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Heat stress (HS) results from excessive heat load on animals such that all adaptive mechanisms used to dissipate the heat do not return the body to normal body temperature. In pigs, HS results in increased fat deposition compared with pair-fed animals in a thermoneutral environment. Although there is evidence that HS increases activity of lipoprotein lipase (LPL) in adipose tissue of heat stressed pigs, the fundamental causes of the increased adiposity are still unknown. It remains unclear whether HS directly alters metabolism in adipocytes. Therefore, to understand the mechanism of HS effects on porcine adipocytes, we used an in vitro adipocyte differentiation model to characterize cellular responses that occur during differentiation of pig adipocytes. Preadipocytes (stromovascular cells) were differentiated for 9 d at a normal (37°C) or HS (41.5°C) temperature under 5% CO. Expressions of HS genes such as heat shock proteins (HSP; HSP27, HSP60, HSP70, and HSP90), adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), fatty acid synthase (FAS), adipocyte protein 2 (aP2), fatty acid translocase 36 (CD36), fatty acid transport protein 4 (FATP4), fatty acid transport protein 6 (FATP6), LPL, glucose transporter protein type 4 (GLUT4), phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C), and glycerol kinase (GK) and adipokines (adiponectin and leptin) were determined by real-time-PCR and immunoblotting or ELISA. Cellular triglyceride (TAG) and ATP concentrations were also determined. As expected, HS increased ( < 0.05) the expressions of HSP genes. There was no HS treatment effect on the level of PPARγ, although C/EBPα was induced ( < 0.05) in HS. So it remains unclear whether HS affects adipocyte differentiation. However, HS leads to increased expressions of genes involved in fatty acid uptake and TAG synthesis (FAS, aP2, CD36, FATP4, FATP6, LPL, GLUT4, PCK1, and GK). This is supported by increased cellular TAG under HS. Therefore, HS promotes increased adipocyte TAG storage, perhaps through upregulation of genes involved in fatty acid uptake and TAG synthesis.

Pyruvate carboxylase (PC) catalyzes the rate-limiting step in gluconeogenesis from lactate and is a determinant of tricarboxylic acid cycle carbon flux. Bovine PC 5' untranslated region (UTR) mRNA variants are the products of a single PC gene containing 3 promoter regions (P3, P2, and P1, 5' to 3') that are responsive to physiological and nutritional stressors. The objective of this study was to determine the direct effects of thermal stress on PC mRNA and gene expression in bovine hepatocyte monolayer cultures, rat hepatoma (H4IIE) cells, and Madin-Darby bovine kidney epithelial (MDBK) cells. Hepatocytes were isolated from 3 Holstein bull calves and used to prepare monolayer cultures. Rat hepatoma cells and MDBK cells were obtained from American Type Culture Collection, Manassas, VA. Beginning 24 h after initial seeding, cells were subjected to either 37°C (control) or 42°C (thermal stress) for 24 h. Treatments were applied in triplicate in a minimum of 3 independent cell preparations. For bovine primary hepatocytes, endogenous expression of bovine PC mRNA increased (P < 0.1) with 24 h of thermal stress (1.31 vs. 2.79 ± 0.49, arbitrary units, control vs. thermal stress, respectively), but there was no change (P ≥ 0.1) in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Similarly, exposure of MDBK cells to thermal stress increased (P < 0.1) expression of bovine PC mRNA without altering (P ≥ 0.1) PEPCK-C mRNA expression. Conversely, there was no effect (P ≥ 0.1) of thermal stress on endogenous rat PC (0.47 vs. 0.30 ± 0.08, control vs. thermal stress) or PEPCK-C (1.61 vs. 1.20 ± 0.48, arbitrary units, control vs. thermal stress, respectively) mRNA expressions in H4IIE cells. To further investigate the regulation of PC, H4IIE cells were transiently transfected with bovine promoter-luciferase constructs containing either P1, P2, or P3, and exposed to thermal stress for 23 h. Activity of P1 was suppressed (P < 0.1) 5-fold, activity of P2 was unchanged (P ≥ 0.1), and activity of P3 was increased (P < 0.1) by 5.4-fold. These data indicate that response of bovine PC gene to thermal stress is through promoter regulation and suggest that there are unique characteristics of bovine PC promoters that may contribute to the physiological response to thermal stress.

