Insulin immuno-neutralization in fed chickens: effects on liver and muscle transcriptome.

Chickens mimic an insulin-resistance state by exhibiting several peculiarities with regard to plasma glucose level and its control by insulin. To gain insight into the role of insulin in the control of chicken transcriptome, liver and leg muscle transcriptomes were compared in fed controls and "diabetic" chickens, at 5 h after insulin immuno-neutralization, using 20.7K-chicken oligo-microarrays. At a level of false discovery rate <0.01, 1,573 and 1,225 signals were significantly modified by insulin privation in liver and muscle, respectively. Microarray data agreed reasonably well with qRT-PCR and some protein level measurements. Differentially expressed mRNAs with human ID were classified using Biorag analysis and Ingenuity Pathway Analysis. Multiple metabolic pathways, structural proteins, transporters and proteins of intracellular trafficking, major signaling pathways, and elements of the transcriptional control machinery were largely represented in both tissues. At least 42 mRNAs have already been associated with diabetes, insulin resistance, obesity, energy expenditure, or identified as sensors of metabolism in mice or humans. The contribution of the pathways presently identified to chicken physiology (particularly those not yet related to insulin) needs to be evaluated in future studies. Other challenges include the characterization of "unknown" mRNAs and the identification of the steps or networks, which disturbed tissue transcriptome so extensively, quickly after the turning off of the insulin signal. In conclusion, pleiotropic effects of insulin in chickens are further evidenced; major pathways controlled by insulin in mammals have been conserved despite the presence of unique features of insulin signaling in chicken muscle.

Glucokinase activation induces potent hypoglycemia without recruiting insulin and inhibits food intake in chicken.

Glucose homeostasis exhibits several peculiarities in chickens (in short, presence of high glycemia and resistance to high doses of exogenous insulin). Though the full chicken glucokinase gene sequence is still lacking, several results suggest its existence. The functionality of chicken glucokinase (GK) has been further investigated using an activator of mammalian GK (GKA). In vitro, GKA decreased GK's S0.5(a) in a glucose-dependent manner in liver homogenates from either fasted or fed chickens; it also increased GK Vmax(a) in homogenates from fed chickens. In vivo, acute oral GKA administration (10-100 mg/kg) induced a potent and dose dependent hypoglycemic effect in fed chickens (starting between 15 and 45 min with a maximum effect at 40 mg/kg, P<0.0001). At this dose, plasma insulin levels showed erratic and minor changes in the early times (an increase at 5 min and a decrease at 10 min, P<0.05). At 90 min, when hypoglycemia had developed plasma insulin levels decreased under controls and plasma pancreatic glucagon levels increased over controls. Also at 40 mg/kg, GKA transiently inhibited food intake at about 3h (P<0.0001). In conclusion, GKA is a potent activator of chicken GK evidencing that the structure and the activity of chicken GK are similar to those of mammalian GK. At variance with results obtained in mammals, the potent GKA hypoglycemic action appears to rely mostly on an effect on liver GK in chicken. This fits with previous results and further support the hypothesis of a "deficient coupling" between Β-cell metabolism and insulin release in this species.

Early steps of insulin receptor signaling in chicken and rat: apparent refractoriness in chicken muscle.

The early steps of insulin receptor (IR) signaling (tyrosine phosphorylation of IR beta-subunit, IRS-1 and Shc and PI 3'-kinase activity) have been characterized in two target tissues in the chicken: liver and muscle. The signaling cascade appeared to depend on nutritional status in the liver, but not in muscle (with a possible exception for a minor tyrosine phosphorylation of the 52 kDa Shc isoform). In this study, we compared the responses of the liver and muscle to exogenous insulin (10 or 1000 mU/kg) in chickens and rats. In the liver, IRS-1 and Shc proteins were present in smaller amounts and the regulatory subunit p85 of PI 3'-kinase was present in larger amounts in chickens than in rats. In the basal state (saline injection), the level of tyrosine phosphorylation of IR was lower, and that of Shc higher, in chickens than in rats. PI 3'-kinase activity in chickens was half that in rats. Insulin activated all components of the cascade in a dose-dependent manner in both species. A different pattern was observed in the muscle. In the basal state, the levels of tyrosine phosphorylation of IR and of PI 3'-kinase activity were much higher in chickens than in rats (by factors of 2 and 30, respectively). Insulin strongly activated all components of the cascade in rats (but with no significant increase in the phosphorylation of Shc). No activation was observed in chickens (with only a slight but significant increase in the tyrosine phosphorylation of Shc). The insulin cascade therefore appears to respond normally in chicken liver but to be refractory in chicken muscle. The large amount of p85 and high levels of PI 3'-kinase activity in muscle may contribute to this situation, making chicken muscle an interesting model of insulin resistance.

Acute cold- and chronic heat-exposure upregulate hepatic leptin and muscle uncoupling protein (UCP) gene expression in broiler chickens.

