Inflammatory tone in liver and adipose tissue in dairy cows experiencing a healthy transition from late pregnancy to early lactation.

The transition from late pregnancy (LP) to early lactation (EL) in dairy cows is characterized by a major reorganization of the metabolic activities of liver and adipose tissue in support of milk synthesis. This reorganization has been attributed in large part to variation in the plasma concentration and actions of growth hormone, insulin, and other metabolic hormones. A role for the immune system has also been suggested by a near-universal rise in circulating levels of liver-derived acute-phase proteins (APP) in early lactating cows. However, less attention has been devoted to the possibility that resident macrophages of liver and adipose tissue adopt a proinflammatory state (referred herein as inflammatory tone) in parallel with the rise in plasma APP. We addressed this question by measuring the expression of genes expressed predominantly in the resident macrophage population of liver and adipose tissue and indicative of a proinflammatory (tumor necrosis factor α, IL-6, IL-12, resistin, and cluster of differentiation 80 [CD80]) or anti-inflammatory state (IL-10 and chitinase-3-like protein 1 [CHI3L1]). In a first group of cows, none of these inflammatory gene markers were regulated in liver between LP on d -29 (relative to parturition) and on d 8 of EL despite 1.7 to 5.6-fold upregulation in the expression of the APP (haptoglobin, serum amyloid α, and orosomucoid 1). In a second group of healthy cows, expression of the inflammatory gene markers did not differ between livers with low (<5.3%) or high (>11.5%) triglyceride content on d 7 of EL. In adipose tissue, a modest increase in inflammatory tone was suggested between LP and EL by increased CD80 expression and decreased CHI3L1 expression in EL. To assess the possibility that inflammatory tone would be more prominent if assayed in a cell compartment enriched with macrophages, adipose tissue was obtained in LP on d -28 and in EL on d +10 from cows experiencing a healthy transition period and fractionated into its adipocyte and stromal vascular cell (SVC) compartments. Expression of inflammatory gene markers was higher in SVC than adipocytes but remained unregulated in SVC between LP and EL. Overall, these results suggest little change in the inflammatory tone of resident macrophages in liver and adipose tissue of healthy transition dairy cows and do not support a role for the local immune system in the reorganization of metabolism in these tissues at the onset of lactation.

The acute phase protein orosomucoid 1 is upregulated in early lactation but does not trigger appetite-suppressing STAT3 signaling via the leptin receptor.

Dairy cows consume inadequate amounts of feed in early lactation and during conditions and diseases such as excessive fatness, heat stress, and infectious diseases. Affected cows often experience increases in plasma concentrations of acute phase proteins consistent with the negative effect of inflammation on appetite. The acute phase protein orosomucoid 1 (ORM1), also known as alpha-1-acid glycoprotein, was recently reported to reduce appetite in the mouse through its ability to bind the full-length leptin receptor (Ob-Rb) and activate appetite-suppressing signal transducer and activator of transcription 3 (STAT3) signaling. These observations raise the possibility that ORM1 exerts appetite-suppressing effects in dairy cattle during periods of increased inflammatory tone. The applicability of this model was assessed in 2 ways. First, we asked whether ORM1 is regulated during periods of inadequate appetite such as the transition from late pregnancy to early lactation and periods of increased inflammatory tone. Plasma ORM1 was invariant in late pregnancy but increased 2.5-fold between parturition and d 7 of lactation. Gene expression studies showed that liver was the major source of this elevation with little contribution by adipose tissue or mammary gland. Additional studies showed that plasma ORM1 was not increased further by excessive fatness or by reproductive dysfunction in early lactation and was completely unresponsive to inflammatory stimuli such as heat stress or intravascular administration of the endotoxin lipopolysaccharide during established lactation. Second, we tested the ability of ORM1 to trigger STAT3 signaling through Ob-Rb using Chinese hamster ovary K1 (CHO-K1) cells transfected with a STAT3 expression plasmid. In this configuration, CHO-K1 cells did not express Ob-Rb and were incapable of leptin-induced STAT3 phosphorylation. Leptin responsiveness was conferred by co-transfecting with bovine Ob-Rb, with leptin causing increases of 5.7-fold in STAT3 phosphorylation and 2.1-fold in the expression of the STAT3-dependent gene, SOCS3. In contrast, neither bovine or human ORM1 triggered STAT3 phosphorylation irrespective of dose and period of incubation tested. In summary, bovine ORM1 is not increased during periods of increased inflammatory tone except in early lactation and is incapable of Ob-Rb-dependent STAT3 signaling. Overall, these data are inconsistent with ORM1 mediating the appetite-suppressing effects of inflammation in cattle through Ob-Rb.

Fibroblast growth factor-21 (FGF21) administration to early-lactating dairy cows. I. Effects on signaling and indices of insulin action.

