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In mammals, insulin regulates blood glucose levels and plays a key regulatory role in appetite via the hypothalamus. In contrast, chickens are characterized by atypical glucose homeostasis, with relatively high blood glucose levels, reduced glucose sensitivity of pancreatic beta cells, and large resistance to exogenous insulin. The aim of the present study was to investigate in chickens the effects of 5 h fasting and 5 h insulin immuno-neutralization on hypothalamic mRNA levels of 23 genes associated with food intake, energy balance, and glucose metabolism. We observed that insulin immune-neutralization by administration of anti-porcine insulin guinea pig serum (AI) significantly decreased food intake and increased plasma glucose levels in chickens, while 5 h fasting produced a limited and non-significant reduction in plasma glucose. In addition, 5 h fasting increased levels of NPY, TAS1R1, DIO2, LEPR, GLUT1, GLUT3, GLUT8, and GCK mRNA. In contrast, AI had no impact on the levels of any selected mRNA. Therefore, our results demonstrate that in chickens, food intake inhibition or satiety mechanisms induced by insulin immuno-neutralization do not rely on hypothalamic abundance of the 23 transcripts analyzed. The hypothalamic transcripts that were increased in the fasted group are likely components of a mechanism of adaptation to fasting in chickens., (© The Author 2017. Published by Oxford University Press on behalf of Poultry Science Association.)

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.

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.

We investigated whether a change in peripheral glucose homeostasis, a local change in the insulin-related ovarian regulatory system, or both occurred in ad libitum-fed broiler breeder hens compared with feed-restricted counterparts. Feed-restricted (R, from 5 to 16 wk of age) and ad libitum-fed (A) hens from a standard commercial line (S) and an experimental dwarf genotype (E) were studied. Basal and stimulated plasma insulin and glucose concentrations were measured during the prebreeding and laying periods. In the basal state (after 16 h fasting) plasma glucose concentrations were significantly lower in SA chickens (-5% at 17 wk, -7.5% at 32 wk) compared with EA, SR, and ER chickens, with no difference in plasma insulin concentrations (n = 16). In 17-wk-old SA birds, 30 min after oral glucose loading, plasma glucose concentrations increased significantly compared with the basal state and were also significantly lower as compared with SR but did not differ significantly from EA and ER. Plasma insulin concentrations did not differ significantly between genotypes or regimens (n = 16). A potential modification of intracellular mediators involved in the regulation of cell growth and survival in small follicles that were overrecruited in SA compared with SR was also investigated in SA and SR hens at 32 wk. There was no effect of food restriction in phospho-Akt, Akt, phospho-ERK, and phospho-S6 in the small white ovarian follicles (n = 6) in the basal state and after 30 min of refeeding. In conclusion, the present study does not demonstrate any evidence of glucose intolerance during the prebreeding period, specific change in the ovarian small follicle insulin signalling pathway, or both, in laying broiler breeders fed ad libitum compared with feed-restricted hens.

When overfed at their maximum (intensive overfeeding) or at only 80% (moderate overfeeding) of food intake capacity, Mule ducks developed strong liver steatosis, whereas Pekin ducks showed very marked extrahepatic fattening. During overfeeding, evolution of plasma glucose and triacylglycerol concentrations suggested a very strong increase in the hepatic lipogenesis as well as genotype- and diet-independent lipoprotein secretion. In contrast, lipoprotein-lipase activity was dependent on alimentary status (the intensive overfeeding induces the highest activities), and Pekin ducks showed higher lipoprotein-lipase activity than Mule ducks, which could favor extrahepatic fattening to the detriment of hepatic steatosis. In Pekin ducks, plasma pancreatic hormone concentrations are related to diet levels and blood sugar. With similar food intake, Mule ducks (moderately overfed) showed global blood insulin lower than that of Pekin ducks (intensively overfed) despite similar blood sugar levels, suggesting a trend towards reduced pancreas response to glucose in Mule ducks. This may result from their lower lipoprotein-lipase activity as previously shown in these two ducks overfed at only 60% of their maximal food intake capacity (unpublished results). These results suggest that high plasma insulin concentrations may be necessary to induce an optimum lipoprotein-lipase activity in overfed ducks.

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.

The plasma glucose-insulin relationships and thyroid status were investigated in two lines of adult cockerels divergently selected for high (R+) or low (R-) residual food consumption (RFC). For a given body weight, R+ birds had a 74% higher food intake than R- birds. Plasma glucose concentrations were significantly lower in the R+ line compared with the R- when fasted, whereas R+ birds exhibited a significantly lower plasma insulin concentration than R- birds either in fed or fasted state. After an overnight fast, R+ birds also exhibited a higher sensitivity to exogenous insulin in view of its more pronounced hypoglycaemic effect. After an oral glucose load, the glucose disposal of R+ cockerels was faster despite lower glucose-induced plasma insulin concentration. Whilst plasma triacylglycerol concentrations were lower in the R+ line when fed, plasma non-esterified fatty acid concentrations were higher in fasted R+ than R- cockerels (684 v. 522 mumol/l). Higher plasma triiodothyronine concentrations were observed in fed R+ compared with R- birds (3.0 v. 2.1 nmol/l respectively). The higher plasma concentrations of triiodothyronine associated with lower concentrations of insulin could account for the leanness and the elevated diet-induced thermogenesis previously observed in the R+ line.