Pyruvate carboxylase (PC; EC 6.4.1.1) is critical in gluconeogenesis from lactate and maintenance of tricarboxylic acid cycle intermediates. Whereas increases in PC mRNA have been observed during feed restriction, the mechanism of regulation is unknown; however, coinciding increases in circulating NEFA concentrations suggests that fatty acids may contribute to regulation of gene expression during feed restriction. The objective of this study was to examine the direct effect of exposure to serum from full-fed control cows with serum from cows that were restricted to 50% of ad libitum intake for 5 d on PC expression in vitro. Rat hepatoma (H4IIE) cells were transiently transfected with bovine promoter-luciferase constructs containing bovine PC promoter 1 and treated with serum from control cows, serum from feed-restricted cows, or modified serum. Modified serum pools were generated by supplemented serum from control cows with C14:0, C16:0, C18:0, C18:1n-9 cis, C18:2n-6 cis, and C18:3n-3 cis to match the total NEFA in serum from feed-restricted cows (1.3 mM) in the relative proportion found in serum from control or feed-restricted cows. Exposure of cells to serum from feed-restricted cows increased (P < 0.05) PC promoter 1 activity 2.2-fold compared with cells exposed to control cow serum. Exposure to serum from control cows with fatty acids added to a NEFA concentration of 1.3 mM to reflect the fatty acid profile of control and feed-restricted cows increased (P < 0.05) promoter 1 activity 2.1- and 2.5-fold, respectively, compared with cells incubated with control cow serum. There was no difference (P ≥ 0.05) in promoter 1 activity in cells treated with modified serum compared with serum from feed-restricted cows. These data indicate that promoter 1 is activated by fatty acids found in serum of feed-restricted cows. These data suggest a role of NEFA to regulate expression of bovine PC mRNA through specific activation of PC promoter 1.

Pyruvate carboxylase (PC; EC 6.4.1.1) is a critical enzyme for gluconeogenesis and maintenance of tricarboxylic acid (TCA) cycle intermediates, and expression of PC mRNA is increased at calving and during feed restriction. The bovine PC gene contains 3 promoters (3, 2, and 1 from 5' to 3') that produce 6 mRNA 5' variants (A through F). Products of promoter 1, untranslated region (UTR) variants A, B, C, and F are specifically expressed in glucogenic and lipogenic tissues. The objective of this study was to develop a quantitative PCR-based assay for bovine PC 5' UTR variants that would permit simultaneous characterization of PC variant expression and to determine the pattern of PC variant expression during the transition to lactation and during feed restriction. Primer combinations specific to the coding region of PC and the 5' UTR for variants D, E, and F were used with Taqman probes in a real-time PCR multiplex assay to simultaneously determine total PC mRNA and expression of each UTR variant. The intraassay and interassay CV were less than 2 and 10%, respectively. Total PC mRNA and PC 5' UTR variant profile was determined for liver biopsy samples collected from Holstein cows (n = 8) at -28, +1, and +28 d relative to calving (DRTC) and from mid-lactation Holstein cows subjected to either feed restriction (n = 8) or fed for ad libitum intake (n = 8). The expression of PC mRNA corresponding to the coding region of PC and PC 5' UTR variant regions A, B, C, and F increased (P < 0.05) 4-fold with feed restriction and 6-fold at calving. Nuclei isolated from liver biopsy samples and used to determine the rates of PC gene transcription indicate changes in the abundance of PC 5' mRNA variants A, B, C, and F that are due to corresponding changes in the rate of transcription of the bovine PC gene. The data support the use of the multiplex assay described here as a proxy measure of the activity of bovine PC promoter 1. The increased PC mRNA expression observed at calving and during feed restriction is the result of specific increases in 5' UTR variants A, B, C, and F due to increased transcriptional activity of promoter 1 of the PC gene.

The bovine pyruvate carboxylase (PC) gene is expressed as 6 alternatively spliced variants that share a common open reading frame but that differ within their 5' untranslated regions (UTR). The PC 5' UTR variants (A through F) contain 6 combinations of 5 exons and are 68, 253, 363, 89, 226, 178 bp in length, respectively. The objective of this experiment was to determine whether or not the bovine PC mRNA variants exhibit different translational efficiencies. Each bovine PC 5' UTR variant was linked to the firefly luciferase coding region, and the resulting constructs were transcribed and translated in a rabbit reticulocyte lysate assay. All constructs resulted in synthesis of luciferase protein. The abundance of luciferase protein synthesized from the UTR of bovine PC 5' D was greater (P < 0.05) than synthesis from either PC 5' UTR C or E, and the abilities of UTR D, A, B, and F to drive protein translation were similar. The disproportionate contribution to protein synthesis of the PC 5' D UTR compared with UTR variant C or E indicates a complexity of control for PC enzyme synthesis in the bovine that is dependent on the profile of PC variants. These observations are consistent with differences in PC variant expression that have been observed in vivo and indicate that when PC mRNA is elevated, the pattern of variants directs an increase in PC activity through augmented PC enzyme synthesis.