Modern dairy cows rely on hormonally driven mechanisms to coordinate the metabolic adaptations needed to meet the energy and nutrient deficits of early lactation. In the case of glucose, dairy cows cope with its scarcity during early lactation via reduced plasma concentrations of insulin and the insulin sensitizing hormone adiponectin and increased insulin resistance. Reduced insulin action promotes diversion of available glucose to the mammary gland but increases susceptibility to diseases if excessive. In earlier work, we reported that the insulin sensitizing hormone fibroblast growth factor-21 (FGF21) is increased in periparturient dairy cows and identified liver and adipose tissue as possible targets. These observations raised the possibility that FGF21 acts directly on these tissues to limit the insulin resistance of early lactation. To test this hypothesis, dairy cows were randomly assigned on d 12.6 ± 2.2 (± standard error) of lactation to receive either excipient (n = 6) or recombinant human FGF21 (n = 7), first as an FGF21 bolus of 3 mg/kg of body weight, followed 2 d later by a constant i.v. infusion of FGF21 at the rate of 6.3 mg/kg of metabolic body weight for 9 consecutive days. Biopsies of liver and adipose tissue were collected during the bolus phase of the experiment and used to analyze FGF21 signaling by Western blotting and expression of its receptor components by quantitative PCR. Bolus FGF21 administration caused a 4-fold increase in p44/42 MAPK (ERK1/2) activation in adipose tissue but had no effect on AKT and signal transducer and activator of transcription-3 (STAT3) signaling. The liver expressed negligible levels of the preferred FGF21 receptor FGFR1c and failed to mount any FGF21 signaling response. The FGF21 administered as a bolus had no effect on plasma glucose or insulin and did not stimulate an acute release of adiponectin from adipose tissue. Similarly, FGF21 infusion had no effect on plasma levels of glucose or insulin measured over the 9-d infusion or on glucose disposal during an i.v. glucose tolerance test performed on d 8 of infusion. Finally, the chronic FGF21 infusion had no effect on indices of adiponectin production, including plasma adiponectin and adipose tissue mRNA abundance of adiponectin and the endoplasmic reticulum chaperones ERO1A and DSBA-L involved in the assembly of adiponectin into multimeric complexes. These data show that human FGF21 does not act as an insulin sensitizer during the energy and glucose deficit of early lactation but do not rule out such a role in other physiological states.

Fibroblast growth factor-21 (FGF21) administration to early-lactating dairy cows. II. Pharmacokinetics, whole-animal performance, and lipid metabolism.

Dairy cows cope with severe energy insufficiency in early lactation by engaging in intense and sustained mobilization of fatty acids from adipose tissue. An unwanted side effect of this adaptation is excessive lipid accumulation in the liver, which in turn impairs hepatic functions. Mice experiencing increased hepatic fatty acid flux are protected from this condition through coordinated actions of the newly described hormone fibroblast growth factor-21 (FGF21) on liver and adipose tissue. The possibility of an analogous role for FGF21 in dairy cows is suggested by its rapid increase in plasma levels around parturition followed by chronically elevated levels in the first few weeks of lactation. To test this hypothesis, dairy cows were randomly assigned on d 12.6 ± 2.2 (± standard error) of lactation to receive either an excipient (control; n = 6) or recombinant human FGF21 (n = 7), first as an FGF21 bolus of 3 mg/kg of body weight (BW) followed 2 d later by a constant i.v. infusion of FGF21 at a rate of 6.3 mg/kg of metabolic BW for 9 consecutive days. After bolus administration, human FGF21 circulated with a half-life of 194 min, and its constant infusion increased total plasma concentration 117-fold over levels in excipient-infused cows. The FGF21 treatment had no effect on voluntary feed intake, milk yield, milk energy output, or net energy balance measured over the 9-d infusion or on final BW. Plasma fatty acids circulated at lower concentrations in the FGF21 group than in the control group for the 8-h period following bolus administration, but this reduction was not significant during the period of constant i.v. infusion. Treatment with FGF21 caused a 50% reduction in triglyceride content in liver biopsies taken at the end of the constant i.v. infusion without altering the mRNA abundance of key genes involved in the transport, acyl coenzyme A activation, or oxidation of fatty acids. In contrast, FGF21 treatment ablated the recovery of plasma insulin-like growth factor-1 seen in control cows during the 9-d i.v. infusion period despite a tendency for higher plasma growth hormone. This effect was associated with increased hepatic mRNA abundance of the intracellular inhibitor of growth hormone receptor trafficking, LEPROT. Overall, these data confirm the ability of FGF21 to reduce lipid accumulation in bovine liver and suggest the possibility that FGF21 does so by attenuating the hepatic influx of adipose tissue-derived fatty acids.

Time-dependent effect of trans-10,cis-12 conjugated linoleic acid on gene expression of lipogenic enzymes and regulators in mammary tissue of dairy cows.

