Effects of the central melanocortin system on feed intake, metabolic hormones and insulin action in the sheep.

Voluntary feed intake is insufficient to meet the nutrient demands associated with late pregnancy in prolific ewes and early lactation in high-yielding dairy cows. Under these conditions, peripheral signals such as growth hormone and ceramides trigger adaptations aimed at preserving metabolic well-being. Recent work in rodents has shown that the central nervous system-melanocortin (CNS-MC) system, consisting of alpha-melanocyte-stimulating hormone (α-MSH) and agouti-related peptide (AGRP) acting respectively as agonist and antagonist on central MC receptors, contributes to the regulation of some of the same adaptations. To assess the effects of the CNC-MC on peripheral adaptations in ruminants, ewes were implanted with an intracerebroventricular cannula in the third ventricle and infused over days with artificial cerebrospinal fluid (aCSF), the α-MSH analog melanotan-I (MTI), or AGRP. Infusion of MTI at 0.03 nmol/h reduced intake, expressed as a fold of maintenance energy requirement (M), from 1.8 to 1.1 M (P < 0.0001), whereas AGRP at 0.3 nmol/h increased intake from 1.8 to 2.0 M (P < 0.01); these doses were used in all subsequent experiments. To assess the effect of MTI on plasma variables, sheep were fed ad libitum and infused with aCSF or MTI or pair-fed to MTI-treated sheep and infused with aCSF (aCSFPF). Feed intake of the MTI and aCSFPF groups was 40% lower than the aCSF group (P < 0.0001). MTI increased plasma triiodothyronine and thyroxine in an intake-independent manner (P < 0.05 or less) but was devoid of effects on plasma glucose, insulin, and cortisol. None of these variables were altered by AGRP infusion in sheep fed at a fixed intake of 1.6 M. To assess the effect of CNS-MC activation on insulin action, ewes were infused with aCSF or MTI over the last 3 d of a 14-d period when energy intake was limited to 0.3 M and studied under basal conditions and during hyperinsulinemic-euglycemic clamps. MTI had no effect on plasma glucose, plasma insulin, or glucose entry rate under basal conditions but blunted the ability of insulin to inhibit endogenous glucose production during hyperinsulinemic-euglycemic clamps (P < 0.0001). Finally, MTI tended to reduce plasma leptin in sheep fed at 0.3 M (P < 0.08), and this effect became significant at 0.6 M (P < 0.05); MTI had no effect on plasma adiponectin irrespective of feeding level. These data suggest a role for the CNC-MC in regulating metabolic efficiency and peripheral insulin action.

Highly productive ruminants face short-term nutritional deficits during demanding phases of their life cycle. They remain productive and healthy during these periods through a series of metabolic adaptations. Current models in ruminant biology attribute the coordination of these adaptations to circulating hormones and bioactive metabolites but have not considered the possibility that the central nervous system (CNS) is also involved. The latter appears likely given recent work in rodents implicating the CNS-melanocortin system in the regulation of some of these adaptations. To test this possibility, mature ewes were surgically implanted with a cannula accessing the brain allowing chronic infusion of melanocortins, and used in experiments assessing peripheral effects. These experiments showed that the CNS-melanocortin system regulates the circulating concentrations of some metabolic hormones as well as the ability of insulin to regulate glucose production. Overall, these studies suggest a role for the CNS-melanocortin system in regulating metabolic adaptations in ruminants.

Fibroblast growth factor-21 improves insulin action in nonlactating ewes.

During metabolically demanding physiological states, ruminants and other mammals coordinate nutrient use among tissues by varying the set point of insulin action. This set point is regulated in part by metabolic hormones with some antagonizing (e.g., growth hormone and TNFα) and others potentiating (e.g., adiponectin) insulin action. Fibroblast growth factor-21 (FGF21) was recently identified as a sensitizing hormone in rodent and primate models of defective insulin action. FGF21 administration, however, failed to improve insulin action in dairy cows during the naturally occurring insulin resistance of lactation, raising the possibility that ruminants as a class of animals or lactation as a physiological state are unresponsive to FGF21. To start addressing this question, we asked whether FGF21 could improve insulin action in nonlactating ewes. Gene expression studies showed that the ovine FGF21 system resembles that of other species, with liver as the major site of FGF21 expression and adipose tissue as a target tissue based on high expression of the FGF21 receptor complex and activation of p44/42 extracellular signal-regulated kinase (ERK1/2) following exogenous FGF21 administration. FGF21 treatment for 13 days reduced plasma glucose and insulin over the entire treatment period and improved glucose disposal during a glucose tolerance test. FGF21 increased plasma adiponectin by day 3 of treatment but had no effect on the plasma concentrations of total, C16:0-, or C18:0-ceramide. Overall, these data confirm that the insulin-sensitizing effects of FGF21 are conserved in ruminants and raise the possibility that lactation is an FGF21-resistant state.

