Heat stress increases apical glucose transport in the chicken jejunum

热应激增加肉鸡空肠顶端葡萄糖转运ABSTRACT：In chickens, elevated environmental temperature reduces food intake We

have previously reported that, during heat stress, the intestinal mucosa has an increased capacity to take up sugars. To investigate whether the effects of warm environment on sugar uptake are an intestinal adaptation to lower energy intake or a response attributable to heat stress, we examined the glucose transport kinetics of apical and basolateral membranes of the jejunum and the mucosal morphology of broiler chickens maintained in climatic chambers for 2 wk. Experimental groups were 1) control ad libitum (CAL), fed ad libitum and in thermoneutral conditions (20°C); 2) heat stress ad libitum (HSAL), fed ad libitum and kept in a heated environment (30°C); and 3) control pair-fed (CPF), maintained in thermoneutral conditions and fed the same amount of food as that consumed by the HSAL group. Both the CPF and the HSAL groups showed reduced body weight gain, but only the HSAL chickens had lower plasma thyroid hormones and higher corticosterone than CAL and CPF groups. The fresh weight and length of the jejunum were only reduced in the HSAL group. The activity and expression of apical sodium-dependent glucose transporter 1 (SGLT-1) were increased by 50% in the HSAL chickens, without effects in the CPF group. No changes in Kd or in SGLT-1 and glucose transporter-2 Km were observed in the pair-fed and heated birds. These results support the view that increased intestinal hexose transport capacity is entirely dependent on adaptations of apical SGLT-1 expression to heat stress and is not due to reduced food intake.

在肉鸡中，提高环境温度会减少饲料采食量。我们以前报道过在热应激时，肠黏膜摄取糖类的能力会加强。为了研究高温对糖类的这种影响是小肠对能量摄入量变低的调节还是对热应激的一种调节，我们检验了在气候仓中处理了 2周的肉鸡的空肠绒毛顶端和基底膜外侧葡萄糖转运动力学和空肠粘膜形态学。处理组分为：1 对照组，随意采食，温度 20度：2 热应激组随意采食，30度高温处理： 3 采食对照组，温度 20度，饲喂量为热应激组的采食量。热应激组和采食量对照组体增重都下降，但只有热应激组血浆中甲状腺激素降低和较高的皮质酮。只有热应激组的空肠长度和重量降低。热应激组中 SGLT-1的活性和表达量有大约 50%的升高采食量配对组没有变化。Kd和 SGLT-1、葡萄糖转运载体 2在采食量配对组中也没有变化。这些结果显示提高小肠己糖转运能力完全依赖于高温影响 SGLT-1 在绒毛顶端的表达而不是采食量的减少。the small intestine constitutes a highly dynamic interface with the external environment through the delivery processing and absorption of nutrients. The intestinal mucosa is capable of rapid and extensive morphological and functional adaptation in response to evolutionary, genetic, and ontogenetic

demands (12), as well as to environmental and nutritional challenges (25). In the domestic fowl, intensive artificial genetic selection has resulted in commercial meat breeds (broiler chickens) whose growth rates and food conversion efficiencies greatly exceed those of their genetic predecessors (21). It may be proposed that the increased growth of "demanding" tissues, such as skeletal muscle, should be accompanied by appropriate adaptations in structure and function of "support" tissues, such as the gastrointestinal tract (38). Indeed, previous studies have demonstrated such adaptations in the small intestine of the highly selected broiler chicken in terms of crypt cell dynamics and enterocyte migration rates (40). Such birds, however, appear to have an intestinal mucosal compartment, which is relatively smaller, compared with

body size, than genetically unselected lines (36). Thus it is proposed that functional adaptations in terms of nutrient absorption at the enterocyte level have supported the increased demands of elevated growth rate (36). In fact, it is now considered that nutrient absorption and adaptations therein may represent the rate-limiting step in further genetic improvements in broiler

chicken growth rates (9).

小肠通过传递和吸收营养与外界环境构成高级动力学界面。肠黏膜能快速而广泛的进行形态学和功能适应性调节来适应进化，生殖，个体发育需要以及环境和营养变化。在饲养家禽中，加强人工选择产生了商业肉仔鸡，它的生长率和食物转化率都显著提高了。这可能是因为需求高的组织的生长增加（比如骨骼肌）应该伴随着适当的基础组织在结构和功能的调节，例如胃肠道。事实上，前期的学习在高度选择的肉鸡小肠隐窝细胞动力学和肠细胞的移动速率证实了这种调节。这些鸟类好像比未经过遗传学选择的有相对较小的肠黏膜间隔。因此它可能是在肠囊肿水平上的营养吸收功能的调节产生了较高的生长速率的要求。事实上，现在认为营养吸收和调节点可能是在进一步提高肉鸡生长率的遗传选择的限速步骤。It is well established that chronic heat stress reduces growth rate and the feed conversion efficiency in broiler chickens (19, 41). While these effects are, in part, attributable to the hyperthermia-induced decrease in food intake, growth depression may also be mediated directly by the associated metabolic and

