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.

ERAD deficiency promotes mitochondrial dysfunction and transcriptional rewiring in human hepatic cells.

Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.

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.

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.

Trans-10, cis-12 CLA dose-dependently inhibits milk fat synthesis without disruption of lactation in C57BL/6J mice.

BACKGROUND: Trans-10, cis-12 conjugated linoleic acid (10,12 CLA) is a potent inhibitor of milk fat synthesis in mammals. In the cow, 10 g/d of 10,12 CLA specifically and reversibly inhibits mammary lipogenesis, whereas substantially higher doses are not specific and cause a generalized inhibition of milk synthesis. OBJECTIVE: The objective of this study was to validate a lactating mouse model by establishing the dose response, specificity, and reversibility of the inhibition of milk fat synthesis by 10,12 CLA. METHODS: Lactating mice (C57BL/6J) received daily doses of 0 (control), 7, 20, or 60 mg of 10,12 CLA for 5 d during established lactation. A second group of lactating mice was treated with 20 mg/d of 10,12 CLA for 4 d and followed post-treatment to evaluate reversibility. RESULTS: CLA decreased pup growth with a 49% decrease occurring with 60 mg/d of CLA. Milk fat percentage was decreased 11% and 20% with the 7 and 20 mg/d dose, respectively, and all CLA treatments had a decreased concentration of de novo synthesized fatty acids (FAs) in milk fat. In agreement, 20 mg/d of 10,12 CLA decreased the lipogenic capacity of mammary tissue by 30% and mammary expression of FA synthase (Fasn), sterol response element binding protein 1 (Srebf1), and thyroid hormone responsive spot 14 (Thrsp) by 30-60%, whereas milk protein percentage and mammary expression of α-lactalbumin (Lalba) were unaltered. This dose of CLA reduced pup growth by nearly 20% and milk de novo synthesized FAs by >35%, and these effects were completely reversed 5 d after 10,12 CLA treatment was terminated. CONCLUSION: Inhibition of mammary lipogenesis by 10,12 CLA is dose-dependent in the mouse, with a specific and reversible reduction in milk fat synthesis at the 20 mg/d dose and additional nonspecific effects on milk synthesis at higher CLA doses.

Supplementation with fish oil as a source of n-3 fatty acids does not downregulate mammary lipogenesis in lactating mice.

The very long chain n-3 (ω-3) polyunsaturated fatty acids (VLCn3PUFAs) are potent regulators of hepatic lipid synthesis, but their effect on lipid synthesis in the lactating mammary gland is less well investigated. The objective of the present study was to examine effects of fish oil (FO) supplementation on mammary lipogenesis and the expression of lipogenic genes in mammary and hepatic tissues of lactating mice. Beginning on day 6 of lactation and continuing for 7 d, female C57BL/6J mice (n = 8/diet) were fed 1 of 3 dietary treatments: a 5%-fat diet containing mainly saturated fatty acids (FAs) (low-fat control) or 2 10%-fat diets, 1 enriched with FO as a source of VLCn3PUFAs and the other enriched with a safflower/palm oil mixture (high-fat control) as a source of oleic acid. Mammary lipogenic capacity, measured by (14)C-glucose incorporation into FAs by mammary explants, was similar among treatments, and there were no treatment effects on the proportion of de novo synthesized FAs in milk fat or on litter weight gain, a proxy for milk energy secretion. Also, there were no treatment effects on mammary mRNA abundance for key lipogenic enzymes and proteins involved in the regulation of milk lipid synthesis. In contrast, there was a treatment effect on hepatic lipogenesis, with FO resulting in a decrease of ~50% in hepatic lipid content and a similar downregulation of lipogenic gene expression compared with the 2 control diets. Overall, there were tissue-specific differences in dietary VLCn3PUFA effects on lipid synthesis with no observed effects for mammary lipogenic variables but marked reductions occurring in hepatic lipogenesis.

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.

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.

Dietary Fat Does Not Overcome trans-10, cis-12 Conjugated Linoleic Acid Inhibition of Milk Fat Synthesis in Lactating mice.

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.

Leptin concentrations and the immune-mediated reduction of feed intake in sheep infected with the nematode Trichostrongylus colubriformis.

The hypothesis that increases in the concentration of the anorectic peptide leptin may be responsible for the immune-mediated reduction in feed intake (FI) during gastrointestinal parasitism in sheep was investigated. In a 2 x 2 x 2 factorial design, the first factor was age at the start of infection (5 months old v. 17 months old). The second factor was parasite infection (no infection v. administration of eighty L3 infective Trichostrongylus colubriformis larvae/kg live weight (LW) per d three times per week for 77 d). The third factor was immunosuppressive therapy with a corticosteroid (no therapy or weekly intramuscular injection of 40 mg methylprednisolone acetate/30 kg LW). Relative to their uninfected counterparts, a 20 % reduction in FI per unit LW (FI/LW; g DM/kg LW) was observed in infected non-suppressed 5-month-old lambs from 21 to 63 d post-infection (P < 0.001) but not in comparable17-month-old ewes or in corticosteroid-treated lambs or ewes (P>0.05 for all), allowing the suggestion that the anorexia was a consequence of the developing immune response. The reduction in FI/LW in 5-month-old lambs was not associated with an increase in plasma leptin concentration. Furthermore, plasma leptin concentrations were greater in corticosteroid-treated animals (P < 0.001) and in 17-month-old animals (P < 0.001), none of which displayed an infection-induced reduction in FI/LW. Plasma leptin was positively correlated with carcass fat percentage in both 5-month-old (P = 0.016) and 17-month-old (P < 0.001) animals and did not appear to provide a direct feedback mechanism that restricted energy intake. The results do not support the hypothesis that an increase in circulating leptin is directly responsible for the immune-mediated anorexia in lambs during T. colubriformis infection.

