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Energy balance controls the expression of the leptin receptor (Lepr) in the ruminant hypothalamus but whether similar regulation occurs in peripheral tissues is unknown. To address this issue, we measured Lepr expression in the liver and adipose tissue of dairy cows during the transition from late pregnancy (LP) to early lactation (EL). This period is characterized by the development of a profound state of energy insufficiency and is associated with reduced plasma insulin and leptin and with increased plasma growth hormone. Hepatic expression of the short (Lepr-a) and long (Lepr-b) isoforms was 40% higher during EL (8 days postpartum) than LP (30 days prepartum). A similar effect was observed when negative energy balance was induced in nonpregnant, late-lactation dairy cows by food restriction, implicating energy insufficiency as a specific cause in EL. The stimulation of hepatic Lepr expression was reversed after a 48-h period of hyperinsulinemic euglycemia in EL. Changes in hepatic Lepr expression during chronic elevation of plasma leptin in EL or plasma growth hormone in nonpregnant, late-lactation cows did not support a role for these hormones in mediating the effects of energy insufficiency on hepatic Lepr expression. In adipose tissue, Lepr expression was increased 10-fold during the transition from LP to EL. Overall, these data indicate that hypoinsulinemia is partly responsible for the induction of Lepr expression in the liver, and perhaps adipose tissue, of energy-deficient dairy cows. bovine; energy balance; liver; adipose

After parturition, increased growth hormone (GH) secretion is important to preserve the metabolic homeostasis of energy-deficient dairy cows. Elevated plasma GH promotes lipid mobilization from adipose tissue, but paradoxically, is associated with depressed concentration of insulin-like growth factor-I (IGF-I), a growth factor produced in a GH-dependent fashion in liver. Primary factors regulating GH responses of liver and adipose tissue are poorly understood in periparturient dairy cows. Consistent with insulin being such a factor, its plasma concentration declined concomitantly with net energy balance (EB) and with plasma IGF-I in a group of 9 periparturient dairy cows. To test the role of insulin in regulating cellular determinants of GH responsiveness, hyperinsulinemic-euglycemic clamps were performed on 6 dairy cows in late pregnancy (28 d prepartum) before the reductions in EB, insulin, and IGF-I were initiated, and when they were completed in early lactation (10 d postpartum). Infusion of insulin nearly doubled the plasma concentration of IGF-I (P < 0.001) and hepatic levels of IGF-I mRNA during both states (P < 0.05). In liver, these responses were associated with increased abundance of the GH receptor protein (GHR; P < 0.05), whereas the abundance of intracellular mediators of GH actions (JAK2, STAT5, or STAT3) remained unaffected. Insulin also doubled GHR abundance in adipose tissue (P < 0.01), indicating that this effect is not liver specific. These results raise the possibility that insulin regulates the efficiency of GH signaling in liver and adipose tissue of dairy cows by acting as a rheostat of GHR synthesis. KEY WORDS: * pregnancy * lactation * hyperinsulinemic-euglycemic clamp * insulin-like growth factor-I

In juvenile and mature animals, the plasma concentration of leptin is regulated by adiposity and nutrition. However, the timing of these influences on plasma leptin, and their relative importance in early postnatal life, are unknown. We investigated these plasma leptin influences in sheep, a species characterized during fetal life by leanness and insensitivity of leptin to variation in maternal nutrition. Small and large neonatal lambs were randomly assigned to either a diet sustaining an average daily weight gain (ADG) of 148 g/d (Low plane) or ate ad libitum a diet sustaining an ADG of 337 g/d (High plane). A subset of animals were slaughtered at 7.5, 10, 15 and 20 kg of body weight. Birth size had no effect on plasma leptin concentrations and adiposity at birth or at later times. Plasma leptin concentrations increased within 6 d of birth in the High plane lambs (P < 0.01) and continued to rise over time. In contrast, plasma leptin concentrations never changed in the Low plane lambs despite increasing adiposity. The positive association between plasma leptin concentration and adiposity was greater in the High plane than in the Low plane lambs, suggesting an independent effect of nutrition. Consistent with this finding, lipid accretion rates, a variable that is mostly independent of adiposity, was a strong predictor of plasma leptin concentrations only in the High plane lambs ([R.sup.2] = 0.77, P < 0.01). A positive association between plasma insulin and leptin developed over time in the High plane lambs ([R.sup.2] = 0.75, P < 0.01 on d 40), but was not seen in the Low plane lambs. These data indicate that both nutrition and adiposity regulate plasma leptin synthesis in early postnatal life, but in contrast to adulthood, the effects of nutrition appear to be predominant. KEY WORDS: * leptin * fat * neonatal * sheep * nutrition * insulin