endocrine responses (29), as indicated by paired feeding studies. Broiler chickens are more susceptible to heat stress than slower growing domestic fowl, and both their adaptive and pathological responses to extended thermal challenge involve multiple organs and systems (1). It is, therefore, possible that both inanition and changes in metabolic and endocrine status will induce adaptive responses in intestinal absorptive function.

有研究报道长期热应激会减少肉鸡生长率和饲料转化效率。从饮食配对组显示，代谢和内分泌可能联合直接介导影响过热导致的采食量减少和生长下降。肉鸡比生长慢的禽类对热应激更敏感，他们对热应激的适应性和病理性反应的调节包括多种器官和系统。因此，代谢和内分泌变化与营养不良会导致小肠吸收功能的适应性反应。In the domestic fowl, the capacity to absorb nutrients depends both on the mucosal surface area of the small and large intestines and on the functional properties of the specific nutrient transporters present in the brush-border and the basolateral membranes (2). There are specific transport systems for the major dietary hexoses. Glucose is absorbed across the apical sodium-dependent glucose transporter 1 (SGLT-1) system, expressed along the small and large intestine (13, 15); fructose is taken up by the apical facilitated glucose transporter (GLUT)-5-type system (16); and both sugars are transported to the interstitial compartment through the basolateral GLUT-2 transporter (15).

在禽类中，吸收营养的能力依赖于小肠、大肠粘膜表面积和特殊营养转运蛋白在刷状缘和基底膜外侧的表达的功能。肉鸡体内有主要饮食己糖的特殊转运系统。葡萄糖依靠在小肠和大肠表达的 SGLT-1系统；果糖通过GLUT-5系统；所有的糖类都通过GLUT-2转运到细胞区室间隙。Previous studies have shown that exposure of chickens to elevated

environmental temperature for 2 wk markedly reduced food intake and that the associated lower growth rate was accompanied by increased in vivo uptake of galactose and methionine when measured on a tissue dry weight basis (33). This apparently enhanced absorption capacity was confirmed in in vitro studies in which enterocytes from chronically heat-adapted birds showed a 50%

increase in galactose accumulation ratio compared with cells from control chickens (34). However, the precise mechanisms of this adaptation and the contributions of reduced food intake associated with heat stress and hyperthermia per se are not clear. The present study was designed to address these areas and specifically to characterize the transport properties of SGLT-1 and GLUT-2 transporters in isolated membrane vesicles from the small intestine of broiler chickens during heat stress adaptation. To distinguish between the adaptive responses attributable to prolonged heat stress per se or to the reduced food intake, our experiment employed paired feedin

前期的研究表明将肉鸡置于高温环境 2周可以减少采食量；当用干组织重为指标时，体内吸收半乳糖和甲硫氨基酸会增加，而生长率会下降。这种明显加强的吸收能力在体内实验（经过长期热应激调节的鸟类肠细胞比对照组鸡的细胞高出 50%的半乳糖堆积量）中被证实。但是，这种调节的精确机制和热应激减少饲料摄入量和体温较高都不清楚。现在的研究的目的是定位这些区域以及明确的表示在热应激调节时期肉鸡小肠中的离体囊泡 SGLT-1 and

GLUT-2载体的运输性质。为了区别调节反应对自身延长热应激或减少摄入量，我们设计了饮食配对组。

MATERIALS AND METHODS：Animal model. Male broiler chickens (Gallus gallus domesticus), 28 days old, were randomized into three experimental groups and maintained in climatic chambers for 2 wk, with free access to water. Birds were fed a diet formulated at the Roslin Institute containing (per kg diet) 183 g crude protein, 43 g lipid, and 398 g carbohydrate;

the metabolizable energy content was 12.35 MJ/kg. The experimental groups were as follows: 1) control ad libitum (CAL), chickens fed ad libitum and exposed to thermoneutral conditions (20°C, 50% relative humidity); 2) heat stress ad libitum (HSAL), chickens fed ad libitum and exposed to heat stress conditions (30°C, 70% relative humidity); and 3) control pair-fed (CPF), chickens exposed to thermoneutral conditions and fed with the same amount of food consumed by the HSAL group. Initially, each experimental group consisted of 12 birds, but natural losses and culls reduced the final numbers in the HSAL and CPF groups. Body weight and rectal temperatures were monitored throughout the 14-day experimental period (between 0900 and 1000 on measurement days to avoid circadian variations).

