Expression Of Pyruvate Carboxylase mRNA Research Articles

Abstract 2327: Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions

Abstract Breast cancer is the most diagnosed cancer in women, and metastasis accounts for 90% of cancer related deaths. In order to metastasize, cancer cells must survive in conditions where they are detached from the extracellular matrix (ECM). Untransformed cells decrease energy metabolism when detached from ECM, but oncogenic transformation results in rescued energy metabolism of detached cells. Expression of the anaplerotic enzyme pyruvate carboxylase (PC) is required for breast to lung metastasis, and may play a role in promoting ECM detached cell survival by supporting metabolic plasticity. PC activity results in the carboxylation of pyruvate to synthesize the TCA cycle intermediate oxaloacetate (OAA). We hypothesize that PC expression is required for cell survival in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. ECM detachment was modeled using Poly-HEMA coated plates in combination with MTT assays to measure cell viability. qPCR was used to quantify mRNA abundance of metabolic genes. Protein expression was quantified using western blot. PC activity was determined using [13C6]-glucose flux analysis by comparing 3 carbon labeled (M+3) citrate, malate, and aspartate. Results show a 94% increase in PC mRNA expression and a 90% increase in PC protein expression, in detached cells compared to attached cells. In addition PC knockdown decreases viability in ECM detached conditions significantly by 15%, while PC overexpression increases viability by 17%. Further, replenishing the TCA cycle with OAA, the product of the PC reaction, rescues PC knockdown cells by 14%. Increased PC activity in detached cells was determined by a 23% increase in M+3 malate and a 61% increase in M+3 aspartate. However, an 11% decrease in M+3 citrate was observed, suggesting that detached cells do not utilize increased PC expression to replenish the TCA cycle intermediate, citrate. Given the larger relative increase in PC activity demonstrated by M+3 aspartate, we hypothesized that PC activity is required for detached cell survival to provide substrate for aspartate synthesis. Providing aspartate (2.5 mM), a downstream product of PC, in detached cells with PC knockdown rescued cell viability with no benefit of aspartate supplementation in detached PC expressing cells. Further, the aspartate synthesizing enzyme, glutamate OAA transferase 2 (GOT2) mRNA expression was unchanged in detached cells. In summary PC expression and activity is increased in detached cells compared to attached cells, and OAA produced by the PC reaction is utilized by cells to synthesize aspartate and maintain cell viability in detached conditions. PC may be a useful target for future treatments aimed at preventing metastasis. Citation Format: Madeline P. Sheeley, Dorothy Teegarden. Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2327.

Pretreatment with saturated and unsaturated fatty acids regulates fatty acid oxidation in Madin-Darby bovine kidney cells

Metabolic fates of fatty acids in tissue may be influenced by extracellular concentration and profile of fatty acids. Previous work has demonstrated the ability of C18:3n-3 cis to ameliorate the effects of C16:0- or C18:0-induced depression of pyruvate carboxylase (PC) mRNA expression. Pyruvate carboxylase catalyzes oxaloacetate synthesis and connects gluconeogenesis from lactate and fatty acid metabolism. Our objective was to determine the effects of co-presence of saturated and unsaturated fatty acids on cellular partitioning of [1-14C]C16:0 metabolism to CO2 or acid-soluble products (ASP) in Madin-Darby bovine kidney cells and the role of PC in this relationship. We hypothesized that the ratio of saturated to unsaturated fatty acid pretreatments regulates [1-14C]C16:0 partitioning to CO2 or ASP. Cells were exposed for 21 h to either individual fatty acids, C16:0, C18:0, C18:1n-9 cis, or C18:3n-3 cis, or to fatty acid combinations in 10:90, 25:75, 50:50, 75:25, or 90:10 ratios for 3 combinations: C16:0/C18:3n-3 cis, C18:0/C18:3n-3 cis, or C18:1n-9 cis/C18:3n-3 cis. Total fatty acid concentration was 1.0 mM during the 21-h pretreatment phase. Following the 21-h incubation phase with fatty acid combinations, cells were incubated in the presence of 1.0 mM [1-14C]C16:0 for 3 h to determine the rate of metabolism to CO2 and ASP collection (per µg DNA-1·h-1). Pretreatment with either C16:0 or C18:0 alone significantly depressed subsequent oxidation of [1-14C]C16:0 to ASP by 62.7 and 41.2%, respectively, compared with C18:3n-3 cis pretreatment. Similar patterns were observed for [1-14C]C16:0 oxidation to CO2. Expression of PC mRNA was significantly decreased with exposure to either C16:0 or C18:0 compared with expression after exposure to either C18:3n-3 cis or control 1% BSA in Dulbecco's modified Eagle's medium. Expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA followed a similar pattern. Fatty acid treatments containing C18:1n-9 cis did not alter PC or PCK1 expression from control or C18:3n-3 cis results. Pearson coefficient correlations were determined for PC mRNA expression and rate of [1-14C]C16:0 metabolism to CO2 or ASP, including ketones, and for PCK1 mRNA expression and rate of [1-14C]C16:0 metabolism to CO2 or ASP. Production of CO2 from [1-14C]C16:0 was positively correlated (r = 0.63) with PC expression, whereas ASP production from [1-14C]C16:0 only tended to positively correlate (r = 0.51) with PC mRNA expression. Production of CO2 or ASP from [1-14C]C16:0 were both positively correlated (r = 0.80 and r = 0.69, respectively) with PCK1 expression. Results show a regulation of ketone production by Madin-Darby bovine kidney cells in response to saturated and unsaturated fatty acid pretreatments.