Emerging evidence showed that variations in environmental temperature affect both leptin and uncoupling protein (UCP) gene expression in mammals, whereas a little is known about such interactions in birds. Thus, we conducted the present study to investigate the influence of acute (2 hours) cold (4 degrees C) and chronic (10 days) heat (32 degrees C) exposure on hepatic leptin and muscle UCP gene expression in 5-wk-old broiler chickens. Both cold- and heat-exposure significantly (P < 0.05 to P < 0.001) upregulated hepatic leptin (by 35 and 46%, respectively) and muscle UCP mRNA levels (by 71 and 71%, respectively) compared to the thermoneutrality (22 degrees C). This result suggests that leptin and UCP may be involved in the thermoregulation response of chickens to extreme climate (cold and hot temperatures). The upregulation of hepatic leptin gene expression was accompanied by an increase in plasma leptin levels, indicating that leptin may be regulated at transcriptional level. The increase of leptin and UCP mRNA abundance, and leptinemia we report here were not related to plasma glucose or insulin levels. In conclusion, the exposure of broiler chickens to extreme ambient temperatures (cold and heat) increases hepatic leptin and muscle UCP gene expression.

Insulin immuno-neutralization in chicken: effects on insulin signaling and gene expression in liver and muscle.

In order to evaluate the role of insulin in chicken, an insulin immuno-neutralization was performed. Fed chickens received 1 or 3 i.v. injections of anti-insulin serum (2-h intervals), while fed or fasted controls received normal serum. Measurements included insulin signaling cascade (at 1 h in liver and muscle), metabolic or endocrine plasma parameters (at 1 and 5 h), and qRT-PCR analysis (at 5 h) of 23 genes involved in endocrine regulation, metabolisms, and transcription. Most plasma parameters and food intake were altered by insulin privation as early as 1 h and largely at 5 h. The initial steps of insulin signaling pathways including insulin receptor (IR), IR substrate-1 (IRS-1), and Src homology collagen and downstream elements: phosphatidylinositol 3-kinase (PI3K), Akt, GSK3, ERK2, and S6 ribosomal protein) were accordingly turned off in the liver. In the muscle, IR, IRS-1 tyrosine phosphorylation, and PI3K activity remained unchanged, whereas several subsequent steps were altered by insulin privation. In both tissues, AMPK was not altered. In the liver, insulin privation decreased Egr1, PPAR gamma, SREBP1, THRSP alpha (spot 14), D2-deiodinase, glucokinase (GK), and fatty acid synthase (whereas D3-deiodinase and IGF-binding protein 1 transcripts were up-regulated. Liver SREBP1 and GK and plasma IGFBP1 proteins were accordingly down- and up-regulated. In the muscle, PPAR beta delta and atrogin-1 mRNA increased and Egr1 mRNA decreased. Changes in messengers were partly mimicked by fasting. Thus, insulin signaling in muscle is peculiar in chicken and is strictly dependent on insulin in fed status. The 'diabetic' status induced by insulin immuno-neutralization is accompanied by impairments of glucagon secretion, thyroid axis, and expression of several genes involved in regulatory pathways or metabolisms, evidencing pleiotropic effects of insulin in fed chicken.

Insulin and amino acid availability regulate atrogin-1 in avian QT6 cells.

New evidence has demonstrated that the expression of major genes, termed atrogenes, controls the ubiquitin-proteasome proteolytic pathway. The present work aimed to study the impact of insulin and amino acids on the expression of one of these atrogenes, the E3 ubiquitin ligase Muscle Atrophy F box (MAFbx, also called atrogin-1), in quail muscle (QT6) fibroblasts. First, we characterized atrogin-1 in QT6 cells and demonstrated the insulin sensitivity of these cells. Second, we showed that insulin reduced atrogin-1 mRNA via the phosphatidylinositol-3'kinase (PI3K)/protein kinase B (PKB or AKT)/target of rapamycin (TOR) pathway. Atrogin-1 expression also depended on the availability of an individual amino acid, i.e., methionine. Moreover, the amino acid-induced reduction of atrogin-1 was inhibited by rapamycin, indicating the involvement of the TOR pathway in such regulation. In conclusion, expression of the ubiquitin ligase atrogin-1 is regulated by both insulin and amino acids through the TOR pathway.

Insulinotropic agent ID-1101 (4-hydroxyisoleucine) activates insulin signaling in rat.

ID-1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin-stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID-1101 on insulin signaling and action besides its previously described insulinotropic action. Insulin-sensitizing effects of ID-1101 were investigated in rat in vivo by three different approaches: 1) using euglycemic hyperinsulinemic clamps in two different rat models of insulin resistance, i.e., Zucker fa/fa rats and rats fed a sucrose-lipid diet; 2) measuring liver and muscle phosphatidylinositol (PI) 3-kinase activity after an acute injection of ID-1101 in normal and insulin-resistant diabetic rats; and 3) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID-1101 can improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid-fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID-1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, chronic ID-1101 treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID-1101 can reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary, ID-1101, besides its insulinotropic effect, directly improves insulin sensitivity, making it a potentially very valuable therapeutic agent for diabetes treatment.