At 42 days, birds were killed in the morning by cervical dislocation without previous starvation. The jejunum was removed, immediately flushed with ice-cold saline（用冰盐水冲洗空肠）, opened lengthwise, frozen in liquid nitrogen, and then stored at –80°C. The brush-border (BBMV) and basolateral membrane vesicles (BLMV) were prepared according to Garriga et al. (15). Manipulation and experimental procedures were in accordance with Spanish regulations for the use and handling of experimental animals, and the protocol was approved by the Ethical Committees of both the Universitat de Barcelona and the Roslin Institute.

Blood sampling. Blood samples (2.5 ml) were obtained by simple venepuncture静脉穿刺 of the brachial vein from 9–12 birds in each group at 42 days of age. All samples were taken between 0900 and 1000 and placed in heparinized tubes in ice. Plasma samples were immediately obtained by centrifugation at 1,500 g and at 5°C for 10 min and stored at –20°C. Packed cell volume 红细胞容积 or

hematocrit血细胞比容 was determined as described by Maxwell (31).

血糖 Plasma glucose. Plasma concentration of glucose was measured by using a commercially available kit (Wako glucose) obtained from Alpha Laboratories

(Hampshire, UK) and modified for use in an automated plate-reading

spectrophotometer (Titertek 2, Autoflow Laboratories).

左甲状腺纳素和三碘甲状腺原氨酸Thyroxine and tri-iodothyronine. Plasma 甲状腺thyroxine (T4) concentration was measured by radio immunoassay with the use of a commercially available kit (Gamma B T4; IDS, Tyne and Wear, UK). The 化验分析 assay sensitivity was 2.0 ng/ml. Plasma tri-iodothyronine (T3) concentration was measured with the use of a commercially available ELISA assay (T3 Microwell EIA; IDS). The assay sensitivity was 0.4 ng/ml. Both assays were adapted for use with avian plasma, and the standard concentrations were

adjusted appropriately.

升血糖素Glucagon. Plasma glucagon concentration was measured by radio immunoassay (Linco Research), as previously described (4, 18). The sensitivity

of the assay was 20 pg/ml. Because the primary antibody is directed at a mammalian glucagon molecule, the values are expressed as glucagon-like immunoreactivity for 鸟类 avian plasma, as previously described (4, 18).

皮质酮Corticosterone. Plasma corticosterone concentration was determined by radio immunoassay (Gamma B 125I-labeled corticosterone; IDS) following

extraction of the plasma with dichloromethane, as described by Mitchell et al. (35). The assay sensitivity was 0.2 ng/ml.

小肠形态测定和光学显微镜 Intestinal morphometry and light microscopy. For the morphometric study, the entire jejunum was removed and immediately flushed with ice-cold saline. The length was determined as described by Mitjans et al.

(37). For the light microscopy study, two pieces 10 mm long were excised from the proximal and distal ends of the segment. Samples were fixed with Bouin

solution, dehydrated in a graded series of ethanol, and finally embedded in paraffin wax (37). Five sections 10 µm thick were obtained and stained with hematoxylin and eosin. Villous lengths were obtained for at least 10 structures in each section, and the average value was calculated for each chicken; therefore, in RESULTS, "n" refers to the number of animals processed.

电子显微镜 Electron microscopy. Pieces 2 mm long were taken from the jejunum for transmission electron microscopy. Samples were fixed in a mixture of 2.5%

glutaraldehyde and 2% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4, 4°C) for 2 h, as described by Mitjans et al. (37), and then washed in 0.2 mol/l phosphate buffer 磷酸盐缓冲液. The tissues were then processed for routine transverse electron microscopy at the Servei de Microscòpia Electrònica of Serveis Científicotècnics of the Universitat de Barcelona. Quantitative analysis was performed on high-magnification photomicrographs. The lengths of longitudinal sections of microvilli were measured in 20–26 micrographs per chicken group.

Membrane vesicle preparation. BBMV were prepared by MgCl2 precipitation (15). The vesicle suspension was obtained in a medium containing 300 mM mannitol, 0.1 mM MgSO4, 0.41 µM LiN3, and 20 mM HEPES-Tris (pH 7.4), with a protein concentration of 15–25 mg/ml.

BLMV were prepared according to the study by Garriga et al. (14). The vesicles were suspended in a medium containing 300 mM mannitol, 20 mM HEPES-Tris (pH 7.5), 0.1 mM MgSO4, and 0.41 µM LiN3 with a protein concentration of 15–20 mg/ml.

