R.W. Rosebrough

Dietary protein regulates in vitro lipogenesis and lipogenic gene expression in broilers.

The purpose of this experiment was to determine the possible relationship between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 21 or 30% protein ad libitum. In addition, another group of birds was fed on a regimen consisting of a daily change in the dietary protein level (12 or 30%). This latter group was further subdivided such that one-half of the birds received each level of protein on alternating days. Birds were sampled from 28 to 30 days of age. Measurements taken included in vitro lipogenesis, malic enzyme activity the expression of the genes for malic enzyme, fatty acid synthase and acetyl coenzyme carboxylase. In vitro lipogenesis and malic enzyme activity were inversely related to dietary protein levels (12-30%) and to acute changes from 12 to 30%. In contrast, expression of malic enzyme, fatty acid synthase and acetyl CoA carboxylase genes were constant over a dietary protein range of 12-21%, but decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out, however, that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.

Effect of pulsatile or continuous administration of pituitary-derived chicken growth hormone (p-cGH) on lipid metabolism in broiler pullets.

1. The effects of pulsatile and continuous intravenous administration of exogenous, pituitary-derived chicken growth hormone (p-cGH) on lipid metabolism and endocrine/metabolite levels of broiler-strain pullets were studied. 2. Eight-week-old pullets were administered p-cGH or vehicle over a 10 min period every 90 min for 7 days. 3. Pullets were also administered the same daily amount (123 micrograms/kg of body weight/day) continuously for 7 days. 4. Feed intake, body weight gain, in vitro lipogenesis and hepatic enzyme activities were determined with certain hormones identified with the control of growth. 5. Pulsatile p-cGH administration for 7 days lacked effect on weight gain, feed efficiency, muscle or bone development. 6. Abdominal fat pad size was decreased (P less than 0.05) by pulsatile but not continuous administration of p-cGH. Pulsatile p-cGH administration also decreased (P less than 0.05) in vitro lipogenesis. Liver malic enzyme and isocitrate dehydrogenase activities were increased (P less than 0.05) by pulsatile but not continuous administration of p-cGH. In contrast, glutamic oxaloacetic transaminase activity was increased by a continuous infusion of p-cGH. 7. Plasma concentrations of T4 corticosterone and triglycerides were decreased (P less than 0.05) by a pulsatile but not a constant infusion of p-cGH. 8. Plasma T3 and GH were increased (P less than 0.05) by pulsatile p-cGH compared to both a continuous infusion of p-cGH and the saline controls. 9. This study is the first to prove that in the broiler chicken, the pattern of exogenous p-cGH administration is a factor influencing in vitro responses to the hormone.

Characterization of the chicken muscle insulin receptor

Insulin receptors are present in chicken skeletal muscle. Crude membrane preparations demonstrated specific 125I-insulin binding. The nonspecific binding was high (36-55% of total binding) and slightly lower affinity receptors were found than are typically observed for crude membrane insulin binding in other chicken tissues. Affinity crosslinking of 125I-insulin to crude membranes revealed insulin receptor alpha-subunits of Mr 128K, intermediate between those of liver (134K) and brain (124K). When solubilized and partially purified on wheat germ agglutinin (WGA) affinity columns, chicken muscle insulin receptors exhibited typical high affinity binding, with approximately 10(-10) M unlabeled insulin producing 50% inhibition of the specific 125I-insulin binding. WGA purified chicken muscle insulin receptors also exhibited insulin-stimulated autophosphorylation of the beta-subunit, which appeared as phosphorylated bands of 92- and 81K. Both bands were immunoprecipitated by anti-receptor antiserum (B10). WGA purified membranes also demonstrated dose-dependent insulin-stimulated phosphorylation of the exogenous substrate poly(Glu,Tyr)4:1. However, unlike chicken liver, chicken muscle insulin receptor number and tyrosine kinase activity were unaltered by 48 hr of fasting or 48 hr of fasting and 24 hr of refeeding. Thus, despite the presence of insulin receptors in chicken muscle showing normal coupling to receptor tyrosine kinase activity, nutritional alterations modulate these parameters in a tissue-specific manner in chickens.

