Prenatal tolbutamide treatment alters plasma glucose and insulin concentrations and negatively affects the postnatal performance of chickens

Prenatal tolbutamide treatment alters plasma glucose and insulin concentrations and negatively affects the postnatal performance of chickens

Domestic Animal Endocrinology 52 (2015) 35–42 Contents lists available at ScienceDirect Domestic Animal Endocrinology journal homepage: www.domestic...

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Domestic Animal Endocrinology 52 (2015) 35–42

Contents lists available at ScienceDirect

Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com

Prenatal tolbutamide treatment alters plasma glucose and insulin concentrations and negatively affects the postnatal performance of chickens L. Franssens a, J. Lesuisse a, Y. Wang a, B. De Ketelaere b, E. Willems a, A. Koppenol a, c, X. Guo d, J. Buyse a, *, E. Decuypere a, N. Everaert a, e a

Laboratory of Livestock Physiology, Department of Biosystems, KU Leuven, Leuven 3001, Belgium Division of MeBioS, Department of Biosystems, KU Leuven, Leuven 3001, Belgium c Animal Sciences Unit, Instituut voor Landbouw- en Visserijonderzoek, Melle 9090, Belgium d College of Animal Science and Technology, Jiangxi Agricultural University, Jiangxi 330045, China e Animal Science Unit, Gembloux Agro-Bio Tech, University of Liège, Gembloux 5030, Belgium b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 June 2014 Received in revised form 19 January 2015 Accepted 19 January 2015

To examine the relationship of insulin and glucose, broiler embryos were subjected to acute or prolonged hypoglycemia during the late embryonic phase by, respectively, injecting once (at embryonic day [ED] 16 or 17) or on 3 consecutive days (ED 16, 17, and 18) with tolbutamide (80 mg/g embryo weight), a substance that stimulates insulin secretion from the pancreas. After 1 tolbutamide injection, a prolonged (32 h) decrease of plasma glucose and a profound acute increase in plasma insulin were observed. The 3 consecutive tolbutamide injections induced hypoglycemia for 4 days (from ED 16 to ED 19). The postnatal performance after 3 consecutive tolbutamide injections in broiler embryos was also investigated. Body weight was lower in tolbutamide-treated chickens from hatch to 42 d compared with sham (P ¼ 0.001) and control (P < 0.001) chickens. Feed intake was lower in the tolbutamide group from hatch to 42 d as compared with sham (P ¼ 0.007) and control (P ¼ 0.017) animals. In addition, at 42 d, plasma glucose concentrations, after an insulin injection challenge (50 mg/kg body weight), were higher in tolbutamide-treated chickens compared with the sham and the control group as were their basal glucose levels (P value of group effect <0.001). In conclusion, tolbutamide treatment during the late embryonic development in broilers resulted in prolonged hypoglycemia in this period and negatively influenced the posthatch performance. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Embryonic injections Hypoglycemia Feed intake Body weight Insulin tolerance test

1. Introduction In vertebrates, glucose is an essential fuel for most cells, and the transport and utilization of this substrate by these cells contribute to body glucose homeostasis in which insulin plays an important role. Interestingly, avian species have blood glucose levels that are twice as high as those

* Corresponding author. Tel.: þ32 16 328525. E-mail address: [email protected] (J. Buyse). 0739-7240/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2015.01.004

commonly observed in mammals [1]. In addition, they have a poor responsiveness to insulin in adult life, despite the presence of normal insulin concentrations (2.02 ng/mL) [2] and the presence of a functionally conserved insulin signaling pathway [3]. In contrast to rats, the expression of some genes of the insulin signaling pathway in muscle and adipose tissue of broiler chickens did not change under experimental conditions (feed deprivation, insulin immune neutralization, and injection of exogenous insulin) [4–10]. Moreover, the major insulin-responsive glucose transporter (GLUT) 4 gene is absent in broiler chickens both during the