Abstract 2571: 1,25-dihydroxyvitamin D regulation of pyruvate carboxylase in MCF10A-ras cells decreases viability in extracellular matrix detached conditions

Abstract Metastasis is the main cause of most breast cancer related deaths. Throughout the process of metastasis, cancer cells strive to survive in conditions where they are detached from the extracellular matrix (ECM). Vitamin D may play a role in preventing breast cancer metastasis by modulating metabolism in breast cancer cells detached from ECM. The anaplerotic enzyme pyruvate carboxylase (PC) is essential for breast to lung metastasis and the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), downregulates PC expression through a vitamin D response element. PC replenishes the TCA cycle with the intermediate oxaloacetate (OAA), and lower expression of PC following 1,25D treatment may cause impaired energy metabolism via the TCA cycle. We hypothesize that the 1,25D mediated decrease in PC expression decreases breast cancer cell viability in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) treated with vehicle (ethanol) or 10 nM 1,25D and maintained in varying nutrient concentrations were utilized for these studies. Poly-HEMA coated plates were used to model ECM detachment paired with MTT assays to measure cell viability. mRNA expression of metabolic genes is quantified using qPCR. Results show that when compared to attached cells, detached cells have a 94% increase in PC mRNA expression, while 1,25D treatment decreases PC mRNA expression in both attached by 75% and detached conditions by 63%. In addition 1,25D treatment and PC knockdown decreases viability in ECM detached conditions significantly by 16% and 11%, with no additional effect when combined. Glutamine deprivation also decreases viability by 40% in detached conditions, however, the combination of 1,25D treatment and glutamine deprivation results in a 71% decrease in viability. These results suggest that while glutamine is required for ECM survival, regulation of glutamine metabolism is not the mechanism by which 1,25D affects ECM survival. Replenishing the TCA cycle with OAA rescues glutamine depleted cells by 84% as well as PC knockdown cells by 14%. Further, when stressed with low glutamine conditions, cells in attached conditions increase mRNA expression of PC by 311%, suggesting that changes in PC expression may be a mechanism that cancer cells use to adapt to energy related stress. These results suggest that 1,25D regulation of metabolism may sensitize breast cancer cells to ECM detachment death via downregulation of PC, reducing the required flux of substrates into the TCA cycle which are needed to survive ECM detached conditions during metastasis. Citation Format: Madeline Sheeley, Dorothy Teegarden, Katie Wong. 1,25-dihydroxyvitamin D regulation of pyruvate carboxylase in MCF10A-ras cells decreases viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2571.

Postpartum supplementation with fermented ammoniated condensed whey altered nutrient partitioning to support hepatic metabolism

