Avian Liver Explants Research Articles

Insulin, glucagon and catecholamine interactions in avian liver explants

1. 1. A mechanical tissue chopper was used to obtain liver explants (35–75 mg) from 2- to 3-week-old chickens to determine both tissue sensitivity and metabolic effects of isoproterenol, avian insulin and glucagon. 2. 2. Avian insulin had no effect on lipogenesis; however, lipogenesis was decreased by dibutyryl cyclic AMP. Insulin did not overcome a decrease in lipogenesis caused by catecholamines. Therefore, this control mechanisms is not modulated by insulin. 3. 3. Preincubation in the presence of glucagon decreased in vitro lipogenesis. Preincubation in the presence of a 19–29 amino acid construct that approximated the radioimmune site for glucagon did not result in a similar effect. Therefore, this site does not relate to the biopotency of the hormone. 4. 4. A previously noted catecholamine induced decrease in in vitro lipogenesis was verified, showing that points of in vitro regulation are under phosphorylation-dephosphorylation control. 5. 5. Preincubation of slices (1 hr) with propranolol blocked the inhibition of lipogenesis caused by α and β adrenergic agonists (arterenol or isoproterenol) during a subsequent 2-hr incubation. 6. 6. Preincubation of slices with either of these agonists decreased lipogenesis even following an extensive washout. 7. 7. Inhibition could be overcome with propranolol, a β adrenergic antagonist.

Glucose production and glycogen cycle enzyme activities in avian liver explants: Procedural optimization

1. 1. There are few studies which both describe the in vitro regulation of glycogen metabolism in nonmammalian animals and list methods to optimize assay conditions for these studies. 2. 2. A mechanical tissue chopper was used to obtain 25 65 mg liver explants from 14- to 28-day old domestic turkeys to determine assay conditions (substrates, buffers, time), regulators (metals, salts and hormones) and points of endogenous regulation of glycogenolysis (protein phosphorylation and enzyme activity). 3. 3. High- and low-bicarbonate-based buffers (Earle's balanced salts, EBSS and Hanks' balanced salts, HBSS, respectively) were used to determine buffer effects. 4. 4. Glucose release into the incubation environment was greater in HBSS than EBSS. Adding HEPES to further buffer conditions did not change release rates. Adding bicarbonate to HBSS resulted in release rates similar to EBSS. 5. 5. Calcium increased glycogenolysis in the presence or absence of equimolar concentrations of EGTA; potassium had no effect. 6. 6. Porcine insulin (100 ng/ml) did not inhibit glycogenolysis; however, glucose release was increased by dibutyryl cyclic AMP. A noted catecholamine-induced increase in in vitro glycogenolysis and phosphorylase activity indicates that points of regulation are under phosphorylation-dephosphorylation regulation.

Measurement of glucose and lipid metabolism in avian liver explants

1. 1. A mechanical tissue chopper was used to obtain 35–75 mg explants from 21 - to 28-day-old chick liver to determine assay conditions (substrates, buffers, time), regulators (metals and hormones) and points of endogenous regulation of de novo lipogenesis (ATPase, reductive potential and protein phosphorylation). High- and low-bicarbonate-based buffers (Earl's balance salts, EBSS and Hanks' balanced salts, HBSS; respectively) were used in conjunction with sources and types of bovine serum albumin (BSA), divalent cations (Mg 2+ or Ca 2+), substrate (glucose or acetate) and hormones (insulin and catecholamines). 2. 2. Neither EBSS nor HBSS changed in vitro lipogenesis, CO 2 or glucose production when 20 mM HEPES was added to these salts. 3. 3. Neither the presence nor the source of BSA (Sigma or Armour) affected metabolism. In contrast, reducing the vessel reaction surface area (5.1 vs 10.5 cm 2) decreased metabolic rates. 4. 4. Acetate was more readily utilized than glucose as an in vitro fatty acid precursor. Use of glucose was complicated by production of glucose from endogenous precursors and by label recycling. Divalent cations (Mg 2+ or Ca 2+) had little affect upon lipogenesis. 5. 5. Chicken insulin (50 ng/ml) did not affect lipogenesis; however, incorporation of acetate into fatty acids was decreased by dibutyryl cyclic AMP. A catecholamine-induced decrease in vitro lipogenesis indicates that major points of regulation are under control of phosphorylation-dephosphorylation steps.