Trans-10,cis-12 conjugated linoleic acid (CLA) has been identified as an intermediate of rumen fatty acid biohydrogenation that caused milk fat depression (MFD) in the dairy cow. Previous studies in cows experiencing CLA- and diet-induced MFD have identified reduced mammary expression of the master lipogenic regulator sterol response element transcription factor 1 (SREBF1) and many of its dependent genes. To distinguish between primary mechanisms regulating milk fat synthesis and secondary adaptations to the reduction in milk fat, we conducted a time-course experiment. Eleven dairy cows received by abomasal infusion an initial priming dose of 6.25 g of CLA followed by 12.5 g/d delivered in multiple pulses per day for 5 d. Cows were milked 3×/d and mammary biopsies were obtained under basal condition (prebolus control) and 12, 30, and 120 h relative to initiation of CLA infusion. Milk fat concentration and yield decreased progressively reaching a nadir at 69 h (1.82% and 38.2 g/h) and averaged 2.03 ± 0.19% and 42.1 ± 4.10 g/h on the last day of treatment (±standard deviation). Expression of fatty acid synthase (FASN) and lipoprotein lipase (LPL) were decreased at 30 and 120 h compared with control. Expression of SREBF1 and THRSP were also decreased at 30 and 120 h compared with control. Additionally, we failed to observe changes in other factors, including peroxisome proliferator-activated receptor γ and liver × receptor ß and milk fat globular membrane proteins, during CLA treatment. However, expression of milk fat globular membrane proteins were decreased after 14 d of diet-induced MFD in samples from a previous experiment, indicating a possible long-term response. The rapid decrease in lipogenic enzymes, SREBF1, and THRSP provide strong support for their transcriptional regulation as a primary mechanism of milk fat depression.

Variation in x-box binding protein 1 (XBP1) expression and its dependent endoplasmic reticulum chaperones does not regulate adiponectin secretion in dairy cows.

Adiponectin is an insulin-sensitizing hormone produced predominantly by adipose tissue; it circulates as oligomers of 3, 6, 18, or more units. Plasma adiponectin might be involved in the development of insulin resistance in transition dairy cows because it falls to a nadir around parturition. The possibility that this regulation occurs through a post-transcriptional mechanism was suggested in a previous study that showed unchanged adiponectin mRNA abundance combined with reduced expression of endoplasmic reticulum (ER) chaperones implicated in assembly of adiponectin oligomers. Expression of ER chaperones is controlled by x-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), suggesting a model whereby transcriptional regulation of ER chaperones during the transition period contributes to the regulation of adiponectin production. In support of this model, XBP1 expression in adipose tissue, measured either as the active spliced XBP1 mRNA or as the total of all XBP1 mRNA isoforms, was 45% lower on d 8 of lactation than 4 wk before parturition; ATF6 mRNA abundance remained unchanged over the same period. To assess the functional importance of XBP1, preadipocytes isolated from pregnant cows were differentiated into adipocytes that secrete adiponectin. Infection of differentiating cells with an adenovirus expressing the active spliced version of bovine XBP1 did not alter adiponectin mRNA but increased the expression of ER chaperones 1.5- to 5-fold. Despite the latter, XBP1 overexpression did not affect the total amount of adiponectin secreted in medium. In additional experiments, adiponectin production was dependent on exogenous lipid in the medium and was reduced during incubation with tumor necrosis factor-α (TNFα). Accordingly, we asked whether the repressive effects of these factors on adiponectin production were related to a reduction in the expression of adiponectin or determinants of ER function (XBP1, ATF6, and ER chaperones). Exogenous lipid had no effect on the expression of any of these genes, whereas TNFα repressed adiponectin mRNA abundance by 61% but had little effect on determinants of ER function. Overall, this work shows that XBP1 is a positive regulator of ER chaperone expression in adipose tissue but provides no support for XBP1 and its dependent ER chaperones in the regulation of adiponectin production in bovine adipocytes. Mechanisms accounting for reduced plasma adiponectin in transition cows remain poorly understood.

Effect of hormonal and energy-related factors on plasma adiponectin in transition dairy cows.

In transition dairy cows, plasma levels of the insulin-sensitizing hormone adiponectin fall to a nadir at parturition and recover in early lactation. The transition period is also characterized by rapid changes in metabolic and hormonal factors implicated in other species as positive regulators of adiponectin production (i.e., negative energy balance, lipid mobilization) and others as negative regulators (i.e., reduced leptin and insulin and increased growth hormone and plasma fatty acids). To assess the role of onset of negative energy balance and lipid mobilization after parturition, dairy cows were either milked thrice daily (lactating) or never milked (nonlactating) for up to 4 wk after parturition. Plasma adiponectin was 21% higher across time in nonlactating than lactating cows. Moreover, nonlactating cows recovered plasma adiponectin at similar rates as lactating cows even though they failed to lose body condition. Next, we assessed the ability of individual hormones to alter plasma adiponectin in transition dairy cows. In the first experiment, dairy cows received a constant 96-h intravenous infusion of either saline or recombinant human leptin starting on d 8 of lactation. In the second experiment, dairy cows were studied in late pregnancy (LP, starting on prepartum d -31) and again in early lactation (EL, starting on d 7 postpartum) during a 66-h period of basal sampling followed by 48 h of hyperinsulinemic-euglycemia. In the third experiment, cows were studied either in LP (starting on d -40 prepartum) or EL (starting on d 7 postpartum) during a 3-h period of basal sampling followed by 5 d of bovine somatotropin treatment. Plasma adiponectin was reduced by an average of 21% in EL relative to LP in these experiments, but neither leptin, insulin, or growth hormone treatment affected adiponectin in LP or EL. Finally, the possibility that plasma fatty acids repress plasma adiponectin was evaluated by intravenous infusion of a lipid emulsion in nonpregnant, nonlactating cows in the absence or presence of glucagon for 16 consecutive hours. The intralipid infusion increased plasma fatty acid concentration from 102 to over 570 µM within 3 h but had no effect on plasma adiponectin irrespective of presence or absence of glucagon. Overall, these data suggest that energy balance around parturition may regulate plasma adiponectin but do not support roles for lipid mobilization or sustained changes in the plasma concentration of leptin, insulin, growth hormone, or fatty acids.