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embryonic and posthatch life [11,12]. In addition, the pancreas of both layer and broiler chickens is also relatively insensitive to glucose and D-glyceraldehyde, both potent insulinotropic agents in mammals [13,14]. For example, high glucose concentrations are required to achieve a 2-fold increase in insulin secretion from pancreatic cells of 5- to 6-d-old layer chickens in vitro, whereas a low glucose concentration is already sufficient to enhance insulin secretion in 4- to 6-wk-old mice [14]. These findings suggest that there are considerable differences between mammals and birds regarding the insulin-glucose relationship. In addition, few reports examine the glucose metabolism in chicken specifically during the prenatal period. Tolbutamide is a compound that stimulates insulin secretion from the pancreas [14] and has been successfully used to induce postnatal hypoglycemia in mammals [15] or chickens [9,16–18]. Thorn et al [19,20] have created a hypoglycemia by continuous infusion of intravenous insulin in fetus of ewes for 8 wk during the late pregnancy and suggested that this contributes to embryonic programming of an increased risk for development of uncontrolled hepatic glucose production, hepatic insulin resistance, and diabetes type II in postnatal life. However, in addition to direct effects of increased insulin concentrations in the dam, these hypoglycemic fetuses from manipulated animals are probably exposed to other maternal endocrine factors, which could affect fetal regulatory mechanisms of glucose homeostasis. The chicken embryo, conversely, develops in its egg and is independent of maternal influences during the embryonic development. Hence, the chicken is a unique animal model to study specific physiological imprinting effects. Earlier research from our group provided evidence of the existence of physiological programming in the chicken embryo, which could influence the postnatal performance of chickens. For example, Everaert et al [21] demonstrated that the removal of albumen decreased both embryonic and posthatch body weight (BW) up to day 7 and stimulated the degradation of proteins in the breast muscle. Hence, the chicken with its peculiar relationship between insulin and glucose might provide a valuable animal model to investigate programming effects on the insulin-glucose relationship. The aim of the present study was to create hypoglycemia in broiler embryos, by 1 (experiment 1) or 3 consecutive (experiment 2) injections with tolbutamide (80 mg/g embryo weight) and investigate the physiological programming on posthatch BW and feed intake. Therefore, broilers were reared until commercial slaughter age of 42 d after a prolonged tolbutamide treatment during the late embryonic phase (experiment 3). We hypothesized that a perturbation in the insulin-glucose ontogeny would affect the insulin sensitivity later in life. 2. Materials and methods The experiments were approved by the Ethical Committee for Experiment Use of Animals of the KU Leuven. 2.1. Incubation Fertile Ross 308 broiler eggs were obtained from a commercial hatchery (Belgabroed, Merksplas, Belgium)