Colchicine inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase in cultured avian liver explants

Chicken embryo liver expiants cultured in chemically defined medium in the absence of serum provide a unique system to probe into the mechanism of insulin induction of lipogenic enzymes. Colchicine at concentrations of 0.2 and 1 μ m in the culture medium caused inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase by 50 and 90%, respectively. As measured by immunochemical techniques, the inhibition of the induction of these two enzyme systems resulted from the decreased content of the Δ-terminal desaturase component of the stearoyl-CoA desaturase and the fatty acid synthetase. Colchicine, however, had no effect on the general protein synthesis, nor did it affect the malic enzyme, which is induced by triiodothyronine but not by insulin. Also, colchicine had no influence on the binding of 125I-insulin to isolated plasma membrane. Pretreatment of liver explants with insulin for 0.5–1 h and subsequent incubation in insulin-free media for 48 h resulted in induction of the desaturase and fatty acid synthetase. However, inclusion of colchicine in the media for 3 h subsequent to the treatment with insulin completely abolished the inductive effect of insulin, suggesting that colchicine affects events occurring subsequent to insulin binding to the cell surface membranes. Since lumicolchicine, an inactive isomer of colchicine, had no effect on insulin action, it is suggested that the inhibition of insulin induction of the desaturase and synthetase is related to the depolymerizing action of colchicine. Therefore, in eliciting long-term responses to insulin, microtubular integrity of the cell may be required for the transfer of a putative signal from cell surface insulin receptor to intracellular sites.

An analysis of rates of polypeptide chain elongation in avian liver explants following in vivo estrogen treatment. II. Determination of the specific rates of elongation of serum albumin and vitellogenin nascent chains.

An analysis of rates of polypeptide chain elongation in avian liver explants following in vivo estrogen treatment. II. Determination of the specific rates of elongation of serum albumin and vitellogenin nascent chains.

An analysis of rates of polypeptide chain elongation in avian liver explants following in vivo estrogen treatment. I. Determination of average rates of polypeptide chain elongation.

Average rates of polypeptide chain elongation have been determined in cockerel liver explants on 4 successive days following an in vivo injection of 17 beta-estradiol. Incorporation of [3H]leucine by the explants is linear for at least 24 h, and the rate of protein synthesis increases significantly after estrogen injection. The explants synthesize and secrete serum albumin. B-apolipoprotein, and the phosphoprotein vitellogenin at relative rates which are similar to those reported for liver in vivo. Using this system, changes in the average rates of polypeptide chain elongation have been analyzed as a temporal sequence following a single injection of 17 beta-estradiol into cockerels. For this, average ribosome half-transit times were determined by measuring the kinetics of transfer of labeled polypeptides from polysomal-bound to released polypeptides. The data revealed a dramatic effect of estradiol on the average ribosome half-transit time with a maximum increase of 4.6-fold; however, the average size of polypeptides synthesized by explants at the peak of induction increased only 15% when compared to uninduced liver explants. These findings indicate that injection of estradiol results in large changes in the actual rates at which amino acids are added to the growing nascent polypeptide chain; that is, rates of polypeptide chain elongation. Therefore, translation-level regulation of protein synthesis in cockerel livers plays a significant part in determining the magnitude of the response to hormone stimulation.

62] Cultured avian liver explants for studies of lipogenic enzymes

Publisher Summary This chapter describes the cultured avian liver explants for the studies of lipogenic enzymes. Most primary liver cell culture systems for the study of lipid metabolism involve disassociated hepatocytes and utilize serum that contains many hormones, unknown growth factors, and lipids, and thus makes it difficult to evaluate the role of hormones and lipids in the regulation of lipogenic enzymes. The characterization of an embryonic avian liver explant culture system maintained in a chemically defined culture medium, not containing serum, has made it possible to probe into the mechanism of hormonal induction of lipogenic enzymes, such as fatty acid synthase, stearoyl-CoA desaturase, and malic enzyme. The explants cultured in control medium lacking hormones maintained their initial specific activities of [U- 14 C]glucose oxidation to CO 2 , [ 3 H]uridine incorporation into RNA, and [ 3 H]leucine incorporation into cellular proteins. These results indicate that the functional viability of the liver can be maintained in explant culture for at least 120 h. The liver explant culture system is well suited for the investigation of mechanism by which insulin induces lipogenic enzymes.

Control of hepatic glycogenolysis.

Control of hepatic glycogenolysis.

Hormonal regulation of fatty acid synthetase in cultured avian liver explants. Role of insulin.

Hormonal regulation of fatty acid synthetase in cultured avian liver explants. Role of insulin.

Hormonal regulation of the terminal enzyme of microsomal stearoyl coenzyme A desaturase in cultured avian liver explants. Role of insulin.

Hormonal regulation of the terminal enzyme of microsomal stearoyl coenzyme A desaturase in cultured avian liver explants. Role of insulin.