Leury, Brian J., Lance H. Baumgard, Stephanie S. Block, Nthabisheng Segoale, Richard A. Ehrhardt, Robert P. Rhoads, Dale E. Bauman, Alan W. Bell, and Yves R. Boisclair. Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows. Am J Physiol Regul Integr Comp Physiol 285: R1107-R1115, 2003. First published July 24, 2003; 10.1152/ajpregu.00320.2003.--After parturition, dairy cows suffer from an intense energy deficit caused by the onset of copious milk secretion and an inadequate increase in voluntary food intake. We previously showed that this energy deficit contributes to a decline in plasma leptin. This decline mirrors that of plasma insulin but is reciprocal to the profile of plasma growth hormone (GH), suggesting that both hormones may regulate plasma leptin in periparturient dairy cows. To study the role of insulin, hyperinsulinemic-euglycemic clamps were performed on six dairy cows in late pregnancy (LP, 31 days prepartum) and early lactation (EL, 7 days postpartum). Infusion of insulin (1 [micro]g.kg body [wt.sup.-1].[h.sup.-1]) caused a progressive rise in the plasma concentration of leptin that reached maximum levels at 24 h during both physiological states. At steady states, the absolute increase in plasma leptin was greater in LP than in EL cows (2.4 vs. 0.4 ng/ml). Insulin infusion increased leptin mRNA in adipose tissue during LP but not during EL. During lactation, mammary epithelial cells expressed leptin mRNA but insulin did not increase milk leptin output. In contrast, a 3-day period of GH administration had no effect on plasma leptin during LP or EL. Therefore, insulin increases plasma leptin in LP by stimulating adipose tissue synthesis but has only marginal effects in EL, when cows are in negative energy balance. Other factors, such as increased response of adipose tissue to [beta]-adrenergic signals, probably contribute to the reduction of plasma leptin in early lactating dairy cows. pregnancy; lactation; hyperinsulinemic-euglycemic clamp

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. [ABSTRACT FROM AUTHOR]

To better understand the biology of leptin during prenatal life, the developmental and spatial regulation of leptin was studied in ovine fetuses. Fetal plasma leptin increased steadily between days 40 and 143 postcoitus (PC), but it was unrelated to fetal weight or placental weight at day 135 PC. Leptin gene expression was detected in fetal brain and liver during most of gestation and in fetal adipose tissue after day 100 PC. At day 130 PC, expression in fetal perirenal adipose tissue was ~10% of maternal expression. In contrast, leptin gene expression was never detected in the placenta and other uteroplacental tissues. When ewes were fed 55% of requirements between days 122 and 135 PC, fetal plasma leptin remained constant despite acute reduction in maternal concentration. We conclude that fetal plasma leptin originates mostly from nonadipose tissue in early pregnancy and, in addition, from fetal adipose tissue near term. The role of fetal plasma leptin remains uncertain given the lack of nutritional regulation and association with fetal growth. pregnancy; placenta; nutrition

Our objectives were to assess the effects of chronic administration of recombinant bovine somatotropin (bST) on nutrient utilization and protein turnover in the hindlimb of growing Holstein steers. External iliac vessels were catheterized to allow for hindlimb measurements of arteriovenous differences and blood flow. Animals were used in a single-reversal design with 16-d treatment periods of daily intramuscular injection of either excipient or 120 micrograms/kg body wt of bST. On d 11 and 13 of each period, a primed-continuous infusion of L-(sidechain-2,3-3H)tyrosine was initiated, followed by a 4-h sampling period to assess hindlimb nutrient utilization and protein kinetics. Somatotropin did not alter blood flow or the consumption of acetate and oxygen across the hindlimb. In contrast, glucose uptake was reduced by 22% despite increases in arterial concentrations of glucose and insulin of 10 and 114%, respectively. Treatment with bST increased hindlimb protein accretion (estimated from net uptake of tyrosine) and whole-body N balance, each by approximately 40%. A modest increase (10%) in the absolute rate of protein synthesis seemed to account for the improved N retention in the hindlimb with no change in the rate of protein degradation. Thus, bST reduced the responses of the hindlimb to insulin, and a small alteration in protein synthesis was sufficient to explain substantial improvement in protein deposition.