and individually numbered. The eggs were set with the blunt end up in a forced-draft incubator (PAS Reform, Zeddam, the Netherlands) at a dry bulb temperature of 37.6 C and wet bulb temperature of 29.0 C and were turned 90 at intervals of 1 h during the incubation period. 2.2. Method of tolbutamide injection Eggs were removed from the incubator and candled to locate a distinct blood vessel of the chorioallantoic membrane (CAM). After removing a piece of shell using a forcep, a drop of oil was smeared over the outer shell membrane to render this membrane transparent and to visualize the blood capillaries. Eggs were injected in a blood capillary with 200 mL of a tolbutamide solution (T-0891; Sigma– Aldrich) dissolved in 0.05 N NaOH solution (as tolbutamide is not soluble in water) to get a final dose of 80 mg/g embryo weight (tolbutamide group) or with 200 mL of 0.05 N NaOH solution (sham group), using a 30-G needle (Microlance3; BD) and 1-mL syringe (Plastipak; BD). After the injection, the exposed membrane was disinfected with 70% ethanol, closed with Micropore Medical tape (3M, St. Paul, MN, USA) and then placed back into the incubator. The average embryo weight of 10 eggs per group was determined on embryonic day (ED) 16, ED 17, or ED 18 and averaged 22.5  0.6 g, 27.4  0.6 g, or 32.3  0.5 g, respectively. 2.3. Blood sampling Blood capillaries of the CAM were visualized as described previously. Blood (300 mL) was taken from the CAM using a 30-G needle and 1-mL syringe, collected in a heparinized tube and kept on ice. After blood sampling, eggs were treated as described previously and placed back into the incubator. Blood was centrifuged (750g, 15 min, 4 C), and plasma was collected and stored at 20 C until analysis. 2.4. Experimental design 2.4.1. Experiment 1: single tolbutamide injection on ED 16 or ED 17 Preliminary experiments were performed to determine the injection days, injection dose, and sampling time points postinjection (PI). One hundred eighty-four broiler eggs were divided randomly over the 3 groups (control, sham, or tolbutamide). After injections on ED 16, blood was sampled at 7 time points (16, 20, 24, 28, 32, 36, and 40 h PI). After injections on ED 17, blood was taken at 4 time points (0.5, 1, 4, and 8 h PI), with 8 eggs per treatment (sham or tolbutamide) per time point. On ED 17, a basal blood sample was collected from 8 control embryos. Blood samples were used for determination of plasma glucose (at all sampling time points) and insulin (only at 0, 0.5, 16, and 40 h PI). The experimental setup is illustrated in Table 1. 2.4.2. Experiment 2: 3 consecutive injections of tolbutamide on ED 16, ED 17, and ED 18 One hundred eighty broiler eggs were randomly divided over 3 groups (control, sham, and tolbutamide), with 60

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Table 1 The setup of the first experiment. Time points PI (h)

Injection day

Parameters

ED 16 0 0.5 1 4 8 16 20 24 28 32 36 40

ED 17 Control Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide

Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide

Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose

and insulin and insulin

and insulin

and insulin

Abbreviations: ED, embryonic day; PI, postinjection. The dose of tolbutamide was 80 mg/g embryo weight.

eggs per group. The sham or tolbutamide group was further subdivided into 3 “injection groups” (1, 2, or 3 consecutive injections). One single injection was performed on ED 16, 2 injections on ED 16 and ED 17, and 2 injections on ED 16, ED 17, and ED 18. Blood was taken at 2 time points (8 h and 24 h PI), with 10 eggs per time point (sham or tolbutamide). Basal blood samples (control) were taken from 10 embryos at the same sampling time points (8 h and 24 h PI). All blood samples were used for analysis of plasma glucose and insulin. The setup is presented in Table 2. 2.4.3. Experiment 3: posthatch performance after 3 consecutive injections of tolbutamide 2.4.3.1. Injections. One hundred eight broiler eggs were randomly divided over 3 groups (control, sham, and tolbutamide), with 36 eggs per group. Eggs were injected daily from ED 16 until ED 18 (sham or tolbutamide), and another 36 eggs were not treated and served as the control group.

pen were monitored weekly. Feed conversion (FC) was calculated as feed intake (g) divided by BW gain (g) per pen per week. 2.4.3.3. Insulin tolerance test. Eight chickens per group were subjected to an insulin tolerance test (ITT) on days 21 and 42 after an overnight fast. Chickens were intramuscularly injected using a 21-G needle (Neolus, Terumo) and 1-mL syringe with an insulin solution from bovine pancreas (I5500, Sigma–Aldrich) dissolved in distilled water to obtain a final dose of 50 mg/kg BW. The administration route for insulin and the dose were tested in preliminary experiments, where the dose, although higher, was based on experiments by Simon et al [22]. Blood was collected before injection and 45, 90, 180, and 270 min PI from a wing or neck vein using a 21-G needle and 1-mL syringe, collected in a heparinized tube and kept on ice. After centrifugation (750g,15 min, 4 C), plasma was pipetted and stored at 20 C until determination of plasma glucose concentrations. 2.5. Measurements