Our previously published paper demonstrated that fermented ammoniated condensed whey (FACW) supplementation improved feed efficiency and metabolic profile in postpartum dairy cows. The objective of this study was to further explore the effects of FACW supplementation on liver triglyceride content, hepatic gene expression and protein abundance, and plasma biomarkers related to liver function, inflammation, and damage. Individually fed multiparous Holstein cows were blocked by calving date and randomly assigned to postpartum (1 to 45 d in milk, DIM) isonitrogenous treatments: control diet (n = 20) or diet supplemented with FACW (2.9% dry matter of diet as GlucoBoost; Fermented Nutrition, Luxemburg, WI, replacing soybean meal; n = 19). Liver biopsies were performed at 14 and 28 DIM for analysis of mRNA expression, protein abundance, and liver triglyceride content. There was marginal evidence for a reduction in liver triglyceride content at 14 DIM in FACW-supplemented cows compared with the control group. Cows supplemented with FACW had greater mRNA expression of glucose-6-phosphatase at 14 DIM relative to control. Supplementation with FACW increased mRNA expression of pyruvate carboxylase (PC), but did not alter cytosolic phosphoenolpyruvate carboxykinase (PCK1), resulting in a 2.4-fold greater PC:PCK1 ratio for FACW-supplemented cows compared with control. There was no evidence for a FACW effect on mRNA expression of propionyl-CoA carboxylase nor on mRNA expression or protein abundance of lactate dehydrogenase A or B. Cows supplemented with FACW had lower plasma urea nitrogen compared with control. Plasma l-lactate was greater for FACW-supplemented cows compared with control at 2 h before feeding time at 21 DIM. There was no evidence for altered expression of IL1B or IL10, or blood biomarkers related to liver function and damage. Greater glucose-6-phosphatase and PC gene expression, together with greater blood glucose and similar milk lactose output, suggests that FACW increased the supply of glucose precursors, resulting in greater gluconeogenesis between 3 and 14 DIM. Greater hepatic PC:PCK1 ratio, together with previously reported decreased plasma β-hydroxybutyrate and the marginal evidence for lower liver triglyceride content at 14 DIM, suggests greater hepatic capacity for complete oxidation of fatty acids in FACW-supplemented cows compared with control. Overall, improvements in metabolite profile and feed efficiency observed with postpartum supplementation of FACW may be attributed to increased gluconeogenic and anaplerotic precursors, most likely propionate, due to modulated rumen fermentation.

Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

BackgroundOvercoming systemic dormancy and initiating secondary tumor grow under unique microenvironmental conditions is a major rate-limiting step in metastatic progression. Disseminated tumor cells encounter major changes in nutrient supplies and oxidative stresses compared to the primary tumor and must demonstrate significant metabolic plasticity to adapt to specific metastatic sites. Recent studies suggest that differential utilization of pyruvate sits as a critical node in determining the organotropism of metastatic breast cancer. Pyruvate carboxylase (PC) is key enzyme that converts pyruvate into oxaloacetate for utilization in gluconeogenesis and replenishment of the TCA cycle.MethodsPatient survival was analyzed with respect to gene copy number alterations and differential mRNA expression levels of PC. Expression of PC was analyzed in the MCF-10A, D2-HAN and the 4 T1 breast cancer progression series under in vitro and in vivo growth conditions. PC expression was depleted via shRNAs and the impact on in vitro cell growth, mammary fat pad tumor growth, and pulmonary and non-pulmonary metastasis was assessed by bioluminescent imaging. Changes in glycolytic capacity, oxygen consumption, and response to oxidative stress were quantified upon PC depletion.ResultsGenomic copy number increases in PC were observed in 16–30% of metastatic breast cancer patients. High expression of PC mRNA was associated with decreased patient survival in the MCTI and METABRIC patient datasets. Enhanced expression of PC was not recapitulated in breast cancer progression models when analyzed under glucose-rich in vitro culture conditions. In contrast, PC expression was dramatically enhanced upon glucose deprivation and in vivo in pulmonary metastases. Depletion of PC led to a dramatic decrease in 4 T1 pulmonary metastasis, but did not affect orthotopic primary tumor growth. Tail vein inoculations confirmed the role of PC in facilitating pulmonary, but not extrapulmonary tumor initiation. PC-depleted cells demonstrated a decrease in glycolytic capacity and oxygen consumption rates and an enhanced sensitivity to oxidative stress.ConclusionsOur studies indicate that PC is specifically required for the growth of breast cancer that has disseminated to the lungs. Overall, these findings point to the potential of targeting PC for the treatment of pulmonary metastatic breast cancer.

1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase

Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)₂D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)₂D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)₂D-mediated regulation and hypothesized that 1,25(OH)₂D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)₂D treatment at 2 (73%±6 relative to vehicle) and 5 (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)₂D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)₂D regulates lipid metabolism in malignant breast epithelial cells.