Effects of birth weight and dietary fat on intake, body composition, and plasma thyroxine in neonatal lambs.

Intrauterine growth restriction (IUGR) is often observed in one of the fetuses carried by well-fed prolific ewes. This condition is the result of an insufficient placental size to cover the nutritional needs of the fetus during the near exponential growth phase of the last trimester. After birth, these IUGR offspring have an elevated appetite and lower maintenance energy requirements, suggesting dysregulation of homeostatic systems governing energy metabolism. It is also unknown whether the consequent increase in fatness occurs similarly in both visceral and carcass fractions. To address these questions, lambs differing in birth size (BS, IUGR vs. Normal, 2.6 ± 0.05 vs. 4.2 ± 0.07 kg, P < 0.001) were offered unlimited amounts of a low fat [LF; 22% of dry matter (DM)] or a high fat (HF; 38% of DM) milk replacer and slaughtered on day 14 of postnatal age (n = 7 to 8 for each BS × Diet); a second group of IUGR lambs (n = 3 for each diet) was slaughtered when they reached 8.5 kg, corresponding to the weight of Normal lambs on day 14. When normalized to body weight (BW), the DM and energy intake of IUGR lambs were higher than those of Normal lambs over the first 14 d of life (BS, P < 0.01), but contrary to expectations, the HF diet did not exacerbate these effects of the IUGR condition. Intrauterine growth restricted lambs had increased viscera fat with both diets (BS and Diet, P < 0.05) but increased carcass fat only with the LF diet (BS × Diet, P = 0.08); the fatness promoting effect of the IUGR condition was increased in both body fractions when lamb groups were compared at the fixed BW of 8.5 kg. A subset of metabolic hormones was analyzed, including the metabolic rate-setting hormone thyroxine (T4) and its possible positive regulator leptin. Plasma T4 was lower in IUGR than in Normal lambs at birth (P < 0.05) but then disappeared by day 7 of postnatal life (BS × Day, P < 0.01). On the other hand, the HF diet had no effect on plasma T4 over the first 3 d but caused an increase, irrespective of BS by day 11 (Diet × Day, P < 0.001). Plasma leptin increased with dietary fat and time (P < 0.06) but bore no relation to the effects of BS or Diet on plasma T4. These data show that IUGR and Normal lambs are similarly unable to adjust caloric intake in early life and that the fatness promoting effects of the IUGR condition are more pronounced in the viscera than in the carcass. These data also reveal dynamic regulation of plasma T4 by BS and Diet in neonatal lambs.

Technical note: Effects of pegylation and route of administration on leptin kinetics in newborn lambs1.