2.4.3.2. Rearing. On ED 19, treated (sham or tolbutamide) and untreated (control) eggs were transferred from the turning trays to individual hatching baskets. Newly hatched chicks were weighed, identified with leg tags, and divided over 4 pens per group (7 chicks/pen) in an environmentally controlled room. Wood shavings were used as litter. Water and a commercial broiler (starter, grower, and finisher) feed were available ad libitum. Individual BW and feed intake per

Table 2 The setup of the second experiment. Group

Injection day

Sampling PI (h) ED 16

Control Sham/tolbutamide Sham/tolbutamide Sham/tolbutamide

ED 16 ED 16–17 ED 16–17–18

ED 17

ED 18

8

24

8

24

8

24

x x

x x

x

x

x

x

x

x x

x

Abbreviations: ED, embryonic day; PI, postinjection. The dose of tolbutamide was 80 mg/g embryo weight.

2.5.1. Plasma glucose concentrations Plasma glucose concentrations were determined using a commercial kit (LabAssay Glucose; Wako Pure Chemical Industries Ltd), according to the manufacturer’s protocol. The absorbance was measured at 490 nm (Victor 1420 Multilabel counter; PerkinElmer, MA, USA). 2.5.2. Plasma insulin concentrations Plasma insulin concentrations were analyzed using an ELISA (Ultrasensitive Mouse Insulin ELISA; Mercodia), according to the manufacturer’s instructions. The absorbance was measured at 450 nm (Victor 1420 Multilabel counter; PerkinElmer). Plasma insulin values were calculated using the calibrator curve (0.025–1.5 mg/L). The intra-assay coefficient of variation was 6.8%, calculated from the variation of 15 chicken plasma samples assayed in duplicate within a single assay. The lowest standard was analyzed in each assay to determine an inter-assay coefficient of variation (9.2%). The use of chicken samples on the Mouse ELISA kit was validated. Serially diluted native chicken plasma samples were parallel to the insulin standard curve,

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according to the methods described by Plikaytis et al [23]: within-dilution coefficient of variations were all 15% (2.8%–9.3%) and within assay coefficient of variations were all 20% (3.75%). Values are comparable with those from Lu et al [24]. 2.6. Statistical analysis All data were analyzed with the statistical software package SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). Plasma glucose and insulin concentrations were analyzed using a 2-way ANOVA model. For the first experiment, to verify the effect of the injection method, glucose and insulin concentrations of the control group were compared with those of the sham group with group and sampling time point as categorical variables and their interaction. Next, to verify the effect of tolbutamide injection, glucose and insulin concentrations of the sham and tolbutamide group were compared at each sampling time point with group and sampling time point as categorical variables and their interaction. For the second experiment, plasma glucose and insulin values were evaluated with injection group (1,2, or 3 injections), group (control, sham, and tolbutamide) and sampling time point as categorical variables and their interactions. When there was a significant overall effect, a Tukey’s post hoc test was performed. For the third experiment, BW, feed intake, and FC were analyzed using repeated measurements, containing age and group (control, sham, and tolbutamide) as variables and their interaction. For analysis of the ITT, the method of repeated measurements was used, with sampling time point and group as variables and their interaction. Wing or pen numbers were used for identification. Differences between these variables were analyzed by the specification of estimates based on the significant model. For all parameters, a 95% confidence interval threshold (P < 0.05) was set. Data are shown as mean  standard error of the mean.

Fig. 1. Plasma glucose levels (mg/dL) of sham and tolbutamide-treated embryos before and after injection on ED 16 or ED 17 (n ¼ 8) (experiment 1). Values are given as mean  standard error of the mean. a, b, c, and d refer to significant differences between sampling time points after tolbutamide injection. *means values are significantly different between the tolbutamide and sham group within each time point. ED, embryonic day.