Propionate induces mRNA expression of gluconeogenic genes in bovine calf hepatocytes

Hepatocytes monolayers from neonatal calves were used to determine the responses of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA expression to propionate and direct hormonal cues including cyclic AMP (cAMP), dexamethasone, and insulin. The responses of other key gluconeogenic genes, including mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphotase (G6PC), were also measured. Expression of PCK1 was linearly induced with increasing propionate concentrations in media and 2.5 mM propionate increased PCK1 mRNA at 3 and 6h of incubation; however, the induction disappeared at 12 and 24 h. The induction of PCK1 mRNA by propionate was mimicked by 1 mM cAMP, or in combination with 5 µM dexamethasone, but not by dexamethasone alone. The induction of PCK1 mRNA by propionate or cAMP was eliminated by addition of 100 nM insulin. Additionally, expression of PCK2 and PC mRNA was also induced by propionate in a concentration-dependent manner. Consistent with PCK1, propionate-stimulated PCK2 and PC mRNA expression was inhibited by insulin. Expression of G6PC mRNA was neither affected by propionate nor cAMP, dexamethasone, insulin, or their combinations. These findings demonstrate that propionate can directly regulate its own metabolism in bovine calf hepatocytes through upregulation of PCK1, PCK2, and PC mRNA expression.

Short communication: Regulation of hepatic gluconeogenic enzymes by dietary glycerol in transition dairy cows

Nutritional status and glucose precursors are known regulators of gluconeogenic gene expression. Glycerol can replace corn in diets fed to dairy cows and use of glycerol is linked to increased rumen propionate production. The effect of dietary glycerol on the regulation of gluconeogenic enzymes is unknown. The objective of this study was to examine the effect of glycerol on expression of pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M), and glucose-6-phosphatase. Twenty-six multiparous Holstein cows were fed either a control diet or a diet where high-moisture corn was replaced by glycerol from −28 through +56 d relative to calving (DRTC). Liver tissue was collected via percutaneous liver biopsy at −28, −14, +1, +14, +28, and +56 DRTC for RNA analysis. Expression of PC mRNA increased 6-fold at +1 and 4-fold at +14 DRTC relative to precalving levels. Dietary glycerol did not alter expression of PC mRNA expression. Expression of PEPCK-C increased 2.5-fold at +14 and 3-fold at +28 DRTC compared with +1 DRTC. Overall, dietary glycerol increased PEPCK-C expression compared with that of cows fed control diets. The ratio of PC to PEPCK-C was increased 6.3-fold at +1 DRTC compared with precalving and tended to be decreased in cows fed glycerol. We detected no effect of diet or DRTC on PEPCK-M or glucose-6-phosphatase mRNA, and there were no interactions of dietary treatment and DRTC for any transcript measured. Substituting corn with glycerol increased the expression of PEPCK-C mRNA during transition to lactation and suggests that dietary energy source alters hepatic expression. The observed increase in PEPCK-C expression with glycerol feeding may indicate regulation of hepatic gene expression by changes in rumen propionate production.

Effects of dry period length and dietary energy source on metabolic status and hepatic gene expression of dairy cows in early lactation

In a prior study, we observed that cows with a 0-d dry period had greater energy balance and lower milk production compared with cows with a 30- or 60-d dry period in early lactation. The objective of the current study was to evaluate the influence of dry period length on metabolic status and hepatic gene expression in cows fed a lipogenic or glucogenic diet in early lactation. Holstein-Friesian dairy cows (n=167) were assigned randomly to 3×2 factorial design with 3 dry period lengths (n=56, 55, and 56 for 0-, 30-, and 60-d dry, respectively) and 2 early lactation diets (n=84 and 83 for glucogenic and lipogenic diet, respectively). Cows were fed a glucogenic or lipogenic diet from 10d before the expected calving date and onward. The main ingredient for a glucogenic concentrate was corn, and the main ingredients for a lipogenic concentrate were sugar beet pulp, palm kernel, and rumen-protected palm oil. Blood was sampled weekly from 95 cows from wk 3 precalving to wk 8 postcalving. Liver samples were collected from 76 cows in wk −2, 2, and 4 relative to calving. Liver samples were analyzed for triacylglycerol concentrations and mRNA expression of 12 candidate genes. Precalving, cows with a 0-d dry period had greater plasma β-hydroxybutyrate, urea, and insulin concentrations compared with cows with a 30- or 60-d dry period. Postcalving, cows with a 0-d dry period had lower liver triacylglycerol and plasma nonesterified fatty acids concentrations (0.20, 0.32, and 0.36mmol/L for 0-, 30-, and 60-d dry period, respectively), greater plasma glucose, insulin-like growth factor-I, and insulin (24.38, 14.02, and 11.08µIU/mL for 0-, 30-, and 60-d dry period, respectively) concentrations, and lower hepatic mRNA expression of pyruvate carboxylase, compared with cows with a 30- or 60-d dry period. Plasma urea and β-hydroxybutyrate concentrations were greater in cows fed a lipogenic diet compared with cows fed a glucogenic diet. In conclusion, cows with a 0-d dry period had an improved metabolic status in early lactation, indicated by lower plasma concentrations of nonesterified fatty acids, greater plasma concentrations of glucose, insulin-like growth factor-I, and insulin, and lower mRNA expression of pyruvate carboxylase in the liver, compared with cows with a 30- or 60-d dry period. Independent of dry period length, the glucogenic diet also improved the metabolic status compared with the lipogenic diet.