Chronic energy insufficiency resulting from inadequate feed intake or increased nutrient demand reduces plasma leptin in ruminants. Treatment of energy-deficient ruminants with exogenous leptin has identified some physiological consequences of reduced plasma leptin, but their full complement remains unknown. Additional leptin-dependent responses could be identified by using strategies that interfere with leptin signaling such as administration of leptin mutants that act as competitive antagonists. The effectiveness of these antagonists depends on their fold excess over endogenous leptin, and this condition can be achieved under in vivo conditions by extending the half-life (t1/2) of the antagonist by addition of a polyethylene glycol (PEG) molecule (pegylation). Use of this approach in ruminants, however, is limited by the lack of information on the t1/2 of native and pegylated leptin and on the most effective route of administration. To answer these questions, newborn lambs (n = 3) were injected with an intravenous (i.v.) bolus of 150 µg of human leptin followed by blood sampling over the next 12 h. Analysis of the semilog plasma leptin concentration over time yielded a t1/2 of 43 ± 4.9 min; an i.v. bolus of 276 µg of bovine leptin yielded a comparable t1/2 (P > 0.05). Next, newborn lambs (n = 4) received a single dose of 229 µg/kg of metabolic body weight (BW0.75) of pegylated super human leptin antagonist (PEG-SHLA) via the i.v. or subcutaneous (s.c.) route. Plasma PEG-SHLA concentration reached a peak of 1,528 ± 78 ng/mL after 1 min and a nadir of 71 ± 9 ng/mL after 24 h with the i.v. route versus a peak of 423 ± 43 ng/mL after 300 min and a nadir of 146 ± 22 ng/mL after 24 h for the s.c. route; the t1/2 of PEG-SHLA was 394 ± 29 min for the i.v. route and 433 ± 58 min for the s.c. route. Finally, plasma concentration of PEG-SHLA was modeled when given either i.v. or s.c. at a dose of 229 µg/kg BW0.75 every 12 h. Once a steady state was reached, peak and lowest concentrations PEG-SHLA over the 12-h windows were 2,269 and 403 ng/mL for the i.v. route and 814 and 555 ng/mL for the s.c. route. Weighted PEG-SHLA concentrations over the 12-h period were 1,455 and 713 ng/mL for the i.v. and s.c. route, translating into 364- and 178-fold excess over endogenous plasma leptin. These data confirm the effectiveness of pegylation in extending the t1/2 of leptin antagonists in newborn lambs and in increasing their circulation in fold excess over endogenous leptin.

Intravenous Triacylglycerol Infusion Promotes Ceramide Accumulation and Hepatic Steatosis in Dairy Cows.

Background: Increased plasma free fatty acids (FFAs) impair insulin sensitivity in dairy cows via unknown mechanisms. In nonruminants, saturated FFAs upregulate the hepatic synthesis and secretion of ceramide, which inhibits insulin action. Objective: We aimed to determine whether an increase in plasma FFAs promotes hepatic and plasma ceramide accumulation in dairy cows. Methods: Six nonpregnant, nonlactating Holstein cows were used in a study with a crossover design and treatments consisting of intravenous infusion of either saline (control) or triacylglycerol emulsion (TG; 20 g/h) for 16 h. The feeding level was set at 120% of energy requirements. Blood was collected at regular intervals and liver was biopsied at 16 h. Ceramides, monohexosylceramides (Glc/Gal-Cer), lactosylceramides (LacCer), and sphingomyelins (SMs) in plasma and liver were profiled. Hepatic expression of ceramide synthases was determined. Data were analyzed with the use of mixed models, regressions, and Spearman rank correlations. Results: After 16 h of infusion, plasma FFA concentrations were >5-fold and liver triacylglycerol concentrations were 4-fold greater in TG cows, relative to control. Plasma total and very long-chain ceramide (e.g., C24:0-ceramide) concentrations increased ∼4-fold in TG over control by hour 16 of infusion, while C16:0-ceramide were not modified by TG. Infusion of TG increased plasma Glc/Gal-Cer (e.g., C16:0-Glc/Gal-Cer, 4-fold by hour 16) relative to control, but did not alter LacCer or SM concentrations. Hepatic ceramide concentrations increased with TG relative to control (e.g., C24:0-ceramide by 1.7-fold). Hepatic expression of ceramide synthase 2 was 60% greater after TG infusion compared with the control. Circulating ceramides were related to circulating FFA and hepatic triacylglycerol concentrations (e.g., C24:0-ceramide, ρ = 0.73 and 0.80, respectively; P < 0.001). Conclusion: Hepatic ceramide synthesis is associated with elevations in circulating FFAs and hepatic triacylglycerol during the induction of hyperlipidemia in dairy cows. This work supports the emerging evidence for the role of ceramide during hepatic steatosis and insulin antagonism in cows.

Effect of circulating glucagon and free fatty acids on hepatic FGF21 production in dairy cows.