Enzyme and protein determinations. The activity of the ouabain-sensitive Na+-K+-activated ATPase (Na+-K+-ATPase, EC 3.6.1.3 [EC] ) was routinely assayed as a marker of the basolateral membrane following the study by Colas and Maroux (8). Sucrase ( -D-glucohydrolase, EC 3.2.1.48 [EC] ), the marker of the brush-border membrane, was assayed according to the study by Messer and Dahlqvist (32). The protein content was determined by using the Coomassie brilliant blue method with bovine serum albumin as standard (5).

Transport assays. The uptake of -methyl-D-glucoside ( Glc1Me) and D-

glucose (D-Glc) was measured at 37°C by a rapid filtration 速率 technique (15). In the BBMV studies, Glc1Me is used because it is specific for the

apical SGLT-1 isoform 亚型, thus avoiding the influence of any

basolateral membrane contamination. For the kinetic studies of the

basolateral GLUT-2 isoform, we used D-Glc in the absence of Na+ to avoid any influence of apical contamination. The assays of Glc1Me transport were performed in short-circuit conditions by loading the vesicles (30 min, 37°C) in a medium containing 200 mM mannitol, 50 mM KCl, 20 mM HEPES-Tris (pH 7.4), 0.1 mM MgSO4, and 0.41 µM LiN3. Valinomycin was added to the incubation medium at a final concentration of 45 µM to render the vesicles permeable to K+. The substrate concentrations used for the kinetic analysis of Glc1Me uptake by BBMV were 0.01, 0.05, 0.075, 0.1, 1, 10, 25, 50, and 75 mM. For the kinetic analysis of D-Glc uptake by the basolateral membrane, vesicles were incubated with 0.01, 0.5, 1, 5, 15, 50, 100, 150, and 200

mM D-Glc. The osmolality of intra- and extravesicular media was kept constant at 320 osmol/kg by adjusting the total sugar concentration with mannitol.

Binding measurements. Steady-state phlorizin binding was assayed at 37°C, as described in a previous work (15). The density of specific phlorizin binding sites is expressed in picomoles of phlorizin bound per milligram of protein at 50 µM phlorizin (B50).

Steady-state cytochalasin B binding was assayed at 37°C by the method described by Cheeseman and Maenz (7) with some modifications (15). The density of specific cytochalasin B binding sites was expressed in picomoles of cytochalasin B bound per milligram of protein at 1 µM cytochalasin B concentration (B1).

Western blot analysis of SGLT-1. SGLT-1 protein abundance in jejunal BBMV was measured by using Western blot analysis, as previously described (17). Blots were incubated with a rabbit polyclonal antibody raised against the synthetic peptide, corresponding to amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 at a 1:5,000 dilution for 16 h at 4°C. In simultaneous experiments,

nitrocellulose membranes were incubated with the same antibody

preadsorbed with the antigenic peptide (1 g/l). Hybridization bands were quantified by scanning densitometry.

Chemicals. All unlabeled reagents were obtained from Sigma-Aldrich (St. Louis, MO). -Methyl-D-[14C]glucoside (specific activity 265 mCi/mmol), D-[U-14C]glucose (specific activity 251 mCi/mmol), [3H]phlorizin (specific activity 46 Ci/mmol), and [3H]cytochalasin B (specific activity 15 Ci/mmol) were purchased from New England Nuclear Research Products (Dreieich, Germany). The final activity of labeled substrates in the incubation media was 0.5–2 µCi/ml.

Kinetic analysis. Total Glc1Me and D-Glc fluxes from five independent experiments were analyzed by nonlinear regression by using the Biosoft Enzfitter program (Cambridge, UK). As the errors associated

with experimental fluxes were roughly proportional to their values, it was considered appropriate to apply proportional weighting to the data.

Statistical analysis. Kinetic constants and binding measurements were analyzed by ANOVA using the SPSS-10 software (SPSS, Chicago, IL).

RESULTS： Animal model. Table 1 summarizes the main characteristics of the animal model. Body

weight gain throughout the experimental period was higher (13% increase; P < 0.05) in CAL than in CPF or HSAL chickens. There were no significant differences in final body weight between the CPF and HSAL groups. Food intake was reduced 21% (P < 0.05) in the raised-temperature condition. High environmental temperature significantly increased the rectal temperature in HSAL chickens (1.5–1.8°C higher than the groups kept in thermoneutrality) already from the second day in the climatic chamber and remained stable thereafter.