Dietary protein levels and the responses of broilers to single or repeated cycles of fasting and refeeding

Abstract The present study was designed to study short-term responses accompanying either chronic or acute fasting-refeeding cycles. Seven-day old Shaver broilers were fed diets containing either 120 or 300 g crude protein on either free choice basis or on 7 cycles consisting of 1 day of fasting followed by 2 days of feeding. In addition, birds fed free choice were subjected to the above regimen, but only for one cycle. Birds were bled and killed on day 1, 2 & 3 of the final cycle for each of these experiments. Measurements taken at these intervals included in vitro lipogenesis (IVL), growth and feed consumption, hepatic enzyme activities and plasma triiodothyronine (T 3 ), and thyroxine (T 4 ). Birds fed the lower level of crude protein free choice from 7 to 28 d ate less, were smaller and less efficient in growth. De novo lipogenesis and plasma T 3 were greater and T 4 was less in birds fed the lower protein diet. Birds subjected to repeated fasting-refeeding cycles exhibited striking changes on each day of the cycle. The lowest rate of IVL was noted following a 1 day fast and the greatest after 2 day of refeeding. This pattern was noted in birds fed diets containing either 120 or 300 g crude protein/kg although the responses were exaggerated in birds fed the lower level of protein. Chickens fed a low-protein diet in conjunction with a single fasting-refeeding cycle exhibited responses that were similar to chronic fasting-refeeding. The magnitudes of fasting-refeeding responses were magnified by repeated cycles of fasting-refeeding. Feeding a high level of protein modified some of the effects of a fasting-refeeding cycle.

Effects of early neonatal development and delayed feeding immediately post-hatch on the hepatic lipogenic program in broiler chicks

The embryo to neonate transition is a critical period of development that has significant impact on broiler production. During this time important genetic programs governing metabolism and growth are established. The goal of this work was to study the effects of early post-hatch (PH) development and the time of initiation of feeding on activation of the genetic program regulating hepatic lipogenesis. A comparison of liver total RNA samples at hatch and 7 days PH was performed using oligonucleotide-based (Affymetrix GeneChip®) chicken genome microarrays. During the first week PH there was significant up-regulation of key lipogenic genes including: ATP citrate lyase (ACL), malic enzyme (ME), fatty acid synthase (FAS), acetyl-CoA carboxylase alpha (ACCα), stearoyl-CoA desaturase-1 (SCD-1), sterol regulatory element binding protein-2 (SREBP-2) and thyroid hormone responsive spot 14α (Spot 14α) among others. These findings were confirmed using gene-specific RT-PCR assays. In a follow-up study, we investigated the effects of withholding feed for the first 48 h PH (delayed feeding, DF) on lipogenic gene expression through 8 days PH. Body weight gain was significantly depressed by DF. Plasma levels of the major metabolic hormones that regulate lipogenic gene expression (insulin, glucagon and T(3)) changed significantly during PH development, but were largely unaffected by DF. Plasma glucose was significantly lower in the DF group at 24h PH but recovered thereafter. In general, DF inhibited the up-regulation of lipogenic genes until feeding was initiated. Delayed up-regulation was also observed for the lipogenic transcription factor genes, SREBP-1, SREBP-2 and peroxisome proliferator-activated receptor gamma (PPARγ), but not for carbohydrate response element binding protein (ChREB) or liver X receptor (LXR). Our results offer additional insight into the transcriptional programming of hepatic lipogenesis in response to the transition from high fat (yolk) to high carbohydrate (feed) nutrition that occurs during early PH development.

Metabolic and hormonal effects of feeding chickens thyroxine and diets containing varied calorie to protein ratios

Abstract Indian River male broiler chickens, growing from 7 to 28 d of age, were fed diets containing energy to protein ratios from 43 to 106 MJ/kg of protein + 0 or 1 mg/kg thyroxine (T 4 ) to study both the energetic and metabolic costs of lipogenesis. Denovo lipid synthesis was determined in the presence and absence of ouabain (ATPase inhibitor) to estimate the energetic costs of ion pumping in explants synthesizing lipid. Growth and feed consumption increased (P 4 depressed plasma growth hormone and insulin-like growth factor-I. A large calorie-to-protein ratio increased (P + /K + transport. In addition, plasma thyroid hormone ratios were changed by both energy to protein ratios and T 4 .

A Diabetic-Like Condition of Turkey Embryos Maintained in Shell-Less Culture

Abstract Serum insulin concentration and pancreatic insulin content were determined for turkey embryos incubated in ovo and in long-term shell-less culture (ex ovo). Insulin was undetectable (< 10 pg) in serum from 87% of the ex ovo embryos compared with their in ovo counterparts. This was evident at all incubation ages, although insulin was detectable in more of the ex ovo embryos on Day 24. Insulin increased in the embryos incubated in ovo from 122 (Day 15) to levels exceeding 2000 pg/ml at hatching. Total pancreatic insulin content was greater in the cultured embryos on Days 15, 17, and 22 compared with their in ovo counterparts. Serum glucose was significantly greater (P < 0.05) in the ex ovo embryos at all ages. In response to an infusion of l-arginine, serum insulin increased from 566 to 1256 pg/ml in the in ovo embryos, whereas no change was evident in the ex ovo embryos (233 vs 257 pg/ml). When embryos incubated in ovo were injected with insulin, a significant (P < 0.05) reduction of serum glucose was observed at 60 min after injection. Serum glucose concentrations remained elevated in the embryos incubated ex ovo despite the insulin injection. Liver glucose 6-phosphatase activity, assessed on Days 15 and 22 of incubation, was found to be significantly (P < 0.05) lower in the ex ovo embryos. Turkey embryos incubated in shell-less culture exhibited chronic hyperglycemia in concert with extremely low circulating levels of insulin. The pancreatic beta cells of these embryos were not responsive to arginine or elevated glucose. Taken together these findings suggest the occurrence of a diabeticlike condition in the ex ovo embryos. This defect in insulin secretion may, in part, be responsible for some of the developmental abnormalities characteristic of the turkey embryo cultured ex ovo.