Multiple E-boxes in the distal promoter of the rat pyruvate carboxylase gene function as a glucose-responsive element.

Pyruvate carboxylase (PC) is an anaplerotic enzyme that regulates glucose-induced insulin secretion in pancreatic islets. Dysregulation of its expression is associated with type 2 diabetes. Herein we describe the molecular mechanism underlying the glucose-mediated transcriptional regulation of the PC gene. Incubation of the rat insulin cell line INS-1 832/13 with glucose resulted in a 2-fold increase in PC mRNA expression. Transient transfections of the rat PC promoter-luciferase reporter construct in the above cell line combined with mutational analysis indicated that the rat PC gene promoter contains the glucose-responsive element (GRE), comprising three canonical E-boxes (E1, E3 and E4) and one E-box-like element (E2) clustering between nucleotides –546 and –399, upstream of the transcription start site. Mutation of any of these E-boxes resulted in a marked reduction of glucose-mediated transcriptional induction of the reporter gene. Electrophoretic mobility shift assays revealed that the upstream stimulatory factors 1 and 2 (USF1 and USF2) bind to E1, the Specificity Protein-1 (Sp1) binds to E2, USF2 and the carbohydrate responsive element binding protein (ChREBP) binds to E4, while unknown factors binds to E3. High glucose promotes the recruitment of Sp1 to E2 and, USF2 and ChREBP to E4. Silencing the expression of Sp1, USF2 and ChREBP by their respective siRNAs in INS-1 832/13 cells blunted glucose-induced expression of endogenous PC. We conclude that the glucose-mediated transcriptional activation of the rat PC gene is regulated by at least these three transcription factors.

Effect of leptin on the gluconeogenesis in calf hepatocytes cultured in vitro

We have investigated the effect of leptin on gluconeogenesis in the liver. Hepatocytes were cultured and treated with 0, 2.5, 5, 10, 50, 100 ng/mL of leptin in groups I, II, III, IV, V, and VI, respectively. mRNA expression and enzyme activity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase were determined by real-time fluorescence quantitative RT-PCR and biochemical kits, respectively. Compared with group I, mRNA expression of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in groups III, IV, V, and VI were significantly lower (P < 0.01). Pyruvate carboxylase and phosphoenolpyruvate carboxykinase enzyme activity decreased significantly (P < 0.05) when leptin concentration exceeded 5 and 10 ng/mL, respectively. These results indicate that leptin markedly downregulated mRNA expression and enzyme activity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in hepatocytes, which suggests that high concentrations of LP inhibit hepatocyte gluconeogenesis, thus making negative energy balance more serious.

Short communication: Genetic differences between New Zealand and North American dairy cows alter milk production and gluconeogenic enzyme expression

Continuous selection of dairy cows for production traits may alter the regulation of metabolic pathways. High-producing North American (NA) cows produce more milk and have a larger degree of somatotropic axis uncoupling than less intensively selected New Zealand (NZ) cows. The objective of this study was to determine if production-based selection priorities (i.e., NA cows) have altered the regulation of the gluconeogenic pathway relative to selection priorities based on production traits (i.e., NZ cows). In this study conducted in New Zealand, NZ (n=27) and NA cows (n=27) were monitored from 1 wk before calving to 12 wk post-calving. Cows were pasture-fed and supplemented with 0, 3, or 6kg of concentrate DM/d. Liver biopsy samples were collected at 0, +1, and +4 wk relative to calving (WRTC) for mRNA analysis. Milk production of NA cows was greater during wk 5 to 11 postpartum and concentrate supplementation increased milk production for both NA and NZ cows. No genotype (NA vs. NZ) by diet interaction occurred for blood glucose, NEFA, or insulin. Expression of pyruvate carboxylase (PC) mRNA was increased at +1 and +4 WRTC compared with 0 WRTC (3.04 and 2.42 vs. 1.25±0.13 arbitrary units, respectively: mean ± standard error of the means) and expression of cytosolic phosphoenolpyruvate carboxykinase mRNA was increased at +4 compared with calving and +1 WRTC (4.78 vs. 2.18 and 2.48±1.41 arbitrary units, respectively). Expression of PC mRNA tended to be greater in NZ cows and tended to decrease with concentrate supplementation in both NZ and NA cows. The responses of NZ and NA cows to the transition to lactation and concentrate supplementation appeared to be similar; however, NZ cattle had a higher basal expression of PC.

