High-Throughput Production of Chromium(III) Complexes for Antibody Immobilization.

A robot-assisted high-throughput methodology was employed to produce chromium(III) complexes suitable for the surface modification of the commercially available PerkinElmer Optiplate96 well plate for use in enzyme-linked immunosorbent assays (ELISAs). The complexes were immobilized to the native functionality of the well plate and first screened using a horseradish-peroxidase-tagged (HRP) mouse antibody to quantify binding. The top "hits" were further assessed for their ability to present the antibody in a functional state using an ELISA. "Hits" from the second screen yielded four complexes capable of improving the signal intensity of the ELISA by greater than 500%. The metal/base ratio of these complexes was also investigated, and we isolated the most stable and reproducible candidate, [Cr(OH)6]3-, which was formed from chromium(III) perchlorate and pH adjusted with ethylenediamine. This chromium solution was employed in a clinically relevant setting for the detection of bovine TNFα producing up to a 200% increase in signal intensity.

Continuous low-dose infusion of tumor necrosis factor alpha in adipose tissue elevates adipose tissue interleukin 10 abundance and fails to alter metabolism in lactating dairy cows.

Repeated bolus doses of tumor necrosis factor-α (TNFα) alters systemic metabolism in lactating cows, but whether chronic release of inflammatory cytokines from adipose tissue has similar effects is unclear. Late-lactation Holstein cows (n=9-10/treatment) were used to evaluate the effects of continuous adipose tissue TNFα administration on glucose and fatty acid (FA) metabolism. Cows were blocked by feed intake and milk yield and randomly assigned within block to control or TNFα treatments. Treatments (4mL of saline or 14µg/kg of TNFα in 4mL of saline) were infused continuously over 7d via 2 osmotic pumps implanted in a subcutaneous adipose depot. Plasma, milk samples, milk yield, and feed intake data were collected daily, and plasma glucose turnover rate was measured on d 7. At the end of d 7, pumps were removed and liver and contralateral tail-head adipose biopsies were collected. Results were modeled with the fixed effect of treatment and the random effect of block. Treatment with TNFα increased plasma concentrations of the acute phase protein haptoglobin, but did not alter plasma TNFα, IL-4, IL-6, or IFN-γ concentrations, feed intake, or rectal temperature. Milk yield and composition were unchanged, and treatments did not alter the proportion of short- versus long-chain FA in milk on d 7. Treatments did not alter plasma free FA concentration, liver triglyceride content, or plasma glucose turnover rate. Surprisingly, TNFα infusion tended to decrease liver TNFα and IL-1 receptor 1 mRNA abundance and significantly increased adipose tissue IL-10 protein concentration. Continuous infusion of TNFα did not induce the metabolic responses previously observed following bolus doses delivered at the same rate per day. Metabolic homeostasis may have been protected by an adaptive anti-inflammatory response to control systemic inflammation.

Investigation of HCAR2 antagonists as a potential strategy to modulate bovine leukocytes.

BACKGROUND: Dairy cows experiencing ketosis after calving suffer greater disease incidence and are at greater risk of leaving the herd. In vitro administration of beta-hydroxybutyric acid (BHBA; the primary blood ketone) has inhibitory effects on the function of bovine leukocytes. BHBA is a ligand of HCAR2 and the activation of these receptors promotes an anti-inflammatory response which may be related with immunosuppression observed in transition dairy cattle. The objective of this study was to identify and test antagonists for HCAR2 in bovine immune cells cultured with BHBA. RESULTS: We observed expression of HCAR2 at the protein level within lymphocytes, monocytes, and granulocytes. The proportion of cells expressing HCAR2 tended to be greater in mid-lactation compared to early lactation cows; the increase was a result of increased proportion of T and B cells expressing HCAR2. Stimulation of HCAR2 with niacin or BHBA promoted Ca2+ mobilization in neutrophils and mononuclear cells. Mononuclear cells treated with BHBA had diminished intracellular Ca2+ responses when HCAR2 was knocked down by siRNA silencing, indicating Ca2+ mobilization was mediated by HCAR2 signaling. Two candidate antagonists for HCAR2, synthesized from niacin (NA-1 and NA-5), were tested; monocytes and neutrophils pre-treated with NA-1 and NA-5 had reduced Ca2+ mobilization after incubation with BHBA. Furthermore, NA-5 but not NA-1 prevented BHBA-associated reductions in cyclic AMP. CONCLUSIONS: We demonstrated that HCAR2 is present on bovine leukocytes and has greater expression later in lactation. We confirmed that BHBA and niacin derived HCAR2 antagonists alter bovine leukocyte activity. Our results demonstrate that both BHBA and niacin affect bovine leukocyte Ca2+ mobilization in a HCAR2-dependent manner.