表一表述了动物模型的主要特征。对照组在实验期的体增重显著大于高温组和采食量配对组。高温组和采食量配对组间体增重没有显著差异。高温组的采食量降低了 21%。高温从第二天到最后一天显著增加了高温组的直肠温度。No effect was observed on blood hematocrit (Table 1). The serum glucose concentration was not affected by food restriction but showed a small but significant increase in the HSAL group compared with CAL animals. Hormone determination in plasma showed that chronic heat stress induces a significant increase in plasma corticosterone (90% increase compared with birds kept on thermoneutral conditions) and a reduction in circulating T3 and T4 concentration

(52 and 37%, respectively). The concentration of glucagon was higher in CPF than in CAL chickens, with values in the HSAL group that were in between the controls, without statistical differences. The activity of mucosal enzymes sucrase and Na+-K+-ATPase was the same in all experimental groups。血细胞比容没有显著影响。控制采食量没有影响血糖浓度，但高温组比常温组的血糖浓度有明显的上升。对血浆中荷尔蒙的测定显示长期热应激导致显著的血浆皮质酮浓度上升以及T3、T4 浓度的显著降低。采食量配对组的血糖浓度比对照组的血糖浓度高，高温处理组处于两组中间，没有统计学差异。

Morphometric and ultrastructural studies. The jejunum of animals exposed to heat stress showed a 22% reduction in fresh weight and a 5% reduction in length (see Table 2). These results should be attributed to an effect of high environmental temperature because the pair-fed animals showed values similar

to those of controls (CAL group).

形态测定和超微结构研究：经过热应激的空肠鲜重减少 22%长度减少 5% 。这种现象应归结于高温，因为采食量配对组与对照组相似。The microscopic study of the villous length was performed at the two ends of the jejunum to obtain information from both proximal and distal regions. In the proximal region, there were no differences between villous lengths from all groups. In contrast, in the distal jejunum, the villous length was different in each experimental group: maximum in CAL chickens, medium in CPF birds, and minimum for the HSAL group (Table 2), compatible with the changes observed in jejunum fresh weight. Results from the electron microscopy study showed that microvillous length was higher in CPF and HSAL groups than in CAL chickens (Fig. 1), indicating that food restriction can stimulate microvillous growth.

绒毛长度的微观研究在空肠的两端进行，为了获得近侧区和远侧区的数据。在近侧区，绒毛的高度没有差异。在远侧区，对照组的绒毛高度最长，采食量配对组次之，高温组最低， 与肠道的鲜重和长度一致。电子显微镜观察显示采食量配对组和高温组比对照组微绒毛膜长度长，显示限饲可以刺激微绒毛膜的增长。Characterization of the membrane vesicles. The membrane purity, vesicular orientation, and intravesicular volume of both BBMV and BLMV were determined. In the final BBMV preparation, sucrase activity was 11-fold, and the overall recovery was > 81% (n = 15), without significant differences between

experimental groups. The activity of the basolateral marker Na+-K+-ATPase was 0.8-fold, indicating that the basolateral contamination was negligible. The intravesicular volume, calculated under equilibrium conditions using 0.1 mM Glc1Me, was 0.80 ± 0.09 µl/mg protein, similar to previous results (14).囊泡特征：细胞膜、囊泡、刷状缘膜囊和BLMV中的 intravesicular volume。在刷状缘膜囊的后期准备中， 蔗糖酶活性没有显著差异。基底膜外侧的钠钾 ATP 酶 ，表明基底膜外侧的污染是可以忽略的。 也没有变化。

In BLMV, the enrichment factor for the Na+-K+-ATPase activity was also high (10-fold), and the overall recovery was 84% (n = 15), without significant differences between the three experimental groups. The activity of apical sucrase was 0.9-fold. The intravesicular volume, calculated under equilibrium conditions using 1 mM D-Glc, was 2.10 ± 0.33 µl/mg protein.

在BLMV中，钠钾 ATP 酶的活性较高，总回收率是 84% ，在实验组间没有差异。绒毛尖端蔗糖酶的活性是 0.9-fold。

Transport of Glc1Me across BBMV. The calculated kinetic constants are shown in Table 3. There are no significant differences between the diffusion and Michaelis constants of any experimental group. However, the D-Glc maximal transport rate of the HSAL animals was increased by 50% compared with the control groups (CAL and CPF). Table 3 also shows that the specific binding of phlorizin to SGLT-1 is also increased by 60% in the heat-stressed birds. The calculated kinetic constants are shown in Table 3. There are no significant differences between the diffusion and Michaelis constants of any experimental group.