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.

Endoplasmic Reticulum-Associated Degradation (ERAD) Has a Critical Role in Supporting Glucose-Stimulated Insulin Secretion in Pancreatic ß-Cells.

The molecular underpinnings of ß-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 ß-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 ß-cells impaired ERAD and led to blunted GSIS. Additionally, mice with SEL1L deletion in ß-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 ß-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 ß-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.

Increased degradation of insulin-like growth factor-I in serum from feed-deprived steers.

Severe feed restriction decreases serum insulin-like growth factor I (IGF-I) concentration in animals, and this decrease is thought to be due to reduced IGF-I production in the liver. The objective of this study was to determine whether feed deprivation also increases degradation of serum IGF-I and serum levels of IGF binding protein 3 (IGFBP-3) and acid-labile subunit (ALS), which inhibit IGF-I degradation and increase IGF-I retention in the blood by forming a ternary complex with IGF-I, in cattle. Five steers had free access to pasture, and another five were deprived of feed for 60 h. Serum concentration of IGF-I and liver abundance of IGF-I mRNA at the end of the 60-h period were 50% and 80% lower, respectively, in feed-deprived steers than in fed steers. Less (125)I-labeled IGF-I remained intact after a 45-h incubation in sera of feed-deprived steers than in sera of fed steers, suggesting that serum IGF-I is more quickly degraded in feed-deprived animals. Serum levels of IGFBP-3 and ALS were decreased by 40% and 30%, respectively, in feed-deprived steers compared with fed steers. These decreases were associated with more than 50% reductions in IGFBP-3 and ALS mRNA in the liver, the major source of serum IGFBP-3 and ALS. Taken together, these results suggest that feed deprivation reduces serum concentration of IGF-I in cattle not only by decreasing IGF-I gene expression in the liver, but also by increasing IGF-I degradation and reducing IGF-I retention in the blood through decreasing IGFBP-3 and ALS production in the liver.

Circulating levels of IGF-1 directly regulate bone growth and density.

IGF-1 is a growth-promoting polypeptide that is essential for normal growth and development. In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. Liver IGF-1-deficient (LID) mice and ALS knockout (ALSKO) mice exhibited relatively normal growth and development, despite having 75% and 65% reductions in serum IGF-1 levels, respectively. Double gene disrupted mice were generated by crossing LID+ALSKO mice. These mice exhibited further reductions in serum IGF-1 levels and a significant reduction in linear growth. The proximal growth plates of the tibiae of LID+ALSKO mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, the double gene disruption LID+ALSKO mouse model demonstrates that a threshold concentration of circulating IGF-1 is necessary for normal bone growth and suggests that IGF-1, IGFBP-3, and ALS play a prominent role in the pathophysiology of osteoporosis.

Protein tyrosine phosphatase 1B attenuates growth hormone-mediated JAK2-STAT signaling.

Protein tyrosine phosphatase-1B (PTP-1B) attenuates insulin, PDGF, EGF, and IGF-I signaling by dephosphorylating tyrosine residues located in the tyrosine kinase domain of the corresponding receptors. More recently, PTP-1B was shown to modulate the action of cytokine signaling via the nonreceptor tyrosine kinase JAK2. Transmission of the growth hormone (GH) signal also depends on JAK2, raising the possibility that PTP-1B modulates GH action. Consistent with this hypothesis, GH increased the abundance of tyrosine-phosphorylated JAK2 associated with a catalytically inactive mutant of PTP-1B. GH-induced JAK2 phosphorylation was greater in knockout (KO) than in wild-type (WT) PTP-1B embryonic fibroblasts and resulted in increased tyrosine phosphorylation of STAT3 and STAT5, while overexpression of PTP-1B reduced the GH-mediated activation of the acid-labile subunit gene. To evaluate the in vivo relevance of these observations, mice were injected with GH under fed and fasted conditions. As expected, tyrosine phosphorylation of JAK2 and STAT5 occurred readily in the livers of fed WT mice and was almost completely abolished during fasting. In contrast, resistance to the action of GH was severely impaired in the livers of fasted KO mice. These results indicate that PTP-1B regulates GH signaling by reducing the extent of JAK2 phosphorylation and suggest that PTP-1B is essential for limiting the action of GH during metabolic stress such as fasting.