Prepartum dietary energy intake affects metabolism and health during the periparturient period in primiparous and multiparous Holstein cows.

An experiment was conducted to determine the effect of prepartum plane of energy intake on metabolic profiles related to lipid metabolism and health in blood and liver. Primiparous (n=24) and multiparous (n=23) Holsteins were randomly assigned by expected date of parturition to 1 of 3 prepartum energy intakes. A high energy diet [1.62 Mcal of net energy for lactation (NE(L))/kg; 15% crude protein] was fed for either ad libitum intake or restricted intake to supply 150% (OVR) or 80% (RES) of energy requirements for dry cows in late gestation. To limit energy intake to 100% of National Research Council requirements at ad libitum intake, chopped wheat straw was included as 31.8% of dry matter for a control diet (CON; 1.21 Mcal of NE(L)/kg of dry matter; 14.2% crude protein). Regardless of parity group, OVR cows had greater concentrations of glucose, insulin, and leptin in blood prepartum compared with either CON or RES cows; however, dietary effects did not carry over to the postpartum period. Prepartum nonesterified fatty acids (NEFA) were lower in OVR cows compared with either CON or RES cows. Postpartum, however, OVR cows had evidence of greater mobilization of triacylglycerol (TAG) from adipose tissue as NEFA were higher than in CON or RES cows, especially within the first 10 d postpartum. Prepartum ß-hydroxybutyrate (BHBA) was not affected by diet before parturition; however, within the first 10 d postpartum, OVR cows had greater BHBA than CON or RES cows. Prepartum diet did not affect liver composition prepartum; however, OVR cows had greater total lipid and TAG concentrations and lower glycogen postpartum than CON or RES cows. Frequency of ketosis and displaced abomasum was greater for OVR cows compared with CON or RES cows postpartum. Controlling or restricting prepartum energy intake yielded metabolic results that were strikingly similar both prepartum and postpartum, independent of parity group. The use of a bulky diet controlled prepartum energy intake in multiparous and primiparous cows, improved metabolic status postpartum, and reduced the incidence of health problems. When metabolic profiles are considered collectively, cows overfed energy prepartum exhibited an "overnutrition syndrome" with characteristics of clinical symptoms displayed by diabetic or obese nonruminant subjects. This syndrome likely contributed to metabolic dysfunction postpartum.

Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows.

In most mammals, prolactin (PRL) is essential for maintaining lactation, and yet the short-term suppression of PRL during established lactation by bromocriptine has produced inconsistent effects on milk yield in cows and goats. To assess the effect of the long-term inhibition of PRL release in lactating dairy cows, 5 Holstein cows in early lactation received daily intramuscular injections of 1mg of the PRL-release inhibitor quinagolide for 9 wk. Four control cows received the vehicle (water) only. During the last week of the treatments, one udder half was milked once a day (1×) and the other twice a day (2×). Blood samples were harvested at milking in wk -1, 1, 4, and 8. The daily injections of quinagolide reduced milking-induced PRL release but not the basal PRL concentration. Quinagolide induced a faster decline in milk production, which was about 5.3 kg/d lower in the quinagolide-treated cows during the last 4 wk of treatment. During wk 9, the inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the half milked 1×. Milk production was significantly correlated with the quantity of PRL released at milking. Quinagolide did not affect the release of oxytocin at milking. Serum concentration of insulin-like growth factor-1 was not affected by treatment or correlated with milk production. Serum concentrations of leptin and the calciotropic hormone stanniocalcin were not affected by the treatment. In conclusion, the chronic administration of the PRL-release inhibitor quinagolide decreases milk production in dairy cows. The effect is likely the result of the reduced release of milking-induced PRL and is modulated at the level of the gland by milking frequency.

Effects of prepartum 2,4-thiazolidinedione on insulin sensitivity, plasma concentrations of tumor necrosis factor-α and leptin, and adipose tissue gene expression.