Differential regulation of bovine pyruvate carboxylase promoters by fatty acids and peroxisome proliferator-activated receptor-α agonist

Pyruvate carboxylase (PC) is a critical enzyme in supplying carbon for gluconeogenesis and oxaloacetate for the tricarboxylic acid cycle. The bovine PC (EC 6.4.1.1) gene contains 3 promoter sequences (P3, P2, and P1 from 5′ to 3′). Physiological stressors, including the onset of calving and feed restriction, lead to elevated nonesterified fatty acids and glucocorticoid levels that coincide with an increase in PC mRNA expression. The effects of elevated fatty acids on bovine PC mRNA expression and promoter function have not been determined. The objective of this experiment was to determine the direct effects of stearic, oleic, and linoleic acids, dexamethasone, and Wy14643 (a peroxisome proliferator-activated receptor-α agonist) on bovine PC promoter activity. Promoter-luciferase constructs, containing 1,005 bp of P1, 1,079 bp of P2, or 1,010 bp of P3, were transiently transfected into rat hepatoma (H4IIE) cells. Cells were then treated with 1mM stearic, oleic, or linoleic acids, 1μM dexamethasone, or 10μM Wy14643 for 23h. Activity of P1 was suppressed with exposure to stearic acid (1.58 vs. 6.19±0.81 arbitrary units for stearic vs. control, respectively) and enhanced with exposure to Wy14643 (9.26 vs. 6.19±0.81 arbitrary units for Wy14643 vs. control, respectively). Conversely, stearic acid enhanced P3 activity (2.55 vs. 0.40±0.33 arbitrary units for stearic vs. control, respectively). Dexamethasone, linoleic acid, and oleic acid failed to elicit a response from any of the promoters tested. These data demonstrate the direct role of fatty acids in regulating PC expression and indicate that fatty acids provide promoter-specific regulation of PC promoters.

Identification of the cyclic AMP responsive element (CRE) that mediates transcriptional regulation of the pyruvate carboxylase gene in HepG2 cells

Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis. Here we investigated the effect of various hormones including cAMP, dexamethasone and insulin on the abundance of PC mRNA in the human hepatocyte cell line, HepG2. Treatment of HepG2 cells with 1 μM of glucagon increased the expression of PC mRNA threefold within 72 h. Treatment with 1 mM 8-Br-cAMP caused the abundance of PC mRNA to increase by 2–3-fold by 48 h, peak at fourfold at 72 h, and remain unchanged to 96 h. This is in contrast to phosphoenolpyruvate carboxykinase (PEPCK) for which expression was decreased after 72 h, suggesting a distinct difference in the control of these two enzymes in the long term. Dexamethasone or insulin alone did not affect the abundance of PC mRNA whereas treatment of HepG2 cells with the combination of 1 mM 8-Br-cAMP and 0.5 μM dexamethasone further increased the abundance of PC mRNA, suggesting the predominant role of 8-Br-cAMP over dexamethasone. Transient transfection of the luciferase reporter construct driven by a 1.95 kbp 5′-flanking sequence of the mouse PC gene and a plasmid encoding the human cAMP-responsive element binding protein increased luciferase reporter activity to 7-fold similar to that observed with a PEPCK promoter-luciferase reporter construct. Deletion of the 5′-flanking region of the PC gene to 781 bp resulted in the complete loss of CREB-mediated induction of reporter gene, suggesting the presence of the cAMP-responsive unit is located between 1.95 kbp and 781 bp upstream of the mouse PC gene. Electrophoretic mobility shifted and chromatin immunoprecipitation assays demonstrated that CREB bind to −1639/−1631 CRE of mouse PC gene in vitro and in vivo, respectively.

Changes in Hepatic Key Enzymes of Dairy Calves in Early Weaning Production Systems

The objective of the present study was to describe plasma hormonal and metabolite profile and mRNA expression levels and activities of the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and acetyl-coenzyme A (CoA) carboxylase in the liver of male Holstein calves before (1 and 3wk of age) and after (8, 13, and 19wk of age) weaning at 6wk of age. The mean plasma concentration of acetate and β-hydroxybutyrate increased, and that of plasma lactate and nonesterified fatty acids decreased with week, particularly after weaning. Plasma glucose concentration was lowest at 8wk of age. The mean plasma concentration of insulin and glucagon did not change with time, and that of cortisol was greatest at 1wk of age. In the liver, enzyme activity of PC was greatest at 1wk of age and decreased with time. There was a significant relationship between the activity and the mRNA level for PC. Activity of PEPCK also decreased with week. Acetyl-CoA carboxylase activity tended to decrease with week, and activity at 13wk of age was lower than that at other times. Expression of PC mRNA, but not that of PEPCK and acetyl-CoA carboxylase α, decreased with week. We conclude that the hepatic gluconeogenic enzymes and acetyl-CoA carboxylase activities tend to decrease with age, reflecting changes in plasma metabolites in early weaning production systems.