Evaluation of a semi-automated computer algorithm for measuring total fat and visceral fat content in lambs undergoing in vivo whole body computed tomography.

Computed tomography (CT) is a suitable tool for measuring body fat, since it is non-destructive and can be used to differentiate metabolically active visceral fat from total body fat. Whole body analysis of body fat is likely to be more accurate than single CT slice estimates of body fat. The aim of this study was to assess the agreement between semi-automated computer analysis of whole body volumetric CT data and conventional proximate (chemical) analysis of body fat in lambs. Data were collected prospectively from 12 lambs that underwent duplicate whole body CT, followed by slaughter and carcass analysis by dissection and chemical analysis. Agreement between methods for quantification of total and visceral fat was assessed by Bland-Altman plot analysis. The repeatability of CT was assessed for these measures using the mean difference of duplicated measures. When compared to chemical analysis, CT systematically underestimated total and visceral fat contents by more than 10% of the mean fat weight. Therefore, carcass analysis and semi-automated CT computer measurements were not interchangeable for quantifying body fat content without the use of a correction factor. CT acquisition was repeatable, with a mean difference of repeated measures being close to zero. Therefore, uncorrected whole body CT might have an application for assessment of relative changes in fat content, especially in growing lambs.

Identification of estrogen-responsive genes in the parenchyma and fat pad of the bovine mammary gland by microarray analysis.

Identification of estrogen-responsive genes is an essential step toward understanding mechanisms of estrogen action during mammary gland development. To identify these genes, 16 prepubertal heifers were used in a 2 x 2 factorial experiment, with ovarian status (intact or ovariectomized) as the first factor and estrogen treatment as the second (control or estradiol). Heifers were ovariectomized at approximately 4.5 mo of age, and estrogen treatments were initiated 1 mo later. After 3 days of treatment, gene expression was analyzed in the parenchyma and fat pad of the bovine mammary gland using a high-density oligonucleotide microarray. Oligonucelotide probes represented 40,808 tentative consensus sequences from TIGR Bos taurus Gene Index and 4,575 singleton expressed sequence tags derived from libraries of pooled mammary gland and gut tissues. Microarray data were analyzed by use of the SAS mixed procedure, with an experiment-wide permutation-based significance level of P < 0.1. Considerable differences in basal gene expression were noted between mammary parenchyma and fat pad. A total of 124 estrogen-responsive genes were identified, with most responding only in the parenchyma or the fat pad. The majority of genes identified were not previously reported to be estrogen responsive. These undoubtedly include genes that are regulated indirectly but also include known estrogen-targeted genes and novel genes with potential estrogen-responsive elements in their promoter regions. The distinctive expression patterns regulated by estrogen in parenchyma and fat pad shed light on the need for both tissues to obtain normal mammary development.

Isolation of the cDNA encoding the acid labile subunit (ALS) of the 150 kDa IGF-binding protein complex in cattle and ALS regulation during the transition from pregnancy to lactation.

During the transition from pregnancy to lactation, dairy cows experience a 70% reduction in plasma IGF-I. This reduction has been attributed to decreased hepatic IGF-I production. IGF-I circulates predominantly in multi-protein complexes consisting of one molecule each of IGF-I, IGF binding protein-3 and the acid labile subunit (ALS). Recent studies in the mouse have shown that absence of ALS results in accelerated turnover and severely depressed concentration of plasma IGF-I. These observations suggest that reduced plasma ALS could be a second factor contributing to the fall of plasma IGF-I in peri-parturient cows. This possibility has not been studied due to the lack of bovine ALS reagents. To address this, we isolated the bovine ALS cDNA and used its sequence to develop a ribonuclease protection assay (RPA) and a bovine ALS antiserum. Using the RPA, ALS mRNA abundance was approximately fivefold higher in liver than in lung, small intestine, adipose tissue, kidney and heart, but was absent in muscle and brain. The antiserum detected the highest ALS levels in plasma followed by ovarian follicular fluid, lymph and colostrum. A portion of colostrum and follicular fluid ALS appears to be synthesized locally as ALS mRNA was found in mammary epithelial cells and ovarian follicular cells. Finally, we measured plasma ALS in dairy cows during the peri-parturient period (days -35 and +56 relative to parturition on day 0). Plasma ALS dropped by 50% between late pregnancy and the first day of lactation and returned to prepartum levels by day +56. To determine whether this reflected a change in hepatic expression, ALS mRNA was measured in liver biopsies collected on days -35, +3 and +56. ALS mRNA expression was significantly lower on day +3 than on day -35, but recovered completely by day +56. Finally, we examined the ability of GH to increase plasma ALS abundance at selected times before and after parturition (weeks -5, -2, +1 and +5). GH increased plasma ALS at weeks -5, -2 and +5, but not at week +1. Identical effects of GH were seen when the response considered was plasma IGF-I. We conclude that the decline in plasma ALS after parturition is a consequence of hepatic GH resistance and contributes to the associated reduction of plasma IGF-I.