Administration of peroxisome proliferator-activated receptor gamma (PPARγ) ligands, thiazolidinediones (TZD), to prepartum dairy cattle has been shown to improve dry matter intake and decrease circulating nonesterified fatty acids (NEFA) around the time of calving. The objective of this work was to elucidate mechanisms of TZD action in transition dairy cattle by investigating changes in plasma leptin, tumor necrosis factor-α (TNFα), the revised quantitative insulin sensitivity check index (RQUICKI), and adipose tissue gene expression of leptin, PPARγ, lipoprotein lipase (LPL), and fatty acid synthase (FAS). Multiparous Holstein cows (n=40) were administered 0, 2.0, or 4.0 mg of TZD/kg of body weight (BW) by intrajugular infusion once daily from 21 d before expected parturition until parturition. Plasma samples collected daily from 22 d before expected parturition through 21 d postpartum were analyzed for glucose, NEFA, and insulin. Plasma samples collected on d -14, -3, -1, 1, 3, 7, 14, and 49 relative to parturition were also analyzed for leptin and TNFα. Adipose tissue was collected on d 7 before expected parturition from a subset of cows, and gene expression was examined via quantitative real-time PCR. A tendency for a treatment by time effect on plasma leptin prepartum was observed such that values were similar on d -14 but cows receiving 2.0 mg/kg of BW of TZD tended to have lower circulating leptin as calving approached. Postpartum leptin tended to be increased linearly (2.3, 2.4, and 2.5±0.1 ng/mL for 0, 2.0, and 4.0 mg/kg treatments, respectively) in cows that received TZD prepartum. Plasma TNFα increased linearly (2.6, 3.7, and 4.0±0.1 pg/mL) in response to TZD treatment and decreased through the first week postpartum. Calculation of RQUICKI 1/[log(glucose)+log(insulin)+log(NEFA)] suggested altered insulin sensitivity in cows administered TZD that may depend on day relative to calving. Administration of TZD increased adipose tissue expression of PPARγ mRNA (11.0, 13.3, and 12.8±1.9). Administration of TZD had a quadratic effect on gene expression of leptin (16.2, 10.7, and 17.4±1.6) and no effect on LPL expression, and expression of FAS was lower for TZD-treated cows than for controls (8.2, 4.2, and 6.1±1.8, respectively). Results imply altered expression and plasma concentrations of leptin, increased plasma TNFα concentrations, and increased expression of PPARγ in adipose tissue as potential mechanisms for the effects of TZD administration on transition dairy cattle.

t10,c12-CLA decreases adiposity in peripubertal mice without dose-related detrimental effects on mammary development, inflammation status, and metabolism.

The trans 10, cis 12-conjugated linoleic acid (10,12-CLA) isomer reduces adiposity in several animal models. In the mouse, however, this effect is associated with adipose tissue inflammation, hyperinsulinemia and hepatic lipid accumulation. Moreover, 10,12-CLA was recently shown to promote mammary ductal hyperplasia and ErbB2/Her2-driven mammary cancer in the mouse. Reasons for detrimental effects of 10,12-CLA on the mouse mammary gland could relate to its effect on the mammary fat pad (MFP), which is essential for normal development. Accordingly, we hypothesized that mammary effects of 10,12-CLA were mediated through the MFP in a dose-dependent manner. Female FVB mice were fed 10,12-CLA at doses of 0%, 0.1%, 0.2%, or 0.5% of the diet from day 24 of age, and effects on mammary development and metabolism were measured on day 49. The 0.5% dose reduced ductal elongation and caused premature alveolar budding. These effects were associated with increased expression of inflammatory markers and genes shown to alter epithelial growth (IGF binding protein-5) and alveolar budding (TNF-α and receptor of activated NF-κB ligand). The 0.5% dose also caused hyperinsulinemia and hepatic lipid accumulation. In contrast, the 0.1% 10,12-CLA dose had no adverse effects on mammary development, metabolic events, and inflammatory responses, but remained effective in decreasing adipose weights and lipogenic gene expression. These results show that a low dose of 10,12-CLA reduces adiposity in the mouse without negative effects on mammary development, inflammation, and metabolism, and suggest that previously reported detrimental effects relate to the use of excessive doses.

Effects of heat stress and nutrition on lactating Holstein cows: II. Aspects of hepatic growth hormone responsiveness.

Heat stress (HS) is a multibillion-dollar problem for the global dairy industry, and reduced milk yield is the primary contributor to this annual economic loss. Feed intake declines precipitously during HS but accounts for only about 35% of the decreased milk synthesis, indicating that the physiological mechanisms responsible for decreased milk production during HS are only partly understood. Thus, our experimental objectives were to characterize the direct effects of HS on the somatotropic axis, a primary regulator of metabolism and milk yield. We recently reported no differences in mean growth hormone (GH) concentrations, GH pulsatility characteristics, or GH response to growth hormone releasing factor in HS versus pair-fed (PF) thermoneutral controls. Despite similarities in circulating GH characteristics, plasma insulin-like growth factor (IGF)-I concentrations were reduced during heat stress conditions but not in PF animals, suggesting that uncoupling of the hepatic GH-IGF axis may occur during HS. We investigated this possibility by measuring proximal indicators of hepatic GH signaling following a GH bolus. Heat stress but not PF decreased abundance of the GH receptor and GH-dependent signal transducer and activator of transcription (STAT)-5 phosphorylation. Consistent with reduced GH signaling through STAT-5, basal hepatic IGF-I mRNA abundance was lower in HS cows. Thus, the reduced hepatic GH responsiveness (in terms of IGF-I gene expression) observed during HS appears to involve mechanisms at least partially independent of reduced nutrient intake. The physiological significance of reduced hepatic GH receptor abundance during HS is unclear at this time. Aside from reducing IGF-I production, it may reduce other GH-sensitive bioenergetic processes such as gluconeogenesis.