and their interaction (P ¼ 0.019). Only at 30 min PI, plasma insulin values in tolbutamide-treated embryos were significantly increased from 0.25  0.05 ng/mL in embryos of the sham group to 0.99  0.22 ng/mL (P ¼ 0.005). 3.2. Experiment 2: 3 consecutive injections of tolbutamide on ED 16, ED 17, and ED 18 Plasma glucose values were affected by injection group (1, 2, or 3 injections; P < 0.001), group (control, sham, or tolbutamide group; P < 0.001), sampling time point (8 h or 24 h PI; P ¼ 0.003), the interaction between injection group and group (P ¼ 0.026), and the interaction between group and sampling time point (P ¼ 0.049), but not affected by the interaction between injection group and sampling time

3. Results 3.1. Experiment 1: single tolbutamide injection on ED 16 or ED 17 The injection of 0.05 N NaOH solution (sham group) did not affect plasma glucose levels as compared with the control group (149  1.7 mg/dL). Plasma glucose values were affected by group (sham vs tolbutamide; P < 0.001), sampling time point (P < 0.001), and their interaction (P < 0.001). At all sampling time points, except for 0.5, 1, and 40 h PI, plasma glucose levels of the tolbutamide group were significantly lower compared with those of the sham group (Fig. 1). Plasma glucose levels of the tolbutamide group decreased fast to a minimum level at 16 h PI, after which it increased slowly and was significantly raised at 32 h PI (P < 0.001) compared with the minimum level. Plasma insulin concentrations are presented in Figure 2. The 0.05 N NaOH injection (sham group) did not influence plasma insulin values in embryos, as compared with untreated control embryos (0.14  0.01 ng/mL). Plasma insulin concentrations were influenced by group (sham vs tolbutamide) (P ¼ 0.029), sampling time point (P ¼ 0.005),

Fig. 2. Plasma insulin levels (ng/mL) of sham and tolbutamide-treated embryos before and after injection on ED 16 or ED 17 (n ¼ 8) (experiment 1). Values are given as mean  standard error of the mean. *Refer to significant differences in plasma insulin values between the sham and tolbutamide group per sampling time point. ED, embryonic day.

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Fig. 3. Plasma glucose levels (mg/dL) of the control, sham, and tolbutamidetreated group (n ¼ 10) (experiment 2). The first injection was performed on ED 16, the second on ED 17, and the third on ED 18. Samples were taken at 8 h and 24 h after each injection. Values are given as mean  standard error of the mean. a, b, c, and d refer to significant differences in plasma glucose values between injection days. A and B indicate that plasma glucose values were significantly different between groups. ED, embryonic day.

point (Fig. 3). After each injection, plasma glucose values of the sham group did not differ from those of the control group. Plasma glucose values in tolbutamide-treated embryos were lower than those in the sham group after the first injection (P < 0.001), the second injection (P < 0.001), and the third injection (P < 0.001). In addition, in tolbutamidetreated embryos, plasma glucose concentrations were higher after the third injection in comparison with those obtained after the first (P < 0.001) and second injection (P < 0.001). In sham embryos, plasma glucose values were higher after the third injection compared with those after the first (P ¼ 0.014) and second injection (P ¼ 0.031). Plasma insulin levels (Fig. 4) were influenced by injection group (P ¼ 0.002), group (P < 0.001), and by the interaction between injection group and sampling time point (P ¼

Fig. 4. Plasma insulin levels (ng/mL) of control, sham, and tolbutamidetreated embryos (n ¼ 10) (experiment 2). The first injection was performed on ED 16, the second on ED 17, and the third on ED 18. Values are given as mean  standard error of the mean. a and b refer to significant differences between injection days. A and B indicate that plasma insulin values were significantly different between groups. ED, embryonic day.

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Fig. 5. Body weight of control, sham, and tolbutamide-treated chickens (n ¼ 28) (experiment 3). a and b refer to significant differences within each age. Values are given as mean  standard error of the mean.