在刷状缘膜囊上的运输。动力学常数显示在表 3中。实验组的米氏常数和扩散没有显著差异。热应激组的D-Glc的最大传输速率比对照组提高了 50% 表 3 显示 sglt-1的特异性结合在热应激组中提高了 60%

Transport of D-Glc across BLMV. In the basolateral membrane, calculation of the kinetic constants from D-Glc uptake showed that there are no statistical differences between groups (Table 3), indicating that chronic high environmental temperature does not affect basolateral hexose transport. The

results of specific binding of cytochalasin B confirmed that the number of GLUT-2 transporters is not affected by chronic heat stress.D-GLC在BLMV中的运输。在基底膜外侧，D-GLC动力学常数的计算在处理组间没有显著差异，表明长期热应激不会影响底外侧的己糖运输。细胞松弛素 b 特异性结合证实热应激对GLUT-2没有显著影响。

SGLT-1 immunoblots. The antibody recognized a single band of 75 kDa in BBMV from all groups that could be blocked by preadsorption with antigenic peptide (Fig. 2). The densitometric analysis indicated that SGLT-1 abundance was increased by 60 ± 13% in HSAL chickens (p < 0.05) compared with CAL chickens.

SGLT-1 免疫印迹，抗体验证了一条 75KDa可以被抗荧光源钛阻断的在所有组中 bbmv上。密度计分析显示 SGLT-1在热应激组中比对照组多 60%

DISCUSSION：Chickens maintained on a high environmental temperature (HSAL group) for 2 wk showed increased rectal temperature and decreased body weight. The reduction in body mass was probably due to decreased food intake, because similar final weight values were observed in the CPF group. On the day

of death, the fresh weight of the small intestine in the HSAL chickens was lower (in absolute value, as well as relative to body weight), and the jejunum was shorter than in the CPF animals. This indicates that, in addition to the effects of food restriction (i.e., lower intestinal growth proportional to body weight reduction), high temperature further reduces intestinal fresh weight, consistent with the observations of Mitchell and Carlisle (33) and with the hypothesis that thermal loads depress enterocyte proliferation and growth (27).

在高温中处理 2周的鸡直肠温度升高，体重下降。体重的减少可能是因为采食量的减少，因为相似的结果在采食量配对组也出现了。在杀死鸡的时候，热应激组与采食量配对组相比小肠的鲜重减少，空肠的长度减少。这可能暗示，除此以外，限饲效应、高温进一步降低小肠鲜重，与Mitchell and Carlisle的观察一致，与热应激减少肠细胞增生的假设一致。At the end of the experimental period, HSAL chickens had reduced plasma T3 and increased plasma corticosterone, consistent with the hormonal profile described for the model of heat stress adaptation (19). A similar pattern of responses was described for heat-acclimated desert rodents, that is, reduced intestinal weight and a large decrease in plasma T3 concentration (46). Our results also show that the plasma concentration of T3 in HSAL chickens was lower than in CPF and CAL groups, supporting the view that caloric restriction does not alter thyroid hormone metabolism (19). Acute heat exposure also induces a transient decrease in plasma T3 and a reduction of food intake in young chicks (44). Since thyroid hormones have trophic effects stimulating the growth of the intestinal mucosa (28), we suggest that functional hypothyroidism mediates the reduction of jejunal mass and villous height in the HSAL chickens.

在实验结束时，热应激组鸡血浆中的 T3减少，皮质酮含量增加。与热应激调节模型中描述的激素一致。一个相似的反应模型在热适应的沙漠鼠类上建立，减少小肠重量和血浆中 T3的大量减少。我们的研究结果也显示热应激组鸡血浆中 T3的含量比对照组和采食量配对组都低，支持热限制不会改变甲状腺激素新陈代谢的观点。激烈的热暴露会减少低日龄鸡血浆中 T3 含量和采食量。因为甲状腺激素有营养性刺激肠黏膜生长的功能，我们假设甲状腺功能减退会降低热应激组鸡上空肠长度和绒毛高度。Acclimation to a warm environment did not affect either the passive hexose permeability or the affinity constant in mucosal enzyme markers, indicating that the metabolic changes associated with heat stress do not affect basic functions

of the membrane. The study of the effects of heat stress adaptation on the hexose transport properties in the chicken intestine showed that the capacity to take up hexose across the apical SGLT-1-type transporter was increased, consistent with previous results (33) and with observations from rodents (46), while the kinetic properties of the basolateral GLUT-2-type transporter remained unchanged. These observations were strengthened by the results of binding and Western blot analysis, both confirming that heat stress increases the number of hexose transporters in the apical membrane only. In rodents, diets rich in free simple sugars (glucose, fructose) stimulated the transient recruitment of GLUT-2 by the brush-border membrane (20, 26). The possibility that GLUT-2was expressed in the chicken apical membrane of the jejunum was not addressed in the present study, but this is considered unlikely because our chickens were fed a diet with low concentrations of free sugars. However, apical SGLT-1 kinetics would not be altered by the presence of GLUT-2, because all transport experiments in BBMV were done using Glc1Me, a glucose analog that is selective for SGLT-1 (14).