Impact of postpartum milking frequency on the immune system and the blood metabolite concentration of dairy cows.

The transition from pregnancy to lactation is marked by metabolic, hormonal, and immunological changes that have an impact on the incidence of infectious and metabolic diseases. The aim of this study was to evaluate the effect on immune function and blood metabolite concentration of limiting milk production in early lactation to reduce negative energy balance. Twenty-two multiparous Holstein cows were milked either once a day (1x) or twice a day (2x) for the first week postpartum. All cows were milked twice daily for the rest of lactation. Blood concentrations of nonesterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), calcium, bilirubin, urea, phosphorus, glucose, leptin, stanniocalcin-1, and 17beta-estradiol were determined in samples collected from 5 wk before scheduled calving to 5 wk after calving. Polymorphonuclear leukocytes (PMNL) were isolated from blood to conduct assays for chemotaxis, phagocytosis, and respiratory burst. Peripheral blood mononuclear cells (PBMC) were isolated to evaluate lymphocyte proliferation and cytokine production (tumor necrosis factor-alpha, IL-4, and interferon-gamma). Cows milked 1x produced 31% less milk than cows milked 2x during the first week of lactation. Over the following 13 wk of lactation, the milk production of cows milked 1x during the first week was 8.1% lower than for cows milked 2x. However, because the percentages of fat and protein were greater in the milk from 1x cows, the yields of milk components and energy-corrected milk were similar. Calving induced an increase in the concentrations of NEFA, BHBA, urea, and bilirubin. The increases in levels of NEFA and BHBA were greater in cows milked 2x than in cows milked 1x. During the same period, the serum glucose concentration decreased but remained greater in cows milked 1x. Serum calcium on d 4 and serum phosphorus on d 4 and 5 were greater in cows milked 1x. The differences between the 2 groups persisted beyond treatment until postpartum d 24 for NEFA and glucose and until postpartum d 14 for BHBA. After calving, the concentrations of leptin and stanniocalcin-1 decreased. During the first week postpartum, the decrease of leptin was less marked in cows milked 1x. The immune functions of PBMC and PMNL isolated from experimental cows and incubated using a standard medium did not show clear-cut peripartum immunosuppression. These variables were not significantly affected by the treatments, with the exception of interferon-gamma secretion, which was greater on d 5 and 14 in cows milked 1x. In conclusion, limiting milk production in early lactation had positive effects on metabolite concentration, but larger studies are necessary to establish if this could reduce disease incidence.

Effect of nutrition on the GH responsiveness of liver and adipose tissue in dairy cows.

Dairy cows enter a period of energy insufficiency after parturition. In liver, this energy deficit leads to reduced expression of the liver-specific GH receptor transcript (GHR1A) and decreased GHR abundance. As a consequence, hepatic processes stimulated by GH, such as IGF-I production, are reduced. In contrast, adipose tissue has been assumed to remain fully GH responsive in early lactation. To determine whether energy insufficiency causes contrasting changes in the GH responsiveness of liver and adipose tissue, six lactating dairy cows were treated for 4 days with saline or bovine GH when adequately fed (AF, 120% of total energy requirement) or underfed (UF, 30% of maintenance energy requirement). AF cows mounted robust GH responses in liver (plasma IGF-I and IGF-I mRNA) and adipose tissue (epinephrine-stimulated release of non-esterified fatty acids in plasma, IGF-I mRNA, and p85 regulatory subunit of phosphatidylinositol 3-kinase mRNA). Reductions of these responses were seen in the liver and adipose tissue of UF cows and were associated with decreased GHR abundance. Reduced GHR abundance occurred without corresponding reductions of GHR1A transcripts in liver or total GHR transcripts in adipose tissue. In contrast, undernutrition did not alter the abundance of proteins involved in the early post-receptor signaling steps. Thus, a feed restriction reproducing the energy deficit of early lactation depresses GH actions not only in liver but also in adipose tissue. It remains unknown whether a similar reduction of GH action occurs in the adipose tissue of early lactating dairy cows.

Ontogenic and nutritional regulation of steroid receptor and IGF-I transcript abundance in the prepubertal heifer mammary gland.

In prepubertal cattle, mammary development is characterized by the growth of an epithelial-rich parenchyma (PAR) into the mammary fat pad (MFP). This proliferation and accumulation of mammary epithelial cells require estrogen. Paradoxically, both epithelial cell proliferation and PAR accumulation rate decline with rising plasma estrogen as puberty approaches. The possibility that variation in abundance of estrogen receptors (ERs) in PAR or MFP accounts for a portion of these effects has not been examined in cattle. Additionally, we recently demonstrated that MFP is highly responsive to exogenous estrogen, suggesting that this tissue may play a role in coordinating estrogen's effects on PAR; however, the developing bovine MFP has yet to be studied in detail. To address these hypotheses, Holstein heifers were assigned to planes of nutrition supporting body growth rates of 950 (E) or 650 (R) g/day and harvested every 50 kg from 100 to 350 kg body weight (BW). Post-harvest, their mammary glands were dissected into PAR and MFP compartments. Transcript abundance of genes encoding members of the ER family (ERalpha, ERbeta, and estrogen-related receptor alpha-1 (ERRalpha)) and estrogen-responsive genes (IGF-I and progesterone receptor (PR)) were measured in both mammary compartments by quantitative real-time RT-PCR. Significant expression was detected for all genes in both compartments, with the exception of the ERbeta gene. Transcript abundance of both ERalpha and IGF-I decreased linearly with increasing BW within both compartments. ERRalpha and PR expressions decreased with increasing BW in PAR but not in MFP. Nutrition stimulated ERalpha and ERRalpha expression in the PAR but had no effect on IGF-I or PR in either PAR or MFP. Overall, ERalpha and IGF-I transcript abundance are consistent with the drop in mammary epithelial cell proliferation and PAR accretion observed over development, but do not support a negative effect of nutrition on PAR growth.