Further studies on short-term adaptations in the expression of lipogenic genes in broilers.

This experiment was conducted to determine possible relationships between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens (Gallus gallus) growing from 7 to 28days of age were fed diets containing 12 or 30% protein ad libitum. Both groups were then switched to the diets containing the opposite level of protein. Birds were sampled at 0, 6, 9, 12, 18 and 24h following the switch in protein levels. Measurements taken included in vitro lipogenesis (IVL), malic enzyme (ME), aspartate aminotransferase (AAT) and isocitrate dehydrogenase (NADP) (ICD) activities. In addition, ME, AAT, ICD, fatty acid synthase (FAS), and acetyl coenzyme carboxylase (ACC) gene expression rates were determined. IVL and ME activities were inversely related to dietary protein levels (12 to 30%) and to acute changes from 12 to 30%. In contrast, expression of ME, FAS and ACC genes was decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out; however, that metabolic regulation at the gene level only occurs when feeding very high or very low levels of dietary protein.

Methimzole and thyroid hormone replacement in broilers

Seven-day-old chickens were fed diets containing 18% crude protein + 0 or 1g methimazole/kg to produce either euthyroid or hypothyroid groups of birds at 28 days of age. These two groups were then offered diets containing either 0 or 1mg triiodothyronine (T(3))/kg diet. Birds were sampled at 0, 2, 5, and 8 days following the onset of the T(3) treatment. Measurements taken at these intervals included in vitro hepatic lipogenesis (IVL), growth and feed consumption, hepatic enzyme activities (malic enzyme, ME; isocitrate dehydrogenase, ICD; and aspartate amino transferase, AAT), plasma hormones (T(3); thyroxine, T(4); insulin like growth factors I, IGF-I; and insulin like growth factors II, IGF-II) and metabolites (glucose; fatty acids, NEFA; triglyerides; uric acid). Hypothyroidism decreased IVL and ME at 28 days of age; however, T(3) supplementation for 2 days restored both IVL and ME. Paradoxically, continuing T(3) replenishment for an additional 3-6 days decreased IVL without affecting ME activity. In contrast, supplemental T(3) decreased IVL in euthyroid birds, regardless of the dosing interval, but had no effect on ME activity. Methimazole decreased plasma T(3), T(4), uric acid, and IGF-I, but did not affect IGF-II at 28 days. Giving T(3) to birds previously on methimazole increased plasma IGF-I as did feeding a control diet. Supplemental T(3) increased NEFA in both euthyroid and hypothyroid birds, but only for a short period following the initiation of supplementation (2 days post-supplementation). These data may help to explain some of the apparent reported dichotomies in lipid metabolism elicited by changes in the thyroid state of animals. In addition, most metabolic changes in response to feeding T(3) occurred within 2-5 days, suggesting that changes in intermediary metabolism preceded morphological changes. In conclusion, the thyroid state of the animal will determine responses to exogenous T(3).

Supplemental triiodothyronine, feeding regimens, and metabolic responses by the broiler chicken.

There are conflicting results concerning the role of the thyroid hormones in lipid metabolism. The experiments in this report were designed to examine the role of T(3) in modifying responses obtained by shifting birds from moderate to low protein diets. Birds were grown from 7 to 28 d on a diet containing 18% protein. At this time, birds were switched to a diet containing 12% protein +/- T(3) The switch was accomplished either immediately or after a 24 hr fast. Measurements taken included in vitro lipogenesis (IVL), hepatic enzyme activities and plasma metabolites and thyroid hormones. Simply switching to birds to the low protein diet increased IVL, but rates were similar for three days following the switch. Feeding T(3) in this same regimen resulted in lower, but again, constant rates of IVL. In contrast, although switching protein levels after a 24 hr fast increased IVL, the rate after two days of refeeding was nearly double that following one day. This accentuated response was somewhat attenuated by including T(3) in the diet. Neither fasting nor refeeding altered plasma T(3) relative to ad libitum values. Supplemental dietary T(3) increased plasma T(3) and results were not affected by feeding regimens. Plasma T(4) was greatest in birds fasted for 24 hr and least in birds fed T(3) suggesting that feeding regimens may regulate the conversion of T(4) to T(3) It is suggested from this study that some of the effects of alterations in dietary feeding regimens can be modulated by T(3)