoring the biological relevance of this pathway in the context of liver injury.

Susceptibility to mastitis is highest during the peri-partal (transition) period and is often concomitant with other comorbidities such as ketosis. Although infection with pathogenic microorganisms and immune-dysfunc-tion around calving clearly play key roles in mastitis development, other metabolic factors also contribute. Sirtuin 3 (SIRT3), a mitochondrial deacetylase regu-lating energy and redox homeostasis, antagonizes the lipotoxic effects of nonesterified fatty acids (NEFA). Thus, we hypothesized that increases in circulating NEFA concentrations, as observed in the transition period, provokes inflammatory responses that can be reversed via activation of SIRT3. Here we aimed to study (1) proinflammatory NF-Kappa B signaling and SIRT3 abundance in mammary tissue of ketotic cows and healthy controls, and (2) the effect of SIRT3 on NF-Kappa B activation in bovine mammary epithelial cells (BMEC) treated with high levels of NEFA. The mam-mary gland biopsy samples were from a previous study, which included 15 healthy cows and 15 ketotic cows. Primary BMEC were isolated from 3 healthy Holstein cows with collagenase III digestion. Purified BMEC were incubated with or without SIRT3 overexpression adenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 24 h. Mammary tissue of ketotic cows was associated with lower protein abundance of SIRT3 along with greater NF-Kappa B P65 phosphorylation levels (p-NF-Kappa B P65), p-NF-Kappa B P65: NF -Kappa B P65 ratio, and mRNA abundance of IL1B and IL6. In BMEC, exog-enous NEFA dose-dependently reduced protein abun-dance of SIRT3, but increased p-NF-Kappa B P65, p-NF-Kappa B P65: NF -Kappa B P65 ratio, and mRNA abundance of IL1B and IL6. Compared with green fluorescent protein adenovirus vector + NEFA, overexpression of SIRT3 in NEFA-treated BMEC downregulated p-NF-Kappa B P65 and mRNA abundance of IL1B and IL6. Immuno-fluorescence indicated that overexpression of SIRT3 inhibited nuclear translocation of NF-Kappa B P65. Overall, our data demonstrated that ketosis is associated with a reduction in SIRT3 abundance and activation of NF-Kappa B signaling in the mammary gland. In vitro data pro-vided evidence that high NEFA concentrations inhibit SIRT3, which contributes to enhanced NF-Kappa B signaling including nuclear translocation and a pro-inflammatory response. The data suggest a promising role of SIRT3 as a target for helping alleviate localized inflammation of the mammary gland resulting from exposure to high concentrations of NEFA.