Effect of estrogen on leptin and expression of leptin receptor transcripts in prepubertal dairy heifers.

Plasma leptin concentrations increase as growing dairy heifers approach puberty and have greater plasma estrogen. In intact and ovariectomized rodents, estrogen has been shown to modulate expression of leptin and its receptor (Ob-R). To determine if estrogen regulates the bovine leptin system, prepubertal dairy heifers were ovariectomized at 140 d of age or left intact. A month later, both groups received a subcutaneous injection of excipient or 17beta-estradiol for 3 consecutive days. Neither ovarian status nor 17beta-estradiol injection altered plasma leptin or leptin mRNA abundance in adipose tissue depots. To assess whether these factors affected Ob-R expression, we tested 20 bovine tissues for leptin receptor (Ob-R) by using quantitative real-time PCR assays for the short receptor isoform (Ob-Ra), the long receptor isoform (Ob-Rb), and all receptor isoforms (Ob-R(TOTAL)). Ob-R(TOTAL) was detected in all tissues, with copy numbers covering 3 orders of magnitude between the lowest and highest expressing tissues (kidney cortex vs. liver). The Ob-Rb isoform accounted for 40% of Ob-R(TOTAL) in the hypothalamus, but averaged less than 3% of Ob-R(TOTAL) in peripheral tissues. Reciprocally, Ob-Ra accounted for only 19% of Ob-R(TOTAL) in the hypothalamus and for nearly all of Ob-R(TOTAL) in most peripheral tissues. Finally, we evaluated the effects of ovarian status and 17beta-estradiol on Ob-R expression in selected tissues. Treatment with 17beta-estradiol reduced Ob-R(TOTAL), Ob-Rb, and Ob-Ra expression by 70% in the uterine endometrium and tended to do the same in mammary adipose tissue. There was no effect of 17beta-estradiol on Ob-R in the hypothalamus, liver, soleus muscle, or subcutaneous adipose tissue. We conclude that greater estrogen secretion does not cause increased plasma leptin in prepubertal dairy heifers but estradiol can modulate Ob-R expression in some estrogen-responsive tissues.

Regulation of gene expression in the bovine mammary gland by ovarian steroids.

It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent on progesterone. The effects of these steroid hormones on gene expression in the mammary gland are mediated primarily by their respective nuclear hormone receptors, which function as hormone-bound transcription factors. To gain insight into how estrogen and progesterone regulate mammary gland growth and function in cattle, we and others have characterized the expression patterns of their cognate nuclear hormone receptors in the bovine mammary gland throughout development, pregnancy, and lactation. This work has identified a lack of expression of estrogen receptor beta and a greater abundance of progesterone receptor during lactation in the bovine mammary gland, compared with the rodent gland. We speculate that interactions among the estrogen receptor isoforms that regulate progesterone receptor expression may contribute to these species differences. Further, demonstrated expression of substantial quantities of estrogen receptor within the prepubertal bovine mammary fat pad, along with coordinated insulin-like growth factor-I expression, suggests that this tissue may stimulate parenchymal growth via an estrogen-responsive paracrine mechanism. In addition, the recent availability of bovine genomic sequence information and microarray technologies has permitted the study of global gene expression in the mammary gland in response to the steroid environment. We have identified more than 100 estrogen-responsive genes, of which the majority are novel estrogen gene targets. Estrogen-induced changes in gene expression were consistent with increased mammary epithelial cell proliferation, increased extracellular matrix turnover in parenchyma, and increased extracellular matrix deposition in the fat pad. A comparison of estrogen-responsive genes in the mammary glands of humans, mice, and cattle suggests considerable variation among species, as well as potential differences in regulatory elements in common estrogen receptor gene targets. Continuing studies using advanced molecular techniques should assist in elucidating the complex regulation of mammary function at the transcript level.

Estrogen-dependent responses of the mammary fat pad in prepubertal dairy heifers.

