The store-operated Ca 2+ 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 siO-RAI1 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 Ca 2+ 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.
The Orai calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca 2+ entry mechanism regulating cellular Ca 2+ 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 BTP 2 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 Ca 2+ ionophore ionomycin (raises the intracellular level of Ca 2+ ) 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 Ca 2+ signaling and ORAI1 seem to mediate in part the BHB-induced ER stress in mammary cells.
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.
Hypocalcemia in dairy cows is associated with decreased neutrophil phagocytosis, adhesion capacity, migration, and reactive oxygen species (ROS) production through alterations in ORAI calcium release-activated calcium modulator 1 (ORAI1). Neutrophils can resist the invasion of pathogenic microorganisms by releasing neutrophil extracellular traps (NET). However, the mechanisms controlling NET formation during hypocalcemia are unknown. To address the role of ORAI1 in NET formation, neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (n = 10 per group) diagnosed as clinically healthy (control) or with plasma concentrations of Ca 2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (SCH). A series of ex vivo experiments were conducted as follows: first, neutrophils isolated from both groups of cows were treated with phorbol 12-myristate 13-acetate (PMA) to stimulate NET formation; second, neutrophils from control and SCH were pretreated with or without the ROS scavenger N-acetylcysteine (NAC), the sarcoendoplasmic Ca 2+ ATPase inhibitor thapsigargin, or ORAI1 blocker 2APB and then treated with PMA to stimulate NET formation; and third, neutrophils were transfected with small interfering (si)ORAI1 or nontarget siRNA (siNEG) and then stimulated with PMA to induce formation of NET. A one-way ANOVA was used for statistical analysis of individual experiments. In the first experiment, neutrophils from SCH cows formed NET with fewer DNA filaments, more diffused nuclei, and reduced translocation of myeloperoxidase (MPO) and neutrophil elastase (NE) to the nucleus. Neutro-phils from SCH cows stimulated with PMA had a lower mitochondrial permeability, the state of mitochondrial permeability transition pore was open, ROS production was lower and there was increased mitochondrial damage. In the second experiment, in both control and SCH-PMA stimulated neutrophils, exogenous NAC decreased NET formation (assessed via Hoechst 33342 dye; Beyotime). Furthermore, following the challenge with PMA, thapsigargin increased NET formation and ROS production, but blocking ORAI1 with 2APB decreased NADPH oxidase activation, ROS production, and NET formation. In the third experiment, neutrophils transfected with siORAI1 before stimulation with PMA had lower intracellular concentrations of Ca 2+ , NET formation, and ROS production. Overall, the data indicated that SCH reduces NET formation in neutrophils partly due to damaged mitochondria. The reduction in ORAI1 abundance in neutrophils of dairy cows with hypocalcemia also decreases ROS production.
Hypocalcemia is closely associated with inflammatory diseases in dairy cows. Recent research has underscored the key role of calcium in the adaptations of the innate immune system during this period. The main objective in the present study was to compare the transcriptome profiles and analyze differences in the expression of neutrophil (PMNL) immune function-related genes and calcium binding-related genes in hypocalcemic cows. At 2 days postpartum, a concentration >2.10 mmol Ca2+/L was used to classify cows as controls (CON), and a concentration <2.00 mmol Ca2+/L used to classify cows as low-calcium (LCAL) (n = 8 in each group). A routine medical examination was conducted by the attending veterinarian to ensure there were no other complications and that the blood β-hydroxybutyrate was <1.2 mmol/L. Blood was collected from the tail vein (20 mL) to isolate PMNL, and 5 cows in each group were used for RNA sequencing and statistical analysis of gene expression differences. Transcriptome RNA-seq sequencing analysis was via omicsstudio using the R package edgeR. GO and KEGG enrichment analysis were used for bioinformatics. The remaining 3 cows in each group were used for validation of RNA sequencing data via quantitative PCR, which confirmed the observed responses. Compared with CON, 158 genes in LCAL were significantly up-regulated and 296 genes were down-regulated. The downregulation of Interleukin-12 (CXCL12), Tubulin beta chain (TUBB1), L1 cell adhesion molecule (L1CAM), and Myeloperoxidase (MPO) indicated a decrease in immune function of PMNL in LCAL cows. The decreased expression of calcium-binding pathway-related genes in PMNL of LCAL cows indicated a decrease in immune function of PMNL likely related to calcium ions. For example, cartilage acid protein 1 (CRTAC1) and calcium/calmodulin-dependent kinase 4 (CAMK4) were significantly reduced in LCAL cows. The upregulation of Cyclin dependent kinase inhibitor 1A (CDKN1A), Perforin 1 (PRF1), and Homeodomain interacting protein kinase 3 (HIPK3) indicated that LCAL led to greater cell apoptosis and senescence. Overall, the analyses indicated that the reduction in PMNL immune function during hypocalcemia is associated with downregulation of intracellular Ca2+ related genes and upregulation of genes controlling apoptosis and senescence. Together, these alterations contribute to an immunosuppressive state during the transition period.
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