Anti-inflammatory salicylate treatment alters the metabolic adaptations to lactation in dairy cattle.

Adapting to the lactating state requires metabolic adjustments in multiple tissues, especially in the dairy cow, which must meet glucose demands that can exceed 5 kg/day in the face of negligible gastrointestinal glucose absorption. These challenges are met through the process of homeorhesis, the alteration of metabolic setpoints to adapt to a shift in physiological state. To investigate the role of inflammation-associated pathways in these homeorhetic adaptations, we treated cows with the nonsteroidal anti-inflammatory drug sodium salicylate (SS) for the first 7 days of lactation. Administration of SS decreased liver TNF-α mRNA and marginally decreased plasma TNF-α concentration, but plasma eicosanoids and liver NF-κB activity were unaltered during treatment. Despite the mild impact on these inflammatory markers, SS clearly altered metabolic function. Plasma glucose concentration was decreased by SS, but this was not explained by a shift in hepatic gluconeogenic gene expression or by altered milk lactose secretion. Insulin concentrations decreased in SS-treated cows on day 7 compared with controls, which was consistent with the decline in plasma glucose concentration. The revised quantitative insulin sensitivity check index (RQUICKI) was then used to assess whether altered insulin sensitivity may have influenced glucose utilization rate with SS. The RQUICKI estimate of insulin sensitivity was significantly elevated by SS on day 7, coincident with the decline in plasma glucose concentration. Salicylate prevented postpartum insulin resistance, likely causing excessive glucose utilization in peripheral tissues and hypoglycemia. These results represent the first evidence that inflammation-associated pathways are involved in homeorhetic adaptations to lactation.

Connecting Metabolism to Mastitis: Hyperketonemia Impaired Mammary Gland Defenses During a Streptococcus uberis Challenge in Dairy Cattle.

ß-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1ß and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Proteome dataset of liver from dairy cows experiencing negative or positive energy balance at early lactation.

This article contains raw and processed data related to research published by Swartz et al. [1]. We present proteomics data from liver of postpartum dairy cows that were obtained by liquid chromatography-mass spectrometry following protein extraction. Differential abundance between liver of cows experiencing either negative energy balance (NEB, n = 6) or positive energy balance (PEB, n = 4) at 17 ± 3 days in lactation was quantified using MS1 intensity based label-free. There is a paucity of studies examining the associations of NEB with the liver proteome in early lactation dairy cows. Therefore, our objective was to examine the differences in the liver proteome in periparturient dairy cows experiencing naturally occurring NEB compared to cows in PEB. In this study, multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter energy balance, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). We collected liver biopsies from 10 cows (n = 5 from each milking frequency), that were retrospectively classified according to their energy balance (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics. This novel dataset contains 2,741 proteins were identified, and 68 of those were differentially abundant between NEB and PEB (P ≤ 0.05 and FC± 1.5); these findings are discussed in our recent research article [1]. The present dataset of liver proteome can be used as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle. Data are available via ProteomeXchange with identifier PXD028124.

Characterization of the liver proteome in dairy cows experiencing negative energy balance at early lactation.