Our objectives were to validate a technique to measure protein turnover based on net fluxes of tyrosine radioactivity and mass across the hindlimb, and to describe the impact of nutrition on hindlimb protein kinetics and nutrient utilization in growing Holstein steers. External iliac vessels were catheterized to allow for measurements of hindlimb arteriovenous (AV) differences and blood flow. Animals were used in a single reversal design with 16-d periods. Treatments were either 60% (0.6M) or 220% (2.2M) of the energy required for maintenance. On d 12 and 14 of each period, a primed-continuous infusion of L-(side chain-2,3-3H)tyrosine was initiated, followed by a 4-h sampling period to assess hindlimb nutrient utilization and protein kinetics. Well-fed steers exhibited rates of hindlimb utilization for non-nitrogenous energy substrates and amino acids that were consistent with values previously reported for well-fed ruminants. Underfeeding decreased hindlimb blood flow (62%), oxygen consumption (51%) and utilization of respiratory fuels (64-73%). Protein synthesis was also reduced (49%), leading to net nitrogen loss from the hindlimb. The estimated energy cost of protein synthesis accounted for 13% of hindlimb oxygen consumption across intake levels. Hindlimb protein balance was consistent with whole-body balance, adding credence to derived rates of degradation. Overall, data support the validity of the AV difference technique to estimate components of hindlimb protein turnover.

Trans‐10, cis‐12 conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis in the cow and similarly reduces milk fat in rodents. The objective of this study was to determine whether dietary fat can overcome CLA inhibition of milk fat concentration in lactating mice. Wild type C57Bl/6J mice (n = 31) were fed semipurified diets containing either low fat (LF; 4% fat) or high fat (HF; 23.6% fat) starting 4–6 days postpartum. Dietary fat was increased by inclusion of high oleic sunflower oil. After 2 days on the experimental diets, lactating dams were orally dosed with either water (control) or trans‐10, cis‐12 CLA (20 mg/day) for 5 days. CLA treatment decreased pup growth similarly in both HF and LF diets. Milk fat percent was increased over 16% by the HF diet and decreased over 12% by CLA, but there was no interaction of dietary fat and CLA. Both CLA and the HF diet reduced the proportion of short‐ and medium‐chain fatty acids that originate from de novo synthesis, and there was no interaction of diet and CLA. CLA had no effect on the percent of preformed fatty acids, but the HF diet increased their abundance. Dietary fat and CLA both modified mammary expression of lipogenic enzymes and regulators, but no interactions were observed. In conclusion, CLA reduced milk fat concentration and litter growth, but these effects were not overcome by increased dietary fat from high oleic sunflower oil. CLA inhibition of milk fat in the mammary gland is not substrate dependent, and the mechanism is independent from dietary supply of oleic acid. [ABSTRACT FROM AUTHOR]

The molecular underpinnings of β-cell dysfunction and death leading to diabetes are not fully elucidated. The objective of the current study was to investigate the role of endoplasmic reticulum-associated degradation (ERAD) in pancreatic β-cells. Chemically induced ERAD deficiency in the rat insulinoma cell line INS-1 markedly reduced glucose-stimulated insulin secretion (GSIS). The mechanistic basis for this effect was studied in cells and mice lacking ERAD as a consequence of genetic ablation of the core ERAD protein SEL1L. Targeted disruption of SEL1L in INS-1 cells and in mouse pancreatic β-cells impaired ERAD and led to blunted GSIS. Additionally, mice with SEL1L deletion in β-cells were chronically hyperglycemic after birth and increasingly glucose intolerant over time. SEL1L absence caused an entrapment of proinsulin in the endoplasmic reticulum compartment in both INS-1 cells and mouse pancreatic β-cells. Both folding-competent and folding-deficient proinsulin can physiologically interact with and be efficiently degraded by HRD1, the E3 ubiquitin ligase subunit of the ERAD complex. GSIS impairment in insulinoma cells was accompanied by a reduced intracellular Ca2+ ion level, overproduction of reactive oxygen species, and lowered mitochondrial membrane potential. Together, these findings suggest that ERAD plays a pivotal role in supporting pancreatic β-cell function by targeting wild-type and folding-deficient proinsulin for proteosomal degradation. ERAD deficiency may contribute to the development of diabetes by affecting proinsulin processing in the ER, intracellular Ca2+ concentration, and mitochondrial function. [ABSTRACT FROM AUTHOR]