Effect of peripartum dietary energy supplementation of dairy cows on metabolites, liver function and reproductive variables

Multiparous Holstein cows ( n = 58) were used to study the effects of peripartum dietary supplementation on metabolic status, liver function and reproduction variables. Diets for cows were as follows: (a) no supplementation (CTL), (b) prilled fatty acids as 1.9% of DM (PrFA), (c) calcium salts of long chain n-6 fatty acids as 2.24% of DM (CaLFA) or (d) daily topdressing with 769 g of 65% propylene glycol (PGLY). Supplements were fed during the last 21 days before expected calving except for PGLY that continued until 21 days after parturition. Ovarian activity was monitored by transrectal ultrasonography and days to first ovulation were recorded. Liver biopsies were obtained on day 8 and 21 postpartum and analyzed for triglyceride content and mRNA expression of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, carnitine palmytoyltransferase 1A, and peroxisome proliferator-activated receptor-α. At 71 days following parturition, stage of ovarian cycles was synchronized and at day15 of the cycle oxytocin was injected i.v., blood samples were obtained at frequent intervals, and analyzed for 13,14 dihydro, 15-keto PGF 2α (PGFM). Milk production and milk components were not different among treatment groups. Cows in PGLY gained body condition score (BCS) prepartum and net energy balance prepartum tended to be greater, but was not different postpartum from other groups. PGLY supplementation increased plasma insulin concentration prepartum, but not during the postpartum period. No significant differences were observed in plasma concentrations of glucose, NEFA, and insulin-like growth factor or hepatic triglyceride content, but all supplements tended to decrease β hydroxybutyrate postpartum compared to CTL cows. Abundance of mRNA of gluconeogenic and lipid oxidation genes was not different among treatment groups. Days to first ovulation and uterine PGF 2α production in response to an oxytocin treatment were not significantly different among treatment groups. Peripartum supplementation did not result in the substantial improvement of metabolic profile in early lactation nor significantly affect days to first ovulation and PGFM response to an oxytocin treatment.

Expression of Pyruvate Carboxylase mRNA Variants in Liver of Dairy Cattle at Calving

BackgroundBovine liver expresses six pyruvate carboxylase (PC) transcript variants, bPC5′A, bPC5′B, bPC5′C, bPC5′D, bPC5′E, and bPC5′F, which only differ at the 5′ untranslated region (UTR) and contain a common coding region. The objective of this experiment was to determine the profile and abundance of PC transcripts in bovine liver at calving.Methodology/Principal FindingsA ribonuclease protection assay (RPA) protocol was developed to simplify analysis of these variants and investigate the changes in abundance of each 5′ UTR transcript relative to total PC mRNA. Liver biopsy samples collected from seven cows on +1 d relative to calving were analyzed by RPA to determine the profile in PC 5′ UTR variants. Results show that all six bovine PC 5′ UTR variants are detected at calving. Data indicate that bovine PC 5′ UTR variant A is the most abundant, variants C and E are least abundant and expression of variants B, D and F is intermediate at calving.ConclusionsThis manuscript describes a simplified RPA method that quantifies the abundance of six PC transcripts by using two riboprobes. The lack of uniformity in the pattern of PC 5′ UTR variants at calving suggests an additional complexity for control of bovine PC mRNA expression at calving that may be the result of transcriptional controls, variation in mRNA processing, or a combination of these processes.