Negative energy balance (NEB) is associated with metabolic disorders in early lactation dairy cows. Therefore, our objective was to characterize the liver proteome in cows experiencing either NEB or positive energy balance (PEB). Forty-two multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter EB, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). Liver biopsies were collected from 10 cows (n = 5 from each milking frequency) at 17 ± 3 DIM (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics and Ingenuity Pathway Analysis used to identify key affected canonical pathways. Overall, 2741 proteins were identified, and 68 of those were differentially abundant (P ≤ 0.05 and FC ± 1.5). ENO3 (FC = 10.3, P < 0.01) and FABP5 (FC = -12.5, P = 0.045) were the most dramatically upregulated and downregulated proteins, respectively, in NEB cows. Numerous mitochondrial proteins (NDUFA5, NDUFS3, NDUFA6, COX7A2L, COX6C, and COA5) were differentially abundant. Canonical pathways associated with NEB were LPS/IL-1 mediated inhibition of RXR function, oxidative phosphorylation, and mitochondrial dysfunction. Additionally, cows experiencing NEB had less hepatic IL10 transcript abundance than PEB. Together, NEB was associated with altered hepatic inflammatory status, likely due to oxidative stress from mitochondrial dysfunction. SIGNIFICANCE: Our manuscript describes the associations of negative energy balance with the liver proteome in early lactation dairy cows, when metabolic stress and the incidence of diseases is increased. Specifically, we found associations of negative energy balance with shifts in hepatic protein abundance involved in fatty acid uptake, impaired anti-inflammatory responses, and mitochondrial dysfunction. Moving forward, differentially abundant proteins found in this study may be useful as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle.

Postpartum meloxicam administration alters plasma haptoglobin, polyunsaturated fatty acid, and oxylipid concentrations in postpartum ewes.

BACKGROUND: Postpartum inflammation is a natural and necessary response; however, a dysfunctional inflammatory response can be detrimental to animal productivity. The objective of this study was to determine the effects of a non-steroidal anti-inflammatory drug (meloxicam) on ewe postpartum inflammatory response, ewe plasma polyunsaturated fatty acid and oxylipid concentrations, and lamb growth. RESULTS: After lambing, 36 Hampshire and Hampshire × Suffolk ewes were sequentially assigned within type of birth to control (n = 17) or meloxicam orally administered on d 1 and 4 of lactation (MEL; 90 mg, n = 19). Milk and blood samples were collected on d 1 (prior to treatment) and d 4. Milk glucose-6-phosphate was not affected by MEL. Plasma haptoglobin (Hp) concentrations were less for MEL ewes; control ewes with greater d 1 Hp concentrations had elevated Hp on d 4, but this was not the case for MEL-treated ewes. Treatment with MEL increased plasma arachidonic acid concentration by more than 4-fold in ewes rearing singles but decreased concentrations of 9,10-dihydroxyoctadecenoic acid, prostaglandin F2α, 8-iso-prostaglandin E2, and 8,9-dihydroxyeicosatetraenoic acid. Nine oxylipids in plasma had interactions of treatment with d 1 Hp concentration, all of which revealed positive associations between d 1 Hp and d 4 oxylipid concentrations for CON, but neutral or negative relationships for MEL. MEL decreased 13-hydroxyoctadecadienoic acid:13-oxooctadecadienoic acid ratio and tended to increase 9-hydroxyoctadecadienoic acid:9-oxooctadecadienoic acid ratio (both dependent on d 1 values), indicating progressive metabolism of linoleic acid-derived oxylipids occurred by enzymatic oxidation after MEL treatment. Meloxicam reduced oxylipids generated across oxygenation pathways, potentially due to an improved redox state. CONCLUSIONS: Postpartum MEL treatment of ewes decreased plasma concentrations of Hp and several oxylipids, with the greatest impact in ewes with biomarkers reflecting a greater inflammatory state before treatment. Anti-inflammatory strategies may help resolve excessive postpartum inflammation in some dams.