Modern dairy cows meet the energy demand of early lactation by calling on hormonally driven mechanisms to increase the use of lipid reserves. In this context, we recently reported that fibroblast growth factor-21 (FGF21), a hormone required for efficient use of lipid reserves in rodents, is upregulated in periparturient dairy cows. Increased plasma FGF21 in early lactation coincides with elevated circulating concentrations of glucagon (GCG) and nonesterified fatty acids (NEFA). To assess the relative contribution of these factors in regulating FGF21, two experiments were performed in energy-sufficient, nonpregnant, nonlactating dairy cows. In the first study, cows were injected with saline or GCG every 8 h over a 72-h period. GCG increased hepatic FGF21 mRNA by an average of fivefold over matched controls but had no effect on plasma FGF21. In the second study, cows were infused and injected with saline, infused with Intralipid and injected with saline, or infused with Intralipid and injected with GCG. Infusions and injections were administered intravenously over 16 h and subcutaneously every 8 h, respectively. Intralipid infusion increased plasma NEFA from 92 to 550 µM within 3 h and increased plasma FGF21 from 1.3 to >11 ng/ml 6 h later; FGF21 mRNA increased by 34-fold in liver but remained invariant in adipose tissue. GCG injections during the Intralipid infusion had no additional effects on plasma NEFA, liver FGF21 mRNA, or plasma FGF21. These data implicate plasma NEFA as a key factor triggering hepatic production and increased circulating concentrations of FGF21 in early lactation.

Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.

Mammals meet the increased nutritional demands of lactation through a combination of increased feed intake and a collection of adaptations known as adaptive metabolism (e.g., glucose sparing via insulin resistance, mobilization of endogenous reserves, and increased metabolic efficiency via reduced thyroid hormones). In the modern dairy cow, adaptive metabolism predominates over increased feed intake at the onset of lactation and develops concurrently with a reduction in plasma leptin. To address the role of leptin in the adaptive metabolism of early lactation, we asked which adaptations could be countered by a constant 96-h intravenous infusion of human leptin (hLeptin) starting on day 8 of lactation. Compared to saline infusion (Control), hLeptin did not alter energy intake or milk energy output but caused a modest increase in body weight loss. hLeptin reduced plasma glucose by 9% and hepatic glycogen content by 73%, and these effects were associated with a 17% increase in glucose disposal during an insulin tolerance test. hLeptin attenuated the accumulation of triglyceride in the liver by 28% in the absence of effects on plasma levels of the anti-lipolytic hormone insulin or plasma levels of free fatty acids, a marker of lipid mobilization from adipose tissue. Finally, hLeptin increased the plasma concentrations of T4 and T3 by nearly 50% without affecting other neurally regulated hormones (i.e., cortisol and luteinizing hormone (LH)). Overall these data implicate the periparturient reduction in plasma leptin as one of the signals promoting conservation of glucose and energy at the onset of lactation in the energy-deficient dairy cow.

XBP1 Regulates the Biosynthetic Capacity of the Mammary Gland During Lactation by Controlling Epithelial Expansion and Endoplasmic Reticulum Formation.

Cells composing the mammary secretory compartment have evolved a high capacity to secrete not only proteins but also triglycerides and carbohydrates. This feature is illustrated by the mouse, which can secrete nearly twice its own weight in milk proteins, triglycerides and lactose over a short 20-day lactation. The coordination of synthesis and export of products in other secretory cells is orchestrated in part by the transcription factor X-box binding protein 1 (XBP1). To assess the role of XBP1 in mammary epithelial cells (MEC), we studied floxed XBP1 female mice lacking (wild type; WT) or expressing the Cre recombinase under the control of the ovine ß-lactoglobulin promoter (ΔXBP1(MEC)). Pregnant ΔXBP1(MEC) females had morphologically normal mammary development and gave birth to the same number of pups as WT mice. Their litters, however, suffered a weight gain deficit by lactation day 3 (L3)3 that grew to 80% by L14. ΔXBP1(MEC) dams had only modest changes in milk composition (-21% protein, +24% triglyceride) and in the expression of associated genes in isolated MEC. By L5, WT glands were fully occupied by dilated alveoli, whereas ΔXBP1(MEC) glands contained fewer, mostly unfilled alveoli and retained a prominent adipocyte population. The smaller epithelial compartment in ΔXBP1(MEC) glands was explained by lower MEC proliferation and increased apoptosis. Finally, endoplasmic reticulum ribbons were less abundant in ΔXBP1(MEC) at pregnancy day 18 and failed to increase in abundance by L5. Collectively, these results show that XBP1 is required for MEC population expansion during lactation and its ability to develop an elaborate endoplasmic reticulum compartment.