The effects of duodenal infusion of glucose on the relationship between plasma concentrations of glucose and insulin and on milk composition were investigated in a crossover design. Eight dairy cows were continually infused with water (control) or glucose (1.5 kg/d). Cows received diets consisting of dehydrated whole-plant maize in restricted amounts to equalize the energy supply between treatments. Basal (before meal) plasma concentrations of glucose and insulin were increased, but concentrations of nonesterified fatty acids (NEFA) were decreased, by glucose treatment. During the first 2 h after feed distribution, plasma insulin increased, and plasma glucose and NEFA decreased, in both control and treated cows. Afterward, plasma glucose increased in treated cows but further decreased in control cows. The difference reached 8 mg/100 ml without any change in plasma insulin. During the meal, concentrations of growth hormones in plasma were inhibited to a similar extent in both groups. In response to intravenous glucose or insulin challenges, changes in plasma glucose, NEFA, and insulin stimulated by glucose were also very similar in both groups. In conclusion, duodenal infusion of glucose increased basal plasma concentrations of glucose and insulin, increased postprandial plasma glucose, and decreased NEFA without inducing insulin resistance. Glucose treatment did not change milk yield but decreased milk fat yield, mainly through a decrease in the yield of C18 fatty acids that were derived from circulating fatty acids. In the absence of insulin resistance, the decrease in the yield of C18 fatty acids might be attributed to an inhibition of adipose lipolysis or an increase in adipose lipogenesis.

The effect of 30 minute intravenous infusions of amino acids (90 or 180 g) or glucose (114 g or 228 g) was studied in five non-pregnant lactating dairy cows. At each dose the same amounts of energy were delivered by glucose or amino acids. The nutrients were infused separately or in combination. In response to each nutrient, plasma insulin increased proportionally to the dose infused. The increase induced by amino acids was lower than that induced by glucose, and plasma insulin returned to its baseline value more rapidly after amino acid than after glucose infusion. The hypoglycemia that developed after amino acid infusions partly explains the short-lived amino acid induced in insulin response. The plasma insulin increase which resulted from the glucose-amino acid mix significantly exceeded that obtained by separate but isocaloric infusions of amino acids or glucose. Therefore, amino acids and glucose acted synergistically to increase plasma insulin release. The mechanism leading to the synergistic effect would be partially due to a permissive effect of hyperglycemia. Other possible potentiating mechanisms are discussed.

1. The possibility that 8-bromo cyclic adenosine monophosphate (8-Br-cAMP) or acetylcholine (ACh) potentiates insulin release in chicken pancreas in response to D-glyceraldehyde (D-GA, a weak insulinotropic fuel), and permits an insulin release in response to D-mannose or alpha-ketoisocaproic acid (alpha-KIC) (two non-insulinotropic fuels in chicken pancreas) is examined. 2. 8-Br-cAMP (1 mM) or ACh (1 microM) permitted a sustained although delayed insulin release in response to D-GA (5 and 15 mM). 3. The resistance to D-mannose (50 mM) or alpha-KIC (10 mM) persisted in the presence of 8-Br-cAMP. 4. At 1 or 100 microM, ACh permitted a slight, immediate and transient insulin output in response to alpha-KIC but not to D-mannose (with one unexplained exception). 5. The simultaneous perfusion of 8-Br-cAMP + ACh increased the basal rate of insulin release, and permitted a large and sustained response to D-mannose. It also greatly increased the immediate response to alpha-KIC + ACh. 6. In conclusion, in chicken pancreas fuel nutrients require the activation of cAMP- and/or ACh-dependent pathways to induce insulin release. Whether this peculiarity is related to the high glycemia of chickens awaits further investigation.

1. As previously shown, 14 mM D-glucose, a non-insulinotropic concentration in isolated chicken pancreas, permits an insulin release in response to D-glyceraldehyde, (D-GA; a glycolytic fuel) and L-leucine or alpha-ketoisocaproic acid (alpha-KIC) (non-glycolytic fuels), which alone are not initiators of insulin release in this species. 2. The "permissive" effect of D-glucose was also observed in the presence of D-mannose (which, as shown herein, is not insulinotropic alone). 3. The specificity of glucose for this "permissive" effect was, therefore, subsequently questioned in the presence of 10 mM alpha-KIC by substituting various glycolytic and non-glycolytic fuels to glucose. 4. D-GA (at 5 and 15 mM), D-mannose (30 and 50 mM), or the association of L-glutamine + L-asparagine permitted an insulin release in response to alpha-KIC. 5. The response was, however, delayed with D-GA, only occasionally with 50 mM D-mannose, and required high concentrations and was delayed in the presence of L-glutamine + L-asparagine as compared to that obtained with 14 mM D-glucose + alpha-KIC. 6. In conclusion, the threshold of fuel-induced insulin release is much higher in the chicken than in mammals and this threshold is most efficiently lowered by glucose.