适应热环境不会对被动的己糖通透性和小肠粘膜亲和常数有影响，显示热应激导致的代谢变化不会影响细胞膜的基本功能。研究热应激对调节己糖运输性质在鸡小肠显示 SGLT-1转运载体通过绒毛顶端吸收己糖的能力加强了，基底膜外侧的GLUT-2载体没有变化，与以前的实验结果和鼠类的观察一致。这些结果被 western blot加强，两者都证实热应激只在绒毛顶端增加己糖转运载体。在啮齿类，没有糖类的饮食刺激可以短暂的产生刷状缘膜GLUT-2 聚集反应。现在的研究没有证明 GLUT-2在鸡空肠绒毛顶端表达，但这个观点不太可能，因为我们的鸡饲喂的是 。无论如何，绒毛顶端的 SGLT-1动力学不会被 GLUT-2的表达改变因为所有的BBMV上的转运实验都是用的 Glc1Me,是 SGLT-1的选择性葡萄糖类似物。Previous studies have shown that elevated environmental temperature

increases galactose and methionine uptake in perfused intestine in vivo (33). One signal mediating these effects could be glucagon, as chronic glucagon administration enhances glucose and galactose transport in the rat jejunum in vitro (42), as also happens in our HSAL condition. However, the CPF chickens, albeit with increased plasma glucagon concentration, have an intestinal SGLT-1 expression similar to that of the CAL group, indicating that other signals may be involved in the heat stress response. The effects on nutrient uptake do not seem to be related to changes in circulating T3 either, because this hormone has the

opposite effects as it stimulates brush-border D-Glc Vmax by increasing SGLT-1 activity (10). Therefore, we can conclude that upregulation of SGLT-1 by heat stress is apparently mediated by signals other than thyroid hormones and

glucagon. A plausible candidate to mediate these effects is corticosterone, especially considering that its plasma concentration in the HSAL group is twofold that of the controls. Glucocorticoids play a key role in glucose metabolism, including the trafficking of GLUT-4 to the plasma membrane of hepatocytes (39). They can also regulate glucose transport in intestinal epithelial cells; for example, dexamethasone induces acute expression of SGLT-1 in the rabbit small intestine (23), and Dieter et al. (11) have demonstrated that glucocorticoids induce the expression of serum and glucocorticoid-regulated kinase 1, which enhances glucose transport by increasing SGLT-1 abundance in the cell membrane.

前期的研究显示提高环境温度增加半乳糖和甲硫氨酸在小肠内的吸收。胰高血糖素可能是一个调节信号，因为长期服用胰高血糖素可以加强葡萄糖和半乳糖在小鼠空肠内的转运，在本实验热应激组中也有相同情况。在采食量配对组鸡中，虽然血浆中胰高血糖素含量升高了，但 SGLT-1 的表达与对照组没有差异，显示可能有其他因素参与热应激反应。T3的含量变化可能不影响营养吸收，因为这种激素可以提高刷状缘膜D_GLC上Vmax与提高 SGLT-1活性作用相反。因此，我们可以推论热应激提高 SGLT-1的信号中没有甲状腺激素和胰高血糖素。皮质酮可能是一个信号，特别是当考虑到热应激组中它的含量是对照组的两倍。糖皮质激素在葡萄糖代谢中起关键作用。它也能调节小肠上皮细胞中葡萄糖转运，例如，地塞米松导致强烈增加兔子小肠中的 SGLT-1的表达，Dieter 等指出糖皮质激素导致糖皮质激素调节激酶 1的表达，它们可以通过增加 SGLT-1在细胞膜中的含量而加强葡萄糖的转运。The higher cumulative capacity due to increased number of SGLT-1 transporters will result in stimulation of net transepithelial transport, because the low-affinity/high-capacity kinetic properties of GLUT-2 enable efficient transfer of D-Glc, even at high cytosolic glucose concentrations (2). The HSAL group shows a significant hyperglycemia, an effect repeatedly observed in chickens (33, 43) and rats (6) under chronic heat stress. This effect of heat stress can be attributed to shorter intestinal transit times (43), which will extend the time that nutrients are exposed to the mucosal absorptive epithelium; to increased apical SGLT-1 activity (33; and the present study), which will result in higher rates of absorption; or to increased gluconeogenesis due to enhanced plasma corticosterone, mainly from muscle tissue proteins (3).