Ovaries are absolutely required for development of the mammary parenchyma (PAR) in cattle, reflecting estrogen-dependent epithelial cell proliferation. However, the estrogen receptor (ER) that mediates the mammary estrogen effects, ERalpha, is absent in proliferating epithelial cells. In the mouse, this discrepancy is explained in part by the ability of the mammary fat pad (MFP) to synthesize epithelial cell mitogens such as IGF-I in response to estrogen. Consistent with a similar role for the bovine MFP, 30% of its fibroblasts and adipocytes were immunoreactive for ERalpha in prepubertal dairy heifers. To assess estrogen-dependent gene expression in the MFP, 16 prepubertal dairy heifers were randomly assigned to a 2x2 factorial. The first factor was ovarian status, with heifers undergoing bilateral ovariectomy or left intact at 4.6 months of age. The second factor was applied 30 days after surgery and consisted of injection of estrogen or excipient. After 3 days of injection, heifers were administered an intrajugular bolus of bromodeoxyuridine (BrdU) and slaughtered 2 h later. The estrogen injection, but not ovarian status, caused significant increases in the fraction of epithelial cells labeled with BrdU and produced tissue-specific effects on gene expression. In the PAR, estrogen injection increased IGF-I gene expression by twofold despite reductions of 50% or more in ERalpha mRNA abundance and the fraction of epithelial cells immunoreactive for ERalpha. The estrogen-dependent increase in IGF-I mRNA was greater in the MFP, presumably because estrogen failed to downregulate ERalpha expression in this mammary compartment. Finally, estrogen-responsiveness of the MFP appears unique among the bovine fat depots as estrogen injection did not induce IGF-I expression in its s.c. counterpart. Our data demonstrate that the bovine MFP is highly responsive to exogenous estrogen, consistent with a role for this tissue compartment in communicating its effects on epithelial cell proliferation.

Leptin does not act directly on mammary epithelial cells in prepubertal dairy heifers.

The mammary gland of prepubertal dairy heifers consists of parenchyma expanding into the stroma, a matrix of connective and adipose tissue. High planes of nutrition increase stromal mass, but inhibit growth of parenchyma. The parenchyma consists of epithelial cells proliferating in response to growth factors such as insulin like growth factor-I (IGF-I). These observations have led to the hypothesis that elevated planes of nutrition increase leptin production, which in turn inhibits IGF-I-mediated epithelial cell proliferation. To assess this possibility, heifers were offered planes of nutrition sustaining average daily gains of 715 g/d (normal; NP) or 1,202 g/d (high; HP) from 42 d of age until slaughter at 240 kg. At slaughter, HP heifers had 2-fold greater plasma leptin concentration and 3-fold greater leptin mRNA abundance in mammary stroma and parenchyma. To assess the causal nature between leptin and parenchymal development, the induction of signaling events and functional responses in the MAC-T cell line and in primary mammary epithelial cells by leptin was examined. Leptin did not induce phosphorylation of signal transducers and activators of transcription (STAT)3, STAT5, extracellular signal-regulated kinase (ERK1/2), or AKT/Protein kinase B. Consistent with its inability to signal, leptin did not alter basal- or IGF-I-stimulated thymidine incorporation or increase suppressors of cytokine signaling 3 (SOCS3) expression in these cells. Transcripts corresponding to the short leptin receptor form were present in mammary tissue, but those corresponding to the long signaling form were not detected in either mammary tissue or cells. In conclusion, elevated planes of nutrition increase leptin synthesis in mammary stroma, but leptin does not act directly on bovine mammary epithelial cells.

Growth hormone stimulates transcription of the gene encoding the acid-labile subunit (ALS) of the circulating insulin-like growth factor-binding protein complex and ALS promoter activity in rat liver.

The growth-promoting activity of GH, the principal hormonal determinant of body size, is mediated by insulin-like growth factor I (IGF-I). Most of the IGF-I in plasma circulates in a 150-kDa complex that contains IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). The 150-kDa complex serves as a reservoir of IGF-I and determines its bioavailability to the tissues. Formation of the 150-kDa complex depends upon the synthesis of ALS, which is synthesized primarily in liver and is regulated by GH. The present study demonstrates that GH stimulates ALS gene transcription in rat liver and ALS promoter activity in a rat hepatoma cell line. ALS messenger RNA (mRNA) and ALS nuclear transcripts were decreased to similar extents in the livers of GH-deficient hypophysectomized rats. GH increased hepatic ALS mRNA within 3-4 h to about 65% of the levels seen in sham-operated control rats. To confirm that GH stimulated ALS gene transcription, we transiently transfected an ALS promoter-luciferase reporter gene construct into H4-II-E rat hepatoma cells and primary rat hepatocytes. Recombinant human GH (hGH) stimulated promoter activity about 3-fold. In contrast, basal promoter activity was lower, and GH stimulation was absent when the ALS reporter construct was transfected into GH-responsive 3T3-F442A mouse preadipocyte fibroblasts. GH stimulation of ALS promoter activity in H4-II-E cells was mediated by functional GH receptors; nonprimate (rat and bovine) GH gave identical stimulation to hGH, and stimulation by hGH occurred at physiological concentrations. Reverse transcriptase-PCR analysis indicated that GH receptor mRNA was present in H4-II-E cells at approximately 40% of the level seen in rat liver. GH also induced the expression of the endogenous c-fos gene, indicating that the signaling pathway necessary for the activation of gene expression by GH was intact in H4-II-E cells. Thus, H4-II-E cells are a GH-responsive liver cell line that should provide a useful system in which to study the molecular mechanism of transcriptional regulation by GH of ALS and other hepatic genes.