Daily injection of tumor necrosis factor-{alpha} increases hepatic triglycerides and alters transcript abundance of metabolic genes in lactating dairy cattle.

To determine whether inflammation can induce bovine fatty liver, we administered recombinant bovine tumor necrosis factor-alpha (rbTNF) to late-lactation Holstein cows. Cows (n = 5/treatment) were blocked by feed intake and parity and randomly assigned within block to control (CON; saline), rbTNF at 2 microg/(kg.d), or pair-fed control (saline, intake matched) treatments. Treatments were administered once daily by subcutaneous injection for 7 d. Plasma samples were collected daily for analysis of glucose and FFA and a liver biopsy was collected on d 7 for triglyceride (TG) and quantitative RT-PCR analyses. Data were analyzed using treatment contrasts to assess effects of tumor necrosis factor-alpha (TNFalpha) and decreased feed intake. By d 7, feed intake of both rbTNF and pair-fed cows was approximately 15% less than CON (P < 0.01). Administration of rbTNF resulted in greater hepatic TNFalpha mRNA and protein abundance and 103% higher liver TG content (P < 0.05) without affecting the plasma FFA concentration. Hepatic carnitine palmitoyltransferase 1 transcript abundance tended to be lower (P = 0.09) and transcript abundance of fatty acid translocase and 1-acyl-glycerol-3-phosphate acyltransferase was higher (both P < 0.05) after rbTNF treatment, consistent with increased FFA uptake and storage as TG. Transcript abundance of glucose-6-phosphatase (P < 0.05) and phosphoenolpyruvate carboxykinase 1 (P = 0.09), genes important for gluconeogenesis, was lower for rbTNF-treated cows. These findings indicate that TNFalpha promotes liver TG accumulation and suggest that inflammatory pathways may also be responsible for decreased glucose production in cows with fatty liver.

Dietary Zinc-Amino Acid Complex Does Not Affect Markers of Mammary Epithelial Integrity or Heat Stability of Milk in Mid-Lactating Cows.

Supplying dietary zinc in excess of traditional requirements has clear impacts on the gut epithelium, but little research has explored whether similar impacts on the mammary epithelium may occur. Our objective was to determine the effects of supplemental Zn sources, in excess of minimal requirements, on markers of mammary epithelial integrity in blood and in milk as well as the heat stability of milk in mid-lactation cows. Twelve multiparous Holstein cows (132 ± 21 days in milk and 51 ± 3 kg/day milk) were blocked according to milk yield and enrolled in a replicated 3 × 3 Latin square experiment. Experimental periods were 21 days, with 17 days allowed for diet adaptation and 4 days for sampling. Treatment sequences were randomly assigned to animals and treatments were as follows: (1) 0.97 g Zn/day provided as ZnSO4 (34.5 mg supplemental Zn/kg diet DM; 30-ZS), (2) 1.64 g Zn/day provided as ZnSO4 (56.5 mg supplemental Zn/kg diet DM; 60-ZS), and (3) 0.55 g Zn/day provided as ZnSO4 plus 1.13 g Zn/day provided as a zinc-methionine complex (58.2 mg supplemental Zn/kg diet DM; 60-ZM). Treatments were administered once daily as an oral bolus containing all supplemental trace minerals. Rumen-bypass methionine was also included in the 30-ZS and 60-ZS boluses to provide metabolizable methionine equivalent to that provided in 60-ZM rations. Milk samples were assessed for electrolytes, somatic cell transcript abundance of genes related to zinc metabolism, and heat coagulation time. Whole blood samples were analyzed for Na and K concentrations, and plasma samples were analyzed for lactose concentration. Cows fed 60-ZS or 60-ZM had greater zinc intake compared to 30-ZS. Dry matter intake and milk fat content tended to be greater in 60-ZS and 60-ZM cows compared to 30-ZS. Somatic cell linear score was similar among treatments. Treatments neither affected markers of mammary epithelial integrity in blood nor in milk of cows, including plasma concentration of lactose, milk concentrations of Na+ and K+, and SLC30A2 and CLU transcript abundance. Treatments had no effect on milk N fractions or heat coagulation time. This study provided no evidence that supplemental Zn above the established requirements can improve blood-milk epithelial barrier or heat stability of milk in healthy mid-lactation dairy cows.