Hepatic FGF21 production is increased in late pregnancy in the mouse.

Female mammals call on hormonally driven metabolic adaptations to meet the energy demand of late pregnancy and lactation. These maternal adaptations preserve limiting nutrients and promote their transfer to the uterus during pregnancy or mammary gland during lactation. The novel metabolic hormone fibroblast growth factor-21 (FGF21) was recently shown to increase suddenly at the onset of lactation in dairy cows, but whether FGF21 is induced during the reproductive cycle of other mammals is unknown. To start addressing this question, we studied subsets of mice when virgin (V), on day 18 of pregnancy (P18) and on lactation day 1 (L1), L5 and L14. Plasma FGF21 increased from nearly undetectable levels to over 8 ng/ml between V and P18 and returned to V levels by L1. Gene expression studies showed that liver was the major source of plasma FGF21 at P18 with little or no contribution from other known expressing tissues or from the developing placenta and mammary epithelial cells. The increased FGF21 production at P18 was dissociated from plasma nonesterified fatty acids and liver lipids, unlike that seen in fasted V mice. Changes in FGF21 signaling components in target tissues were modest except for reduced ß-Klotho and FGFR1c expression in P18 adipose tissue. The placenta expressed both ß-Klotho and FGFR1c, raising the possibility that it responds to FGF21. In conclusion, maternal FGF21 is increased when products of conception account for ∼ 40% of maternal weight, suggesting that FGF21 orchestrates some of the adaptations needed to meet the energy demand of late pregnancy.

Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.

In rodents and primates, insulin resistance develops during pregnancy and fades after parturition. In contrast, dairy cows and other ruminants maintain insulin resistance in early lactation (EL). This adaptation favors mammary glucose uptake, an insulin-independent process, at a time when the glucose supply is scarce. Reduction in circulating levels of the insulin-sensitizing hormone adiponectin promotes insulin resistance in other species, but whether it contributes to insulin resistance in EL dairy cows is unknown. To address this question, plasma adiponectin was measured in high-yielding dairy cows during the transition from late pregnancy (LP) to EL. Plasma adiponectin varied in quadratic fashion with the highest levels in LP, a maximal reduction of 45% on the day after parturition and a progressive return to LP values over the next 8 wk. Adiponectin circulated nearly exclusively in high molecular weight complexes in LP, and this distribution remained unaffected in EL. The reduction of plasma adiponectin in EL occurred without changes in adiponectin mRNA in adipose tissue but was associated with repression of the expression of proteins associated with the endoplasmic reticulum and involved in assembly of adiponectin oligomers. Finally, EL increased the expression of the adiponectin receptor 1 in muscle and adiponectin receptor 2 in liver but had no effect on the expression of these receptors in adipose tissue and in the mammary gland. These data suggest that reduced plasma adiponectin belongs to the subset of hormonal adaptations in EL dairy cows facilitating mammary glucose uptake via promotion of insulin resistance.

Plasma FGF21 is elevated by the intense lipid mobilization of lactation.

In many mammals, lactation success depends on substantial use of lipid reserves and requires integrated metabolic activities between white adipose tissue (WAT) and liver. Mechanisms responsible for this integration in lactation are poorly understood, but data collected in other conditions of elevated lipid use suggest a role for fibroblast growth factor-21 (FGF21). To address this possibility in the context of lactation, we studied high-yielding dairy cows during the transition from late pregnancy (LP) to early lactation (EL). Plasma FGF21 was nearly undetectable in LP, peaked on the day of parturition, and then stabilized at lower, chronically elevated concentrations during the energy deficit of EL. Plasma FGF21 was similarly increased in the absence of parturition when an energy-deficit state was induced by feed restricting late-lactating dairy cows, implicating energy insufficiency as a cause of chronically elevated FGF21 in EL. Gene expression studies showed that liver was a major source of plasma FGF21 in EL with little or no contribution by WAT, skeletal muscle, and mammary gland. Meaningful expression of the FGF21 coreceptor ß-Klotho was restricted to liver and WAT in a survey of 15 tissues that included the mammary gland. Expression of ß-Klotho and its subset of interacting FGF receptors was modestly affected by the transition from LP to EL in liver but not in WAT. Overall, these data suggest a model whereby liver-derived FGF21 regulates the use of lipid reserves during lactation via focal actions on liver and WAT.