0.041), but not influenced by sampling time point. Plasma insulin values were similar between the control and sham group. In contrast, tolbutamide-treated embryos had significantly higher plasma insulin values than the sham (P ¼ 0.007) and control group. In addition, plasma insulin concentrations after tolbutamide injection were higher on ED 16 than on ED 17 (P ¼ 0.003) and on ED 18 (P ¼ 0.026). 3.3. Experiment 3: posthatch performance after 3 consecutive injections of tolbutamide 3.3.1. BW, feed intake, and FC BW was affected by group (P < 0.001) and age (P < 0.001), but not by their interaction (Fig. 5). The sham group did not differ from the control group. Compared with the control and sham group, tolbutamide-treated chickens had a consistently lower BW at all ages (P < 0.001; P ¼ 0.001, respectively).

Fig. 6. Feed intake per chicken per day of control, sham, and tolbutamidetreated chickens (n ¼ 4 pens) (experiment 3). a and b refer to significant differences within each age. Values are given as mean  standard error of the mean.

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Fig. 7. Insulin tolerance test on day 42 (experiment 3). Plasma glucose levels (mg/dL) of the control, sham, and tolbutamide-treated group after intramuscular injection of insulin (50 mg/kg body weight) (n ¼ 8). Values are given as mean  standard error of the mean. A, B, and C refer to significant differences between sampling time points. a and b indicate significant differences between groups within each time point.

Feed intake was affected by group (P ¼ 0.015) and age (P < 0.001), but not by their interaction. The NaOH solution did not influence feed intake in chickens. Tolbutamidetreated chickens had a lower feed intake compared with the control and sham group at all ages (P ¼ 0.017; P ¼ 0.007, respectively; Fig. 6). FC increased with age (P < 0.001), but was not affected by group nor by the interaction (data not shown). 3.3.2. Insulin tolerance test On day 21, plasma glucose values were affected by sampling time point (P < 0.001) and not affected by group and their interaction (data not shown). At 45 min PI, plasma glucose levels decreased to a minimum level and remained low until 180 min PI. At 270 min PI, they increased slightly, but did not reach basal values. On day 42, plasma glucose values were affected by group (P < 0.001) and sampling time point (P < 0.001), but not affected by their interaction (Fig. 7). Plasma glucose concentrations declined to the minimum level at 90 min PI, remained low until 180 min PI, and then increased slightly at 270 min PI, but did not reach basal values. Plasma glucose levels of the tolbutamide group were higher than those of the control (P < 0.001) and sham group (P < 0.001), which were similar in this respect. 4. Discussion The aim of the present study was to create an acute or prolonged hypoglycemia in broiler embryos, by 1 (experiment 1) or 3 consecutive (experiment 2) injections with tolbutamide (80 mg/g embryo weight), respectively, to investigate the programming effect on posthatch performance and on insulin sensitivity. The prolonged hypoglycemic state from 4 h PI to 36 h PI after a single injection of