Choline Regulates the Function of Bovine Immune Cells and Alters the mRNA Abundance of Enzymes and Receptors Involved in Its Metabolism in vitro.

Dietary choline can impact systemic immunity, but it remains unclear whether this is primarily via direct impacts on immune cells or secondary effects of altered metabolic function. To determine whether increased choline concentrations (3.2, 8.2, 13.2 µM) in cell culture alter the function of bovine innate and adaptive immune cells, we isolated cells from dairy cows in early and mid-lactation as models of immuno-compromised and competent cells, respectively. Phagocytic and killing capacity of isolated neutrophils were linearly diminished with increasing doses of choline. In contrast, lymphocyte proliferation was linearly enhanced with increasing doses of choline. Furthermore, increasing doses of choline increased the mRNA abundance of genes involved in the synthesis of choline products (betaine, phosphatidylcholine, and acetylcholine) as well as muscarinic and nicotinic acetylcholine receptors in a quadratic and linear fashion for neutrophils and monocytes, respectively. Phagocytic and killing capacity of neutrophils and proliferation of lymphocytes were not affected by stage of lactation or its interaction with choline or LPS. In neutrophils from early lactation cows, choline linearly increased the mRNA abundance of muscarinic and nicotinic cholinergic receptors, whereas choline-supplemented monocytes from mid-lactation cows linearly increased the mRNA abundance of several genes coding for choline metabolism enzymes. These data demonstrate that choline regulates the inflammatory response of immune cells and suggest that the mechanism may involve one or more of its metabolic products.

Restricted nutrient intake does not alter serum-mediated measures of implant response in cell culture.

BACKGROUND: During nutritional stress, reduced intake may reduce the efficacy of anabolic implants. This study was conducted to evaluate basic cellular responses to a growth promotant implant at two intake levels. METHODS: Sixteen crossbred steers (293 ± 19.3 kg) were used to evaluate the impact of anabolic implants in either an adequate or a restricted nutritional state. Steers were trained to individual Calan gates, and then randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement. Treatments consisted of: presence or absence of an anabolic growth implant (Revalor-XS, 200 mg TBA and 40 mg estradiol; IMPLANT or CONTROL) and a moderate energy, pelleted, starting cattle diet fed at either 2.0 × or 1.0 × maintenance energy (NEM) requirements (HIGH or LOW). Serum (d 0, 14, and 28) was used for application to bovine muscle satellite cells. After treatment with the serum (20% of total media) from the trial cattle, the satellite cells were incubated for 72 h. Protein abundance of myosin heavy chain (MHC), phosphorylated extracellular signal-related kinase (phospho-ERK), and phosphorylated mammalian target of rapamycin (phospho-mTOR) were analyzed to determine the effects of implant, intake, and their interaction (applied via the serum). RESULTS: Intake had no effect on MHC (P = 0.85) but IMPLANT increased (P < 0.01) MHC abundance vs. CONTROL. Implant status, intake status, and the interaction had no effect on the abundance of phospho-ERK (P ≥ 0.23). Implanting increased phospho-mTOR (P < 0.01) but there was no effect (P ≥ 0.51) of intake or intake × implant. CONCLUSIONS: The nearly complete lack of interaction between implant and nutritional status indicates that the signaling molecules measured herein respond to implants and nutritional status independently. Furthermore, results suggest that the muscle hypertrophic effects of anabolic implants may not be mediated by circulating IGF-1.