SGLT-1转运能力的增加会导致净跨上皮转运的增加，因为GLUT_2的动力学特性低亲和力和高容量使 d_glc高效率转运，甚至是在高细胞溶质葡萄糖浓度中。热应激组有显著的高血糖，我们在小鼠和鸡中反复观测到这一点。热应激的作用可以归结于减少了小肠的过渡期，这个时期可以增长营养在粘膜吸收上皮的接触时间，可以增加 SGLT-1的活性，SGLT-1可以因皮质酮的增加而增加糖异生，主要是从肌肉组织蛋白。At high ambient temperatures, there is a decrease in protein synthesis (19), probably due to reduced plasma amino acid concentration and to lower energy supply (41), as observed in broiler chicken muscle tissue. In addition, heat stress decreases plasma T3 concentration and increased plasma corticosterone, both changes known to reduce protein deposition through alterations in protein

turnover in birds and other species (47). These effects reduce the total muscle mass, as reflected by the body weight decrease, but have little repercussion in the intestine (just a 5% shortening in the jejunum and a small decrease in villous length, restricted to the distal region). Interestingly, the two groups with lower food intake (CPF and HSAL) show increased microvillous length, indicating that the surface brush-border membrane is increased even in a metabolic situation characterized by a general reduction in protein synthesis (19). This effect on apical surface, together with increased activity of SGLT-1, enhances the capacity to absorb D-Glc and can, therefore, be interpreted as physiological adaptations of the chicken jejunum to guarantee energy supply.

在环境温度高时，蛋白质合成下降可能是因为血浆中氨基酸含量减少和供能减少，这些在肉鸡肌肉组织中观察到了。此外，热应激减少 T3 含量和增加皮质酮血浆含量，这两种变化都可以在肉鸡和其他物种的蛋白质转化减少蛋白沉积。这些变化减少了总肌肉量，反应到体重减少上，但小肠中减少量较小。有趣的是，采食量较低的 2个组显示微绒毛的长度增加，暗示刷状缘膜表面细胞量即使在蛋白质合成下降的代谢状态下也会升高。这种情况和 SGLT-1的活性增加一起增加 D-Glc的吸收，而且能解释为肉鸡空肠保证能量供给的生物学调节。The effects of elevated environmental temperatures can be compensated by the kind of adaptations. First, there is a heat acclimation response, an autonomically controlled array of physiological mechanisms that involve reduced metabolic rate, lower temperature thresholds for activation of heat dissipation effectors, and an increased capacity of the evaporative cooling system (22). Second, there is a rapid heat shock response, involving the

synthesis of several families of heat shock proteins of different molecular weights (30). On the one hand, the role of heat shock proteins in birds in the protection of cells from heat stress is well established (45); on the other hand, Garriga et al. (17) have suggested that cytokines induced by administration of Staphyloccocus aureus enterotoxin B are involved in the regulation of SGLT-1 expression in the brush-border membrane of rat small intestine. Furthermore, Ikari et al. (24) have reported that heat shock stress elevated SGLT-1 activity via production of cytokine transforming growth factor- 1 in LLC-PK1 cells. Because

transforming growth factor- 1 and other cytokines can stimulate serum and glucocorticoid-regulated kinase 1 expression (11), this kinase could be a common regulatory step for both local (cytokine) and general (corticosterone) mediators of SGLT-1 expression during heat stress.

增加环境温度的影响可以被各种调节补偿。首先，有一个高温适应反应，是一个自主控制的生理机制，包括减少的代谢速率、散热损失效应活性较低的阈值、蒸发散热系统的加强。第二，有快速热激反应，包括不同分子量的几种热休克蛋白的合成。一方面，热休克蛋白在鸟类中被证实可以在热应激中保护细胞。另一方面，Garriga 等提出，肠霉素B与 SGLT-1的表达调节有关。而且，Ikari 等报道过热应激提高 SGLT-1活性是通过细胞因子转化因子 B在 LLC-

PK。因为生长因子 B与其他细胞因子可以刺激血清和糖皮质激素调节激酶的表达，这种激酶在细胞因子和皮质酮调节中是一个普遍的调节步骤。We conclude that the increased apical hexose transport capacity in chickens maintained on a high thermal load is entirely dependent on adaptations of SGLT-1 expression to heat stress and not on reduced food intake.

我们得出结论，绒毛顶端由高热负荷增加的己糖运输能力完全依赖与 SGLT-1表达的调节，而不是采食量的减少。

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