Introduction: This study aimed to characterise the effects of ketosis on milk yield and composition and digestive capacity in transition dairy cows. Material and Methods: Seven ketotic and seven healthy cows were housed in individual stalls for six days. Samples of plasma, milk, refused total mixed ration, and faeces were collected, and the blood biochemical parameters, milk yield and composition, dry matter intake, and faecal dry matter (FDM) production were determined. Results: Compared with healthy cows, the ketotic cows had significantly higher concentrations of milk fat and citrate, but lower levels of milk protein and lactose. The cows exhibited a need for acid detergent fibre in forage and better digestion of neutral detergent fibre, starch, crude protein, and phosphorus than healthy cows, but more fat and gross energy were excreted in their faeces. Ketotic cows had higher energy-corrected milk yields and lower FDM than healthy cows. Conclusion: Lower feed intake coinciding with the requirement to maintain high milk production is considered to be the cause of ketosis in dairy cows. Ketotic cows exhibited lower dry matter fat digestion.

The store-operated Ca²⁺ entry (SOCE) moiety ORAI calcium release-activated calcium modulator 1 (ORAI1) located in the endoplasmic reticulum (ER) participates in key cellular functions such as protein folding, transport, and secretion, and lipid metabolism. We used an in vitro approach to test whether exogenous fatty acids alter ORAI1 signaling and to explore potential consequences on mitochondrial dysfunction and ER stress. First, hepatocytes isolated from 4 healthy female calves (1 d old, 40-50 kg) were challenged with a 1.2 mM mixture of oleic, linoleic, palmitic, stearic, and palmitoleic acids for 0.5, 1, 3, 6, 9, and 12 h to measure oxidative stress [intracellular reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and hydrogen peroxide] and ER stress (protein abundance of PERK, IRE, ATF6, and GRP78). Concentrations of GSH and SOD decreased at 0.5 h, and MDA and hydrogen peroxide increased at 1 h; ER stress proteins increased at 6 h. To determine whether ER stress was caused by oxidative stress, primary calf hepatocytes were treated with the same 1.2 mM fatty acid mix or the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) for 6 h. We found that NAC prevented an increase in ER stress protein abundance. Next, the role of ORAI1 on ER stress was measured by transfecting hepatocytes with small interfering (si)ORAI1 or the ORAI1 inhibitor BTP2, followed by a challenge with 1.2 mM fatty acids for 3 h. Without inhibiting ORAI1, exogenous fatty acids upregulated ORAI1 mRNA and protein abundance, oxidative stress, ER stress proteins, and protein abundance of marker indicators of an opened mitochondrial permeability transition pore (mPTP). Inhibition with BPT2 or silencing via siORAI1 abrogated oxidative stress, including increased GSH concentration and SOD activity, decreased MDA, hydrogen peroxide, and ROS concentration; ER stress protein abundance was downregulated, and mitochondrial function was restored. Last, changes in markers of mPTP opening were evaluated by culturing hepatocytes for 6 h with the sarcoendoplasmic Ca²⁺ ATPase inhibitor thapsigargin or the calcium ionophore ionomycin. We detected an increase in VDAC1, CLPP, and CypD protein abundance, all of which indicated opening of the mPTP. Overall, data from these in vitro studies suggest that ORAI1 mediates ER stress induced by high concentrations of fatty acids, in part through alleviating mitochondrial dysfunction caused by oxidative stress.