TNFα altered inflammatory responses, impaired health and productivity, but did not affect glucose or lipid metabolism in early-lactation dairy cows.

Inflammation may be a major contributing factor to peripartum metabolic disorders in dairy cattle. We tested whether administering an inflammatory cytokine, recombinant bovine tumor necrosis factor-α (rbTNFα), affects milk production, metabolism, and health during this period. Thirty-three Holstein cows (9 primiparous and 24 multiparous) were randomly assigned to 1 of 3 treatments at parturition. Treatments were 0 (Control), 1.5, or 3.0 µg/kg body weight rbTNFα, which were administered once daily by subcutaneous injection for the first 7 days of lactation. Statistical contrasts were used to evaluate the treatment and dose effects of rbTNFα administration. Plasma TNFα concentrations at 16 h post-administration tended to be increased (P<0.10) by rbTNFα administration, but no dose effect (P>0.10) was detected; rbTNFα treatments increased (P<0.01) concentrations of plasma haptoglobin. Most plasma eicosanoids were not affected (P>0.10) by rbTNFα administration, but 6 out of 16 measured eicosanoids changed (P<0.05) over the first week of lactation, reflecting elevated inflammatory mediators in the days immediately following parturition. Dry matter and water intake, milk yield, and milk fat and protein yields were all decreased (P<0.05) by rbTNFα treatments by 15 to 18%. Concentrations of plasma glucose, insulin, ß-hydroxybutyrate, non-esterified fatty acids, triglyceride, 3-methylhistidine, and liver triglyceride were unaffected (P>0.10) by rbTNFα treatment. Glucose turnover rate was unaffected (P=0.18) by rbTNFα administration. The higher dose of rbTNFα tended to increase the risk of cows developing one or more health disorders (P=0.08). Taken together, these results indicate that administration of rbTNFα daily for the first 7 days of lactation altered inflammatory responses, impaired milk production and health, but did not significantly affect liver triglyceride accumulation or nutrient metabolism in dairy cows.

Toll-like receptor 4 signaling is required for induction of gluconeogenic gene expression by palmitate in human hepatic carcinoma cells.

Saturated free fatty acids (FFA) can activate inflammatory cascades including the toll-like receptor 4 (TLR4) pathway. TLR4 is expressed by hepatocytes and may help link FFA to altered hepatic gluconeogenesis in type 2 diabetes mellitus. This study examined the role of TLR4 in mediating palmitate effects on the expression of phosphoenolpyruvate carboxykinase (PCK1) and the catalytic subunit of glucose-6-phosphatase (G6PC), rate-determining gluconeogenic enzymes. Human hepatocellular carcinoma cells (HepG2 and HuH7) were incubated in media including 2% bovine serum albumin and 250 to 1000 µM palmitate for 24 h. Signaling mediated by TLR4 was blocked by a TLR4 decoy peptide or small interfering RNA knockdown of TLR4. Palmitate induced dose-dependent increases in PCK1 and G6PC mRNA abundance, which were prevented by the TLR4 decoy peptide. Palmitate doubled PCK1 promoter activity, and TLR4 knockdown ablated this response. Lipopolysaccharide and monophosphoryl lipid A also up-regulated G6PC and PCK1 transcript abundance in a TLR4-dependent manner. Addition of oleate attenuated palmitate-induced increases in G6PC and PCK1 mRNA abundance. Palmitate increased nuclear factor κ-light-chain-enhancer of activated B cells reporter gene activity, which was unaffected by TLR4 blockade, but increased mRNA abundance of hepatocyte-specific cyclic AMP response element binding protein, a transcriptional regulator of PCK1, in a TLR4-dependent manner. Finally, TLR4 activation by palmitate increased subsequent cellular uptake of palmitate, and inhibiting ceramide synthesis ablated palmitate effects on PCK1 mRNA abundance and promoter activity. These results suggest that TLR4 signaling could play a critical role in linking elevated saturated FFA to increased transcription of gluconeogenic genes.