tolbutamide, associated with a profound rise in plasma insulin concentrations at 0.5 h PI, is in agreement with in vitro and in vivo studies on layer and broiler chickens at different ages [9,14–18,25–28] and with in vitro and in vivo studies on mice, rats, dogs, and hamsters [29–33]. Indeed, tolbutamide is a potent hypoglycemic agent in mammals and birds and stimulates insulin secretion from pancreatic b-cells, principally by inhibiting adenosine triphosphate– sensitive Kþ channels in the cell membrane, consequently, causing a membrane depolarization and an activation of voltage-gated Ca2þ influx [34–36]. Secreted insulin causes stimulation of glucose uptake, glycolysis and glycogen synthesis, and inhibition of glucose output, primarily through affecting a postreceptor mechanism of insulin action. Indeed, Rideau et al [37] and Seki et al [9] suggested that tolbutamide may stimulate glucose utilization by the chicken liver and some types of chicken muscles. In addition, not only insulin action but also extrapancreatic effects are probably involved. Tolbutamide activates glycolytic and glycogenic pathways and inhibits gluconeogenic pathway through regulating key enzyme activities in the liver [38]. Danby et al [17] demonstrated that in pancreatectomized chickens, the hypoglycemic action of tolbutamide depends on an inhibition of glucose release by the liver. The duration of hypoglycemia in tolbutamide-treated broiler embryos was longer than that in tolbutamidetreated broiler chickens in which glucose levels returned back to basal levels almost at 5 h and 8 h after intubation with 100 mg/kg tolbutamide delivered into the crop [16,18]. However, the difference in the duration of hypoglycemia may not depend on the age but might be because of the treatment method (injection vs intubation). To our knowledge, this is the first study measuring plasma glucose levels after tolbutamide injection in chicken embryos. Because insulin sensitivity is decreasing with age, tolbutamide injections in broiler chickens could lead to new insights on the differences in the insulin signaling pathway between chicken embryos and chickens. The prolonged hypoglycemic state for 4 days after 3 daily tolbutamide injections is in agreement with the study by Seki et al [18] who observed persistent hypoglycemia for 5 d by sequential dosing (3 times a day during 5 d) of tolbutamide (100 mg/kg BW). These authors observed a short-term effect of tolbutamide on plasma insulin levels, which is similar to our findings as higher plasma insulin levels (5-fold) were found 30 min after the single tolbutamide injection (experiment 1). Ball et al [39,40] and McClenaghan et al [41] demonstrated that chronic in vitro exposure of rat pancreatic b-cells to tolbutamide for 18 to 144 h led to a decline in their insulinotrophic activity because of a direct desensitization of pancreatic b-cells and a declined insulin secretion and pancreatic insulin content. Similar findings were also reported in rats after in vivo treatment of tolbutamide [42,43]. As higher insulin levels and lower plasma glucose concentrations in tolbutamidetreated embryos were observed 8 and 24 h after each tolbutamide injection, desensitization of b-cells in broiler embryos did not seem to occur or the dose of tolbutamide was high enough to continue the stimulation of insulin secretion. Alternatively, the prolonged hypoglycemia despite relatively unchanged plasma insulin levels in

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tolbutamide-treated embryos may be partly because of hepatic effects. Indeed, Kaku et al [38] suggested that tolbutamide also could stimulate glycolytic and glycogenic pathways and inhibit gluconeogenic pathways in the liver. The injection of NaOH (sham group) did not influence the plasma glucose and insulin concentrations compared with the control groups, demonstrating little effects of the manipulation itself. Hence, in this study, a prolonged hypoglycemia has been successfully induced in broiler embryos after the tolbutamide treatment. The low concentration of plasma insulin measured in this experiment is in agreement with the study of Lu et al [24] who showed increasing concentrations from ED 10 up to 21 d posthatch and suggested that insulin might be an important promoter for embryonic growth. Injection of tolbutamide increased insulin levels by 5-fold 30 min after the injection, while significantly higher levels could still be observed 8 and 24 h PI in the second experiment and only numerically 16 h PI in the first experiment. This discrepancy and the variability in plasma insulin levels of tolbutamide-treated embryos might be explained by differences in embryo weight, which was estimated by calculating the average embryo weight of a small number of eggs. The embryonic programming after tolbutamide treatment negatively influenced the posthatch performance as reflected in the lower BW of tolbutamide-treated chickens compared with control and sham chickens, as well as the lower feed intake, resulting in the similar FC during this period. Lower BW caused by tolbutamide is in parallel with the studies of Akazawa et al [44] and Smoak and Sadler [45] who showed that rat or mouse embryos, exposed to the hypoglycemic medium, displayed growth retardation. The lower BW at hatch, together with a lower feed intake during the entire rearing period, was responsible for a continued lower BW in the tolbutamide group compared with the sham and control group up to 42 d. How a prolonged hypoglycemia during embryonic development affected control of feed intake remains unknown and might be investigated by measurements of hypothalamic neuropeptides. Although the disturbances in the insulin-glucose metabolism during chicken embryonic development affected posthatch performance negatively, Sato et al [46] showed that a single oral injection of tolbutamide (100 mg/kg) in newly hatched broiler chicks was effective in improving chicken growth performance because of insulin secretion as enhancer of myoblasts proliferation. Therefore, the timing and duration of the hypoglycemic state seem important in the programming of growth performance. The ITT at slaughter age showed that the decrease of plasma glucose concentrations after insulin injection was less pronounced in tolbutamide-treated chickens compared with the sham and control group, which might suggest that the tolbutamide group was less sensitive to insulin administration. This finding is in agreement with several mammalian studies showing that fetal adaptations to hypoglycemia and low birth weight (by lowering maternal glucose supply) could underlie the increased risk for developing insulin resistance in the postnatal life [19,47,48]. However, basal glucose levels before the insulin