Fatty acid accumulation in hepatocytes induced by high concentrations of fatty acids due to lipolysis and the associated oxidative damage they cause occur most frequently after calving. Because of their role in esterification of fatty acids, diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights, we performed in vivo and in vitro analyses using liver biopsies or isolated primary hepatocytes. The in vivo study (n = 5 cows/group) involved healthy cows [average liver triacylglycerol (TAG) = 0.78%; 0.58 to 0.93%, ratio of triglyceride weight to wet liver weight] or cows diagnosed with fatty liver (average TAG = 7.60%; 5.31 to 10.54%). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old, 44 to 53 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with DGAT1 inhibitor or DGAT2 inhibitor for 2 h followed by a challenge with (DGAT1 inhibitor + fatty acids or DGAT2 inhibitor + fatty acids) or without (DGAT1 inhibitor or DGAT2 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data analysis of liver biopsies was compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocyte treatment comparisons were assessed by one-way ANOVA, and multiplicity for each experiment was adjusted by the Holm's procedure. Data indicated that both fatty liver and in vitro challenge with fatty acids were associated with greater mRNA and protein abundance of SREBF1, FASN, DGAT1, and DGAT2. In contrast, mRNA and protein abundance of CPT1A and very low-density lipoprotein synthesis-related proteins MTTP and APOB were markedly lower. However, compared with fatty acid challenge alone, DGAT1 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A and APOB, and greater mRNA abundance of SREBF1 and MTTP. Furthermore, this treatment led to lower mRNA abundance of FASN and DGAT2 and TAG concentrations. Compared with fatty acid challenge alone, DGAT2 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A, MTTP, and APOB, and lower mRNA and protein abundance of SREBF1 and FASN. In addition, compared with control and fatty acids, there was greater protein abundance of GRP78 and PERK in both DGAT1 and DGAT2 inhibitor with or without fatty acids. Furthermore, compared with control and fatty acids, reactive oxygen species concentrations in the DGAT1 inhibitor with or without fatty acid group was greater. Overall, data suggested that DGAT1 is particularly relevant in the context of hepatocyte TAG synthesis from exogenous fatty acids. Disruption of both DGAT1 and DGAT2 altered lipid homeostasis, channeling fatty acids toward oxidation and generation of reactive oxygen species. Both DGAT isoforms play a role in promoting fatty acid storage into TAG and lipid droplets to protect hepatocytes from oxidative damage.

The Orai calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca2+ entry mechanism regulating cellular Ca2+ balance in nonruminants. Alterations in ORAI1 abundance have been associated with endoplasmic reticulum (ER) stress and changes in lipid metabolism in hepatocytes, an important lipogenic organ in nonruminants. Objectives were to (1) determine abundance of ORAI1 and components of the ER stress response in mammary tissue of ketotic cows, and (2) the potential role of ORAI1 on mammary cell responses to high levels of β-hydroxybutyrate (BHB). Healthy (n = 6, plasma BHB < 0.60 mmol/L) and clinically ketotic (n = 6, plasma BHB > 2.0 mmol/L) Holstein cows (days in milk = 10.13 ± 1.90) were used for mammary gland tissue and blood sample collection. Although milk production (22.5 ± 1.26, 33 ± 1.59, kg of milk/cow per day) and dry matter intake (19.5 ± 1.05, 21.9 ± 0.95, kg/d) were lower in ketotic cows, abundance of ORAI1 protein was greater and was associated with greater mRNA abundance of ER stress proteins (PERK, IRE1, ATF6, and GRP78) and lipogenic genes (FASN, SREBP1, and ACACA). Cellular mechanisms to establish links between BHB and mammary cell responses were evaluated using the immortalized cell line bovine mammary epithelial cells (MAC-T). First, a dose response study was performed with 0, 0.6, 1.2, 1.8, 2.4, or 4.8 mM BHB for 24 h. The mRNA abundance of FASN, SREBP1, and ACACA and lipid droplet formation peaked at 1.2 mM BHB. A subsequent study involved transfecting MAC-T with small interfering Orai 1 (siORAI1) or the ORAI1 inhibitor BTP2 for 24 h followed by a challenge with 1.2 mM BHB for 24 h. Transcription and protein abundance of FASN, SREBP1, ACACA, and ER stress proteins returned to basal levels when ORAI1 was silenced or inhibited. Furthermore, the Ca2+ ionophore ionomycin (raises the intracellular level of Ca2+) also increased abundance of ORAI1, FASN, SREBP1, ACACA, and ER stress proteins. Data suggest that the mammary gland experiences ER stress during ketosis, partly due to the greater supply of BHB originating from ketogenesis in the liver. Intracellular Ca2+ signaling and ORAI1 seem to mediate in part the BHB-induced ER stress in mammary cells.

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 μg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 μM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 μg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 μg/mL) for 24 h and then challenged with 150 μg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 μM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 μg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 μg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 μg/mL LPS; concentrations of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1β, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.

Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7-13 d in milk) and cows with fatty liver (n = 6; 7-11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-β-cyclodextrin (MβCD + FA) or 0.147 mg/mL MβCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, β-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MβCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver.