Effects of wheat quality on digestion differ between the D+ and D- chicken lines selected for divergent digestion capacity.

The aim of the experiment was to study the effects of 2 wheat cultivars (Baltimor and Scipion) with different hardness values (75 and 5, respectively) on 2 divergent lines (D+ and D-) of broiler chickens selected on the basis of their digestion ability assessed by AME(n). Wheat was incorporated at 54.6% in diets. The other main ingredients were soybean meal (35.3%) and rapeseed oil (5.5%). Diets were given as pellets from 7 to 26 d. The experimental design was a 2 x 2 factorial design testing 2 wheat cultivars (soft or hard) on 2 selected lines of broiler chickens (high AME(n) or low AME(n)). From 7 to 16 d, D+ line showed lower (P < 0.0001) feed intake and feed:gain ratio than the D- line. At 3 wk of age, the D+ chickens resulted in increased digestibility values (P < 0.01) and 9% increased AME(n) value (P < 0.0001) compared with D-. Wheat cultivar effects on feed efficiency and AME(n) differed between lines. In the D+ line, their values were about 6% higher (P < 0.05) with soft than with hard wheat, whereas they did not differ in the D- line. However, wheat cultivar effect on starch digestibility did not differ between lines; soft instead of hard wheat resulted in about 6% improvement (P < 0.0001) in both lines. In the D- line, soft instead of hard wheat tended to reduce lipid and protein digestibilities, which explained why the starch digestibility improvement due to soft wheat was not converted into a significant AME(n) improvement in D birds. Study of digestive organ size revealed that increased proventriculus and gizzard weight (P < 0.05) could be one of the causes for the better digestion capacity of the D+ line. The pancreas was bigger (P < 0.01) in D- than in D+ birds, which probably came from an adaptation to a digestive disorder in D- birds.

Regulation of neutrophil apoptosis by Mcl-1.

Neutrophils rapidly undergo spontaneous apoptosis, but this process can be considerably delayed by exposure to a variety of agents such as pro-inflammatory cytokines. The anti-apoptotic protein of the Bcl-2 family, Mcl-1, plays a key role in the regulation of neutrophil apoptosis. The protein has some unusual properties compared with other family members, including an extremely high turnover rate. Many factors, such as cytokines and local oxygen concentrations, can regulate cellular levels of Mcl-1 via transcription and post-transcriptional modification, control the survival time of neutrophils within tissues and thereby influence the inflammatory response.

Peripheral leptin effect on food intake in young chickens is influenced by age and strain.

The acute effect of leptin on the regulation of food intake was investigated in layer and broiler chickens. In an initial study, we observed that a single intraperitoneal injection of recombinant chicken leptin (1 mg/kg BW) dramatically reduced (38%) food intake in 56-day-old layer chickens, more moderately reduced (15%) food intake in 9-day-old layer chicks, and had no significant effect in 9-day-old broiler chicks. In a subsequent study, body weight and plasma concentrations of leptin were measured weekly in layer and broiler chicks from day 1 to 35 of age and brain leptin receptor and neuropeptide Y (NPY) mRNA expression were analyzed at 1, 9, and 35 days of age. At day 1 of age, peripheral concentrations of leptin were significantly greater in layer than broiler chicks. Subsequently, despite increases in body weight and differences in growth rates between layer and broiler chicks from day 8 to day 35 of age, peripheral concentrations of leptin were constant and similar in both genotypes. Leptin receptor and NPY mRNA were expressed in brain from day 1 in chicks of both genotypes and increased significantly to day 35 of age. These observations provide evidence that the inhibitory effect of leptin on the regulation of food intake in growing chicks is an age dependent process. Furthermore, acquisition of the anorectic effect of leptin is likely to be associated with greater expression of the leptin receptor and NPY mRNAs than to changes in blood levels of leptin. Finally, this study provides evidence that chickens selected for high growth rates may be less sensitive or responsive to peripheral concentrations of leptin than chickens with low growth rates (layers), suggesting that the faster growth of broiler chicks may be related to a lessened responsiveness to anorexigenic factors.

Leptin and insulin downregulate leptin receptor gene expression in chicken-derived leghorn male hepatoma cells.

In chickens, leptin is expressed mainly in the liver, where its receptor gene expression has also been reported, and in adipose tissue. In view of the key role played by the liver in lipogenesis in avian species, the hepatic expression of leptin may have physiological significance. In this study, we showed that leptin is constitutively expressed and secreted in a chicken-derived hepatoma cell line (LMH). Although insulin regulates leptin expression in vivo, incubation of LMH cells in the presence of 100 nM insulin for 24 or 48 h had no effect on leptin expression or its secretion in the culture medium. In addition, we developed a specific chicken leptin receptor real-time reverse transcription (RT)-PCR, and downregulation of leptin receptor gene expression by homologous and heterologous signals was demonstrated, as relative leptin receptor mRNA levels were significantly decreased after exposure of LMH cells to recombinant chicken leptin or porcine insulin. In conclusion, our results indicate that leptin is probably able to desensitize its own response in the chicken liver. Finally, the ability of insulin and leptin to regulate chicken leptin receptor gene expression suggests a direct role of leptin in the control of hepatic metabolism.

Leptin fully suppresses acetylcholine-induced insulin secretion and is reversed by tolbutamide in isolated perfused chicken pancreas.

So far, there has been no evidence for any direct pancreatic effect of leptin in the chicken. The present study was aimed at detecting chicken leptin receptor (cOb-R) expression in isolated chicken islets of Langerhans and to examine the direct effect of leptin on insulin secretion after stimulation by acetylcholine (1 micro M) + glucose (14 mM) from isolated perfused chicken pancreas. We will show that i) full length cOb-R mRNA was expressed in isolated pancreatic islets of chickens, ii) recombinant chicken leptin (10 nM) or diazoxide (100 micro M) rapidly (within 2 min) and significantly suppressed insulin secretion induced by acetylcholine stimulation without any change in volume outflow rate, iii) tolbutamide (100 micro M) introduced 10 min after leptin and perfused for 10 min fully reversed the suppressive effect of leptin on pre-established acetylcholine-induced insulin release. In conclusion, we found that leptin has a profound inhibitory influence upon insulin secretion in perfused chicken pancreas. The results suggest that leptin inhibits insulin secretion by acting before or at the level of K ATP channels in chicken pancreatic beta-cells. Further studies are warranted to clarify the specific inhibitory mechanism.

BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis.

The human neutrophil spontaneously undergoes apoptosis, but this type of cell death can be delayed or accelerated by a wide variety of agents. There are wide discrepancies in the literature regarding the expression of the Bcl-2 family of proteins in human neutrophils. Here, we show that A1, Mcl-1, Bcl-X(L), and Bad are major transcripts in human neutrophils and that levels of these transcripts are cytokine regulated. However, no Bcl-X(L) protein was detected in Western blots. Protein levels for the proapoptotic proteins Bad, Bax, Bak, and Bik remained constant during culture, despite changes in the levels of mRNA for these gene products. These proapoptotic proteins were extremely stable, having very long half-lives. In contrast, A1 and Mcl-1 transcripts were extremely unstable (with approximately 3-h half-lives), and Mcl-1 protein was also subject to rapid turnover. These results indicate that neutrophil survival is regulated by the inducible expression of the short-lived Mcl-1 and possibly the A1 gene products. In the absence of their continued expression, these prosurvival gene products are rapidly turned over, and then the activity of the stable death proteins predominates and promotes apoptosis.

Plasma glucose-insulin relationship in chicken lines selected for high or low fasting glycaemia.

1. Selected Fat or Lean chickens differ in their plasma glucose insulin relationship: in the fed or fasted state, Fat chickens have a lower glycaemia associated with normal or higher insulinaemia, depending upon the difference in glycaemia. 2. Conversely, chickens selected for low fasting glycaemia (LG) are fattier than their counterparts selected for high fasting glycaemia (HG), although the divergence in fat content is lower than in the Fat-Lean model. 3. The plasma glucose insulin relationship has been investigated in males of the HG and LG lines in the F4 and F5 generations. 4. A difference in glycaemia is suggested during embryonic development and was present at hatching and later on in the fasted or the fed state; insulinaemia did not differ. 5. During refeeding after an overnight fast, glycaemia differed between lines (except at intermediate times); cumulative food intake and insulinaemia were similar. 6. During a glucose tolerance test, glucose disposal rate and insulinaemia were rather similar. 7. Exogenous insulin exerted a very similar hypoglycaemic effect in both lines. 8. Other variables (body temperature, plasma concentrations of potassium and alpha NH2-non protein nitrogen) did not differ between HG and LG chickens. 9. In conclusion, HG and LG chickens do not exhibit any differences in glucose disposal rate, insulinaemia (in various nutritional conditions) or sensitivity to exogenous insulin, which contrasts with Fat or Lean chickens and may explain why HG and LG chickens have diverged to a lesser extent in fat content.

Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins.

The chicken leptin sequence, in contrast to mammalian leptins, contains an unpaired Cys at position 3 of the original cDNA (AF012727). The presence of an extra Cys may confer a different structure and affect the leptin's biological activity. To address this, we studied the effects of wild-type and mutated (C4S) chicken leptins in vitro and in vivo and compared them with mammalian leptin prepared from ovine leptin cDNA. The prokaryotic expression vector pMON, encoding full-size A(-1) chicken leptin (AF012727), was mutated using a mutagenesis kit, yielding the C4S analog. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin C4S upon induction with nalidixic acid. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding three electrophoretically pure fractions, eluted from the column by 100, 125, and 150 mM NaCl, respectively. All three fractions showed a single band of the expected molecular mass (16 kDa) and were composed of >95% monomeric protein. Proper refolding was evidenced by comparing the circular dichroism spectrum of the analog with spectra of nonmutated chicken and ovine leptins. The biological activity of the C4S analog was evidenced by its ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct similar to its nonmutated counterpart, indicating that Cys4 plays no role in leptin activity. The in vitro activity of both wild-type and mutated chicken leptins was approximately 10-fold lower than that of ovine leptin. After intravenous or intraperitoneal injections, C4S analog and the nonmutated chicken and ovine leptins all lowered the food intake of starved 9-day-old broiler or 5-wk-old layer male chickens by 11-34%. Monitoring food behavior revealed that the attenuated food intake resulted not from a decreased number of approaches to the feeders but from a decrease in the average time spent eating during each approach.

An anti-insulin serum, but not a glucagon antagonist, alters glycemia in fed chickens.

Attempts at altering plasma glucose and, as a consequence, food intake were performed in fed broiler chickens by single i.v. injection of des-His1(Glu9) glucagon amide (a glucagon antagonist) or a non-stimulating anti-insulin serum. Plasma glucose level was not altered by des-His1(Glu9) glucagon amide but was rapidly and largely increased (for at least 2 h) by the injection of the insulin-immune serum. Hour and cumulative food intake were unaltered up to 10 h post injection. These results strongly suggest that in fed chickens, plasma glucose is mainly, if not exclusively, controlled by plasma insulin, and that the transient and heavy hyperglycemia evoked by inhibiting insulin action does not alter food intake.

Metabolic differences between genetically lean and fat chickens are partly attributed to the alteration of insulin signaling in liver.

Insulin signaling [tyrosine phosphorylation of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Src homology and collagen protein (Shc) and phosphatidyl inositol 3'-kinase activity (PI 3'-kinase)] was studied in the liver and thigh muscles of fat (FL) and lean (LL) chickens. These lines result from a divergent selection on abdominal fat pad size. The divergence is of metabolic origin. Extreme nutritional states were studied (fed, 48-h starved and 30-min refed). Such conditions significantly altered insulin signaling in chicken liver, but surprisingly not in the muscle (except the phosphorylation of Shc in the refed state). No major differences that could account for this divergence were found in muscle. Liver IR number and Shc protein did not differ between genotypes. Liver IRS-1 (protein and messenger) was lower in the fed state and higher in the starved state in FL compared to that in LL chickens. In the fed state, tyrosine phosphorylation of liver IR, IRS-1 and Shc action was higher in FL than in LL chickens that in the absence of insulin resistance rely on higher plasma insulin levels. In the starved state, phosphorylation of liver IR was lower, but the phosphorylation of IR and IRS-1 were higher in LL than in FL chickens, most likely in response to higher plasma glucose and insulin in the lean genotype. In the refed state, the phosphorylation of liver IR and IRS-1 did not differ between genotypes despite significantly lower plasma insulin in FL chickens. Finally, PI 3'-kinase was not affected by the genotype. A significant activation of early steps of insulin signaling in liver of fed FL chickens may at least partly account for their increased liver lipogenesis and ultimately their fattening.

Corticosterone alters insulin signaling in chicken muscle and liver at different steps.

Chronic treatment with corticosterone evokes insulin resistance in chickens, a species which is already resistant to insulin compared with mammals. The in vivo effects of corticosterone on insulin signaling were investigated in chicken liver and thigh muscle in two nutritional states: basal (overnight fasted) and stimulated (30 min refeeding). Corticosterone significantly decreased specific insulin binding in liver and the amount of insulin receptor substrate-1 (IRS-1) and p85 (regulatory subunit of phosphatidylinositol (PI) 3'-kinase) in both tissues. Insulin receptor (IR) and IRS-1 mRNAs generally varied accordingly. Src homology and collagen protein (Shc) and messenger were not altered. In liver, in the basal state, the tyrosine phosphorylation of IR, IRS-1 and Shc, and the IR-associated PI 3'-kinase activity were largely decreased by corticosterone. Following refeeding the cascade was activated in control but totally inhibited in treated chickens. In muscle, as previously observed, IR and IRS-1 phosphorylation and PI 3'-kinase were not stimulated by refeeding in controls. Only the phosphorylation of Shc was increased. On this background, corticosterone decreased the basal PI 3'-kinase activity and prevented the phosphorylation of Shc in response to refeeding. In conclusion, corticosterone largely impaired insulin signaling in liver and to some extent in muscle. This should contribute to the large impairment of growth. In addition, the present studies further emphasize the peculiarities of insulin signaling in chicken muscle, which needs further investigation.

Effect of nutritional state on the formation of a complex involving insulin receptor IRS-1, the 52 kDa Src homology/collagen protein (Shc) isoform and phosphatidylinositol 3'-kinase activity.

The Src homology and collagen protein (Shc) is tyrosine phosphorylated in response to insulin; however, evidence for its interaction with insulin receptor (IR) in normal tissues is missing. Interactions between IR, Shc and regulatory subunits of the phosphatidylinositol 3'-kinase (PI 3'-kinase) were characterized in the present study in liver and muscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, including the phosphotyrosine binding domain was sequenced. It shows 89% homology with the corresponding human homologue. The amounts of the three Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased by fasting in both liver and muscle. Importantly, Shc was immunoprecipitated by IR antibody (mostly the 52 kDa isoform) or by alphaIRS-1(mostly the 46 kDa isoform). IR-Shc association was decreased by fasting and restored by refeeding. In liver, alphaShc immunoprecipitated the three forms of regulatory subunits of PI 3'-kinase and a PI 3'-kinase activity which was decreased by fasting. In muscle, alphaShc immunoprecipitated only the p85 isoform; the associated PI 3'-kinase activity was not altered by the nutritional state. Conversely, in both tissues anti-p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antibodies to insulin receptor substrate-1 (alphaIRS-1), Shc or IR immunoprecipitated the three regulatory subunits of PI 3'-kinase and an equal PI 3'-kinase activity, without any residual activity left in the supernatants, suggesting the presence of a large complex involving IR, IRS-1, Shc (mainly the 52 kDa isoform) and PI 3'-kinase activity. The presence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3'-kinase activity, is suggested.

Insulin receptor substrate 1 antisense expression in an hepatoma cell line reduces cell proliferation and induces overexpression of the Src homology 2 domain and collagen protein (SHC).

In mammalian cells, the insulin receptor substrate 1 protein (IRS-1) is a specific substrate for insulin and IGF-1 receptor tyrosine kinases which is involved in mediating metabolic and mitogenic actions of insulin and IGFs. In order to determine if IRS-1 is also essential in a chicken derived hepatoma cell line (LMH cells), IRS-1 gene has been invalidated in these cells. For this, we subcloned chicken IRS-1 gene in an antisense orientation into a mammalian expression vector driven by the cytomegalovirus early promoter. LMH cells were stably transfected with this construct or with the empty vector carrying only the neomycin resistance gene and selected for cIRS-1 expression. One subclone, C2, showed a complete repression of cIRS-1 expression at both protein and mRNA levels. Proliferation of C2 cells was dramatically reduced (54%) compared with Neo(r) cells. Furthermore this reduction was accompanied by a decrease in insulin-dependent [3H]thymidine incorporation, indicating a reduction in DNA synthesis. Insulin-dependent [U-14C]glucose incorporation into cellular lipids was also significantly reduced in C2 cell line suggesting an alteration in lipogenesis. In wild type LMH cells, SHC which is involved in Ras pathway, also served as a substrate for insulin receptor tyrosine kinase. In C2 cells, SHC expression, its association with the insulin receptor and its tyrosine phosphorylation were largely increased. Two forms of the regulatory subunit of PI 3-kinase were present: p85 and p55 forms. Furthermore, C2 cells displayed increased basal phosphatidylinositol (PI) 3'-kinase activity. This report demonstrates a role for cIRS-1 in the metabolic and mitogenic actions of insulin in LMH cells. However, the overexpression of cIRS-1 antisense did not completely abolish cell proliferation. This may be explained by the exacerbation of an alternative pathway that only partly compensate for the knocking out of cIRS-1 gene: the overexpression of SHC.

Cloning the chicken leptin gene.

Chicken is characterized by a relative insulin resistance and a physiological hyperglycemia (2g/L) and is also subjected to fattening. Fat deposits in chicken, as in mammals, are regulated by environmental and genetic factors. In mammals, leptin, an adipose cell-specific secreted protein has been characterized that is encoded by ob gene. Leptin regulates satiety through hypothalamic specific receptors, energy balance, energy efficiency and contributes to adaptation to starvation. The leptin gene has been characterized in various mammalian species, and the cloning and sequencing of the chicken leptin gene (ob gene) are reported. Using RT-PCR and primers flanking the coding region of the leptin gene selected from known mammalian sequences, we have successfully amplified a 600-bp fragment from chicken liver and adipose tissue total ARNs. The amplified fragment exhibits a similar size to that of the coding region of the mammalian leptin gene. The sequences of the coding region of chicken liver and adipose tissue are identical and presented 97%, 96% and 83% similarity to the mouse, rat and human sequences, respectively. Finally, this is the first report showing that leptin gene expression in chicken is not exclusively localized in adipose tissue but is also expressed in liver. The expression of leptin in liver may be associated with a key role of this organ in avian species in controlling lipogenesis.

Nutritional state regulates insulin receptor and IRS-1 phosphorylation and expression in chicken.

After insulin binding, insulin receptors (IR) phosphorylate the insulin receptor substrate 1 (IRS-1) on specific motifs and thereby initiate insulin action. The interaction between IR and IRS-1 and their expression were studied in vivo in two target tissues (muscle and liver) in chickens, a species that is insulin resistant. To induce extreme changes in plasma insulin levels, chickens were subjected to three different nutritional states (ad libitum fed, fasted for 48 h, and refed for 30 min after 48-h fast). Liver membrane IR number was significantly increased in fasted compared with fed chickens. This upregulation of IR number was concomitant with the an enhanced expression of IR mRNA as determined by reverse transcription-polymerase chain reaction. In leg muscle, IR mRNA was not altered by the nutritional state. Using specific antibodies directed toward human IR, anti-phosphotyrosines, or mouse IRS-1, we demonstrated that IR and IRS-1 are associated in vivo in liver and muscles. Tyrosine phosphorylation of liver IR and IRS-1 were significantly decreased by prolonged fasting and restored by 30-min refeeding. These alterations were not observed in muscle. Fasting increased IRS-1 mRNA expression in liver but not in muscle. These results are the first evidence showing that chicken liver and muscle express IRS-1. Therefore, the chicken insulin resistance is not accounted for by the lack of IRS-1. The differences observed for the regulation of IR and IRS-1 messengers and phosphorylation between liver and muscle in response to alterations of the nutritional state remain to be explained.

Large-scale preparation of biologically active recombinant chicken obese protein (leptin).

Prokaryotic expression vector pMON3401 encoding full size A(-1) chicken leptin (AF012727) was prepared by PCR of previously described cDNA. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin upon induction with nalidixic acid. The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on a Q-Sepharose column, yielding two electrophoretically pure fractions (leptin-1 and leptin-2), eluted from the column by 100 and 125 mM NaCl. Both fractions showed a single band of the expected molecular mass of 16 kDa and were composed of over 95% of monomeric protein. The biological activity of both fractions, resulting from proper renaturation, was further evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin-receptor construct and by lowering the food intake of starved chicken following intravenous or intraperitoneal injections.

Isolation and characterization of Muscovy (Cairna moschata) duck insulin.

Ducks (Anatidae Family, Anseriform order) are divided in two genera: Pekin duck (Anasplatyrhynchos genus) and Muscovy duck (Cairina moschata genus) and differ for their number of liver insulin receptors (despite rather similar plasma insulin levels). The possibility that the presence of different endogenous insulins account for the difference in insulin receptor number between the two duck species led us to purify, sequence and characterize the binding properties of Muscovy duck insulin. The sequence of Muscovy duck insulin (measured mass: 5729.11) was identical to that described in two other species from the Anseriforme order: Pekin duck or goose. The binding affinity of Muscovy duck insulin for rat liver insulin receptors (either membrane bound or solubilized receptors) was lower than that of porcine insulin (0.3), which most likely accounts for the low biological potency of Pekin duck insulin previously described. In contrast, liver receptors from chicken and both duck species exhibited the same affinity for duck and porcine insulin suggesting the presence of specific changes in the structure of binding sites of bird liver insulin receptors. The decrease in the number of insulin receptors in Muscovy duck liver is not therefore the consequence of a change at the level of the insulin molecule itself. As discussed, among bird insulins, the hypoactive "duck type" insulin would have appeared after the hyperactive "chicken type" insulin during the evolution of Aves.

Biological activity of immunoreactive insulin-like activity extracted from rat submandibular gland.

Earlier studies indicate the presence of an insulin-like immunoreactivity (ILI) in rat submandibular salivary glands (SSG). Previous observations also showed that streptozotocin (STZ)-induced diabetes was accompanied by an increase in SSG ILI concentrations. In the present work we studied the effect of SSG ILI from normal and STZ diabetic rats (ILI-N and ILI-D, respectively) on insulin receptor binding and function in LMH cell line. ILI-N and ILI-D inhibited 125I-insulin binding to intact cells and wheat germ agglutinin (WGA)-purified insulin receptors with a high affinity. Furthermore, ILI-N and ILI-D activated, although weakly, the beta-subunit autophosphorylation of solubilized and WGA-purified insulin receptors. An ATP hydrolytic activity was present in ILI-N and, to a greater extent, in ILI-D extracts, which can at least in part explain their low potency for activating autophosphorylation and kinase activity of insulin receptors in vitro. However, after ILI treatment of intact cells and immunoprecipitation of insulin receptors, ILI induced a dose-dependent tyrosine phosphorylation of the insulin receptor beta-subunit. Finally, ILI-N and ILI-D stimulated amino acid uptake and lipogenesis in LMH cells. These findings suggest that SSG ILI is biologically active and can participate in metabolic regulations.

Corticosterone-induced insulin resistance is not associated with alterations of insulin receptor number and kinase activity in chicken kidney.

Chicken renal insulin receptors have been recently characterized; their number and kinase activities vary in response to altered nutritional status. In the present study, the effect of chronic corticosterone treatment was examined in 5-week-old chickens. The development of an insulin resistance following corticosterone was suggested after 1 and 2 weeks of treatment by a significant increases in plasma insulin levels (1.63 +/- 0.13 vs 0.56 +/- 0.14 ng insulin/ml in controls) and in renal cytosolic phosphoenolpyruvate carboxykinase activity (17.2 +/- 0.8 vs 13.7 +/- 0.7 nm/mn/mg tissue in controls). No significant changes were present at the level of insulin receptor number and kinase activity. Therefore, in kidney and, as previously observed, in muscles, corticosterone can induce insulin resistance at postreceptor steps in the cascade of events leading to insulin action.

Characterization of insulin receptors in chicken kidneys: effect of nutritional status.

In chickens, the kidneys actively contribute to gluconeogenesis. A cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) is present in this tissue but is absent in liver. Cytosolic renal PEPCK is nutritionally and hormonally controlled which indicates a likely contribution of insulin in the control of this enzyme (and other renal functions). The present studies characterize renal insulin receptors in the chicken. The effects of the following nutritional conditions were examined: fed, 48 hr fasted, and 24 hr refed following a 48-hr fast. PEPCK activity was increased by the 48-hr fast and returned to normal after refeeding. Specific binding of 125I-insulin to renal membranes was time-, temperature-, and protein-dependent. Unlabeled insulin was more potent than IGF-1 in inhibiting 125I-insulin binding; the ratio of potencies for insulin and IGF-1, however, was dependent upon the nutritional state. Insulin binding was significantly higher (P < 0.05) following 48 hr fasting and lower (P < 0.05) following refeeding compared to ad libitum feeding. Receptor affinity was similar irrespective of the nutritional state. Solubilized and wheat germ agglutinin purified renal insulin receptors were devoid of ATPase activity in contrast to hepatic receptors. The sizes of alpha- and beta-subunits of renal receptors were similar to those of hepatic receptors: 135 and 95 kDa, respectively. Insulin-stimulated autophosphorylation of the beta-subunit was decreased, although not significantly, by prolonged fasting. Phosphorylation of artificial substrate: poly(Glu-Tyr) 4:1 was significantly decreased by the 48-hr fast at high insulin concentrations (10 and 100 nM). Kinase activities of renal insulin receptors from fed or refed chickens were very similar. In conclusion, typical insulin receptors are present in chicken kidneys. These receptors exhibit a regulation at the level of their number and kinase activity in a fashion similar to that found for hepatic receptors. The present results suggest a role for insulin in chicken renal function.