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injection were higher in the tolbutamide group compared with those in the sham and control group, which makes the interpretation of the test difficult without basal insulin measurements. Nevertheless, these findings indicate perturbations in the insulin-glucose relationship observed on 42 d and not yet on 21 d because of the induced prolonged hypoglycemia during embryonic development, which invites for further investigation on the insulin signaling pathway and on epigenetic modifications of embryonic programming. 5. Conclusions A single tolbutamide injection during the late embryonic development in broiler embryos resulted in a prolonged (32 h) hypoglycemia. This hypoglycemia was preceded by a profound secretion of insulin. Three consecutive tolbutamide injections from ED 16 to ED 18 induced a long-lasting hypoglycemia during 4 days, programming posthatch BW, and feed intake accompanied by an altered glucose homeostasis at 42 d. With this experimental model, differences in insulin signaling pathways between the embryonic and posthatch growing stage in broilers and epigenetic modifications can be further investigated. Acknowledgments The authors thank the technical staff Andre Respen, Marcel Samain, and Daniel Vermeulen of the Laboratory of Livestock Physiology of the KU Leuven. This research was funded by the Fonds Wetenschappelijk Onderzoek, Vlaanderen (FWO nr. G.0566.08N). References [1] Polakof S, Mommsen TP, Soengas JL. Glucosensing and glucose homeostatis: from fish to mammals. Comp Biochem Physiol B Biochem Mol Biol 2011;160:123–49. [2] Simon J, Rideau N, Taouis M, Dupont J. Plasma insulin levels are rather similar in chicken and rat. Gen Comp Endocrinol 2011;171: 267–8. [3] Dupont J, Tesseraud S, Simon J. Insulin signaling in chicken liver and muscle. Gen Comp Endocrinol 2009;163:52–7. [4] Dupont J, Derouet M, Simon J, Taouis M. Nutritional state regulates insulin receptor and IRS-1 phosphorylation and expression in chicken. Am J Physiol 1998;274:E309–16. [5] Dupont J, Derouet M, Simon J, Taouis M. 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. Biochem J 1998;335:293–300. [6] Dupont J, Dagou C, Derouet M, Simon J, Taouis M. Early steps of insulin receptor signaling in chicken and rat: apparent refractoriness in chicken muscle. Domest Anim Endocrinol 2004;26:127–42. [7] Dupont J, Tesseraud S, Derouet M, Collin A, Rideau N, Crochet S, Godet E, Cailleau-Audouin E, Métayer-Coustard S, Duclos MJ, Gespach C, Porter TE, Cogburn LA, Simon J. Insulin immunoneutralization in chicken: effects on insulin signaling and gene expression in liver and muscle. J Endocrinol 2008;197:531–42. [8] Dupont J, Métayer-Coustard S, Ji B, Ramé C, Gespach C, Voy B, Simon J. Characterization of major elements of insulin signaling cascade in chicken adipose tissue: apparent insulin refractoriness. Gen Comp Endocrinol 2012;176:86–93. [9] Seki Y, Sato K, Akiba Y. Changes in muscle mRNAs for hexokinase, phosphofructokinase-1 and glycogen synthase in acute and persistent hypoglycemia induced by tolbutamide in chickens. Comp Biochem Physiol B Biochem Mol Biol 2005;142:201–8.

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