Effect of dietary carbohydrate and monensin on expression of gluconeogenic enzymes in liver of transition dairy cows1

Thirty-four multiparous Holstein cows were used in a randomized block design to evaluate the effects of feeding nonforage fiber sources (NFFS), monensin, or their combination on expression of gluconeogenic enzymes in the liver during the transition to lactation. The addition of 0 or 300 mg/d of monensin to a conventional (CONV) or NFFS prepartum diet was evaluated in a 2 x 2 factorial arrangement of treatments. The NFFS diet was formulated by replacing 30% of the forage component of the CONV diet with cottonseed hulls and soyhulls. The CONV and NFFS basal diets were fed at dry-off and continued through parturition. Monensin was fed from -28 d relative to calving (DRTC) through parturition. At calving, all cows were placed on the same diet. Liver biopsy samples obtained at -28, -14, +1, +14, and +28 DRTC were used to determine pyruvate carboxylase (PC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Feeding NFFS resulted in greater (P < 0.05) prepartum DMI compared with the CONV diet. There was no effect of prepartum diets on postpartum DMI or average milk production to 56 d of lactation. Expression of PC mRNA was elevated (P < 0.05) at 1 d postpartum, but there was no effect of NFFS or monensin on PC mRNA abundance. Expression of PEPCK-C mRNA at calving was increased (P < 0.05) with prepartum monensin feeding. The data indicate that feeding monensin to transition cows induces hepatic PEPCK-C mRNA expression before calving. The increased expression of hepatic PEPCK-C mRNA with monensin feeding suggests a feed-forward mechanism of metabolic control in ruminants that links molecular control of gluconeogenesis with the profile of rumen fermentation end products.

Effect of propionate, pyruvate and β‐hydroxybutyric acid on pyruvate carboxylase mRNA expression of in vitro culture bovine hepatocytes

ABSTRACTThe objective was to detect the effect of propionate, pyruvate and β‐hydroxybutyric acid on pyruvate carboxylase mRNA expression in in vitro culture bovine hepatocytes through a quantity reverse transcription polymerase chain reaction method. Pyruvate carboxylase is a key enzyme in the liver gluconeogenesis of ruminants and plays an important role in the negative energy balance of peri‐parturient dairy cows. The effect of propionate, pyruvate and β‐hydroxybutyric acid on pyruvate carboxylase mRNA expression at cell level is presented in this article. In experiment 1, the various concentrations of propionate from 0 mmol/L to 11.5 mmol/L, were added to the hepatocytes media. The results indicate that the level of pyruvate carboxylase mRNA was significantly enhanced accompanying increased propionate concentrations in the media (P &lt; 0.01) and high concentrations of propionate did not inhibit the levels of pyruvate carboxylase mRNA. In experiment 2, pyruvate in concentrations from 0 mmol/L to 12 mmol/L was added to the media. The results indicate that the levels of pyruvate carboxylase mRNA were significantly enhanced when concentrations of pyruvate increased from 0 mmol/M to 4 mmol/M and decreased when concentrations of pyruvate increased from 8 mmol/M to 12 mmol/M (P &lt; 0.01). In experiment 3, β‐hydroxybutyric acid of various concentrations from 0 mmol/L to 3.0 mmol/L was added to the media. The results indicate that pyruvate carboxylase mRNA levels increased slightly when concentrations of β‐hydroxybutyric acid were less than 1 mmol/L, and pyruvate carboxylase mRNA levels decreased significantly when they exceeded 2 mmol/L or 3 mmol/L (P &lt; 0.01). There results suggest that β‐hydroxybutyric acid inhibits PC mRNA expression.

Feed Restriction Induces Pyruvate Carboxylase but not Phosphoenolpyruvate Carboxykinase in Dairy Cows

The ability of dairy cattle to adapt to changes in nutrient intake requires appropriately responsive expression of several key genes in liver. Holstein cows were used in 2 experiments to determine the effect of short-term feed restriction on expression of mRNA for gluconeogenic and ureagenic enzymes in liver. In experiment 1, cows were fed a total mixed diet for ad libitum intake for a 5-d period followed by 5 d of 50% of their previous 5-d ad libitum intake followed by 10 d of ad libitum feeding. Liver biopsies and blood samples were obtained on d 5, 10, and 20 of the experiment, the last day of each feeding period. Pyruvate carboxylase (PC) mRNA increased with feed restriction, but phosphoenolpyruvate carboxykinase (PEPCK) was unchanged. Expression of carbamoyl phosphate synthetase (CPS-I), argininosuccinate synthetase, and ornithine transcarbamylase mRNA were not altered by feed restriction; however, CPS-I mRNA expression tended to increase during realimentation. In experiment 2, cows were fed for ad libitum intake for 5 d and then fed 50% of previous intake for 5 d. Liver biopsy samples collected on d 5 and 10 were used for PC mRNA, PEPCK mRNA, and in vitro measure of gluconeogenesis from radiolabelled propionate and lactate. The data indicate expression of genes for key metabolic processes in liver of lactating cows is responsive to feeding level. Expression of PC mRNA is part of the adaptive response to feed intake restriction and is matched by increased capacity for gluconeogenesis from lactate.