Plasma glucose and insulin levels were measured in chronically catheterized, freely moving, undisturbed geese, which were offered a free standard meal after an overnight fast. The insulin level markedly rose within the first minute after the start of food ingestion, whereas plasma glucose did not increase. This early insulin response was not correlated with the size of the meal. In contrast, both postabsorptive insulin response and plasma glucose changes were dependent on meal size. When a small amount of food (2-6 g) was eaten, insulin returned to basal level within 30 min, whereas plasma glucose remained unchanged. Larger meals (15-20 g) maintained plasma insulin at a higher level and induced a sustained rise of plasma glucose. These results indicate that there is a cephalic phase of insulin secretion at the beginning of the meal in birds as previously described in mammals.

1. The effects of subcutaneous injection of cysteamine (2-mercaptoethylamine, 300 mg/kg) were investigated in 5-6 week-old chickens. 2. In the short term (1 hr), cysteamine increased plasma levels of glucose, free fatty acids and insulin, and decreased that of alpha-amino non protein nitrogen. 3. In a longer term (17-24 hr), cysteamine increased the plasma level of glucose, did not modify those of alpha-amino non protein nitrogen, insulin and glucagon and decreased that of free fatty acids. 4. The disposal of an oral glucose load was impaired and the glucose-induced inhibition of pancreatic glucagon and stimulation of insulin release were blunted 17 hr after cysteamine administration. 5. Therefore, cysteamine exerts multiple effects on chicken pancreatic islet cells.

In the isolated perfused chicken pancreas, 20 and 40 mM L-leucine or 10-40 mM alpha-ketoisocaproic acid (alpha-KIC) did not initiate insulin release. In the presence of 14 mM glucose (a noninsulinotropic concentration), 20 mM L-leucine and 10 mM alpha-KIC evoked a slight biphasic insulin release. The response to 20 mM L-leucine was further increased when 14 mM glucose was combined with 10 mM L-glutamine (10 mM glutamine alone did not induce insulin release and did not potentiate the response to 10 mM L-leucine). At 1 mM, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BrcAMP) alone caused a slight and progressive increase in insulin secretion but did not sensitize the pancreas to either 20 mM L-leucine or 10 mM alpha-KIC, whereas it facilitated a marked insulin release in response to 14 mM glucose. On the other hand, 10-40 mM K+ or 20 mM L-arginine induced a rapid monophasic insulin output. In conclusion, L-leucine or alpha-KIC, which do not initiate insulin release alone and are not potentiated by 8-BrcAMP, may not be regarded as primary insulinotropic agents in the chicken. This result, together with the previously documented resistance of the chicken pancreas to glucose alone or to D-glyceraldehyde, strongly suggests that the mechanisms initiating insulin secretion are different in chickens and mammals, whereas potentiating mechanisms (low glucose concentration, arginine, acetylcholine, and cAMP) and membrane depolarization events (K+ and arginine) are present in both chickens and mammals.

Insulin release by the isolated-perfused pancreas of 5-8 week-old chickens was studied in response to graded concentrations of D-glyceraldehyde (D-GA) (5-20 mM). D-GA was perfused for 30 min in the absence or in the presence of glucose concentrations (2.8 or 14 mM) which do not evoke insulin release alone. D-GA alone or in combination with glucose does not cause a biphasic insulin release. As compared to rodents the release is reduced and delayed (at least 16 min) and requires higher D-GA concentrations which reveals the insensitivity of the chicken pancreas to D-GA. In the absence of glucose, the release is higher at 15 mM D-GA. Glucose (2.8 or 14 mM) sensitized the pancreas to D-GA by eliciting earlier and higher insulin response but never restored an immediate and biphasic secretory response to D-GA. Glucose amplified the response to low D-GA levels (5 and 10 mM) and inhibited the response to higher concentrations (15 and 20 mM). A transient insulin rise ("off-response") related to the D-GA concentration was observed after withdrawal of D-GA either in the absence or in the presence of glucose. Therefore, in addition to glucose, the chicken pancreas is relatively insensitive to D-GA suggesting that the mechanism leading to insulin release in response to nutrient metabolism is different in chickens as compared to that in mammals.

Insulin release from the perfused pancreas was studied in genetically selected fat and lean chickens. The previously described glucose insensitivity of the chicken pancreas cannot be overcome by 5 or 10 mM D-glyceraldehyde, suggesting that the resistance is not related to glucose metabolism before the triose phosphate step. At 42 mM, glucose induced a biphasic insulin release which was specific, since 42 mM mannitol did not elicit insulin release. Arginine (10 mM) or acetylcholine (0.1-1 microM), which in themselves do not cause insulin release, generated a biphasic insulin release in the presence of a low nonstimulating glucose concentration (14 mM); the effect was synergistic. In contrast to the glucose tolerance test observed in vivo, the pancreas from the fat line chicken in response to glucose or glucose plus arginine released significantly less insulin during the first phase. The significance of this defect awaits further elucidation. On the other hand, acetylcholine, a more potent secretagogue, did not reveal any significant difference between fat and lean chickens.