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.

Hepatocyte monolayers were established from young preruminating (7 to 14 d of age) or older ruminating (11 to 12 wk of age) calves and used to evaluate the effects of insulin and glucagon on incorporation of carbon from 2.5 mM [2-14C]propionate and 2.0 mM [U-14C]lactate into glucose and glycogen. Developmental state (young preruminating vs older ruminating) of the donor calf did not affect the rate of gluconeogenesis from propionate in the absence of hormones. Insulin decreased (P < .05) gluconeogenesis and increased (P < .05) glycogenesis from propionate and lactate in hepatocytes from preruminating calves but had no effect on hepatocytes from ruminating calves. Lactate was poorly metabolized to glucose and was not responsive to glucagon in hepatocytes from ruminating calves compared with hepatocytes from preruminating calves. Hepatocytes responded to glucagon by increasing (P < .05) gluconeogenesis from propionate. Maximal responsiveness to glucagon did not differ between ruminating and preruminating calves, but hepatocytes from preruminating calves responded at lower glucagon concentrations (P < .05). These data demonstrate a similar capacity of hepatocytes from preruminating and ruminating calves to metabolize propionate to glucose, but there was a seven- to eightfold decrease in gluconeogenesis from lactate in ruminating calves that was accompanied by a decreased response to acute changes in insulin and glucagon.

Hepatocytes isolated from male dairy calves were used in monolayer culture or in suspension culture to determine their suitability for the study of hormonal regulation of hepatic gluconeogenesis. The rate of gluconeogenesis (nanomoles of 2.5 mM [2-14C]propionate incorporated into glucose.microgram of DNA-1.hour-1) was higher for monolayers than for suspension cultures. Gluconeogenesis and ureagenesis (nanomoles of urea N formed.microgram of DNA-1.3 hours-1) were similar in monolayers cultured for 24 and 48 h but declined by 120 h. Ureagenesis was barely detectable in suspension cultures. Glucagon (10 nM) increased gluconeogenesis from propionate in monolayers but was without effect on suspension cultures. Actinomycin D (800 nM) and cycloheximide (200 microM) abolished glucagon stimulation of gluconeogenesis, suggesting that glucagon acts to mediate gene expression. Prolonged exposure (45 h) of monolayers to insulin (1,000 nM) decreased basal gluconeogenic rates but did not affect glucagon-stimulated gluconeogenesis. Prior incubation with glucose or valerate did not affect gluconeogenesis. Cells can be successfully maintained in serum-free media for 41 h at the expense of diminished basal gluconeogenic activity. Culture of bovine hepatocytes as monolayers provides a useful tool for the study of chronic and acute hormonal regulation of specific liver functions in the bovine.

Milk-fed calves were used as a source of hepatocytes to establish monlayers to test the effects of chronic (41 h) incubation with no hormone, 100 nM insulin, or 100 nM glucagon on gluconeogenesis (glucose retained as glycogen plus glucose released into the medium) from 2.5 mM [2-(14)C]propionate and 2.0 mM [U-14C]lactate (1.0 mM lactate plus 1.0 mM pyruvate) during a subsequent 3-h (acute) incubation. Media for acute incubations contained no hormone, 0 or 10 nM insulin, or 0, 1, 10, or 100 mM glucagon. Chronic glucagon increased gluconeogenesis from propionate and glyconeogenesis from propionate and lactate compared with chronic exposure to medium with no hormone. A chronic glucagon x acute hormone interaction was manifested as an augmented response to acute glucagon on gluconeogenesis from propionate; a similar potentiation was not evident for gluconeogenesis from lactate. The concentration of glucagon required to acutely stimulate gluconeogenesis was increased by prior incubation with glucagon. Acute glucagon decreased the flux of glucose retained as glycogen regardless of chronic hormone treatment. Chronic incubation with insulin diminished the stimulatory effects of glucagon on gluconeogenesis from lactate. Chronic incubation with insulin did not alter the sensitivity or responsiveness at acute glucagon of gluconeogenesis from propionate. The data demonstrate persisting changes that favor increased basal gluconeogenesis from propionate with chronically elevated glucagon coupled to an increased capacity to respond acutely to glucagon and opposing chronic actions of insulin on lactate metabolism. These data suggest that insulin and glucagon target separate pathways that are unique to metabolism of propionate and lactate.