The full-length isoform of the mouse pleckstrin homology domain-interacting protein (PHIP) is required for postnatal growth.

PHIP was isolated as an insulin receptor substrate 1 (IRS-1) interacting protein. To date, the physiological roles of PHIP remain unknown. Here we show that mice lacking PHIP1, the full-length isoform of PHIP, are born at normal size but suffer a 40% growth deficit by weaning. PHIP1 mutant mice develop hypoglycemia and have an average lifespan of 4-5 weeks. PHIP1-deficient mouse embryonic fibroblasts (MEFs) grow markedly slower than wild-type MEFs, but exhibit normal AKT phosphorylation and an increased cell proliferation in response to IGF-1 treatment. Together these results suggest that PHIP1 regulates postnatal growth in an IGF-1/AKT pathway-independent manner.

Mammary ductal growth is impaired in mice lacking leptin-dependent signal transducer and activator of transcription 3 signaling.

Mice lacking leptin (ob/ob) or its full-length receptor (db/db) are obese and reproductively incompetent. Fertility, pregnancy, and lactation are restored, respectively, in ob/ob mice treated with leptin through mating, d 6.5 post coitum, and pregnancy. Therefore, leptin signaling is needed for lactation, but the timing of its action and the affected mammary process remain unknown. To address this issue, we used s/s mice lacking only leptin-dependent signal transducer and activator of transcription (STAT)3 signaling. These mice share many features with db/db mice, including obesity, but differ by retaining sufficient activity of the hypothalamic-pituitary-ovarian axis to support reproduction. The s/s mammary epithelium was normal at 3 wk of age but failed to expand through the mammary fat pad (MFP) during the subsequent pubertal period. Ductal growth failure was not corrected by estrogen therapy and did not relate to inadequate IGF-I production by the MFP or to the need for epithelial or stromal leptin-STAT3 signaling. Ductal growth failure coincided with adipocyte hypertrophy and increased MFP production of leptin, TNFalpha, and IL6. These cytokines, however, were unable to inhibit the proliferation of a collection of mouse mammary epithelial cell lines. In conclusion, the very first step of postnatal mammary development fails in s/s mice despite sufficient estrogen IGF-I and an hypothalamic-pituitary-ovarian axis capable of supporting reproduction. This failure is not caused by mammary loss of leptin-dependent STAT3 signaling or by the development of inflammation. These data imply the existence of an unknown mechanism whereby leptin-dependent STAT3 signaling and obesity alter mammary ductal development.

The acid-labile subunit is required for full effects of exogenous growth hormone on growth and carbohydrate metabolism.

Normal postnatal growth is dependent in part on overlapping actions of GH and IGF-I. These actions reflect GH stimulation of IGF-I production in liver and extrahepatic tissues, representing respectively the endocrine and autocrine/paracrine arms of the IGF system. Recent experiments in genetically modified mice show that each source of IGF-I can compensate for absence of the other but do not resolve their relative role in postnatal growth. In an effort to address this issue, we studied the GH responsiveness of mice harboring a null mutation of the acid-labile subunit (ALS). Null ALS mice have a substantial reduction in endocrine IGF-I but, unlike other models of plasma IGF-I deficiency, have no obvious additional endocrine defects. Wild type and null ALS mice of both sexes received daily sc injections of saline or recombinant bovine GH between d 35 and 63 of postnatal age. The GH-stimulated body weight gain of null ALS mice was reduced by more than 30% relative to wild type mice, irrespective of sex. Reductions in GH responsiveness were also seen for kidney and linear growth. Absence of ALS eliminated the ability of GH to increase plasma IGF-I despite intact GH-dependent stimulation of IGF-I expression in liver, adipose tissue, and skeletal muscle. GH treatment was also less efficient in antagonizing insulin action in null ALS mice. Overall, these results suggest that the GH effects mediated by endocrine IGF-I depends on ALS, and accordingly null ALS mice are less responsive to exogenous GH therapy.