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Article

Metabolic Differences between Genetically Lean and Fat Chickens Are Partly Attributed to the Alteration of Insulin Signaling in Liver¹

Endocrinologie Moléculaire et Cellulaire du Métabolisme, Station de Recherches Avicoles, Institut National de la Recherche Agronomique, 37380 Nouzilly, France

²To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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

KEY WORDS: • insulin receptor • insulin receptor substrates • tyrosine phosphorylation • chickens • signal transduction • obesity • insulin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The recent cloning of the chicken insulin receptor substrate-1 gene [(IRS-1)³ ; Taouis et al. 1996 ] permits investigation of insulin signaling in the chicken, an insulin-resistant species with a plasma glucose level of 2 g/L and a weak hypoglycemic effect of exogenous insulin (Simon and Taouis 1993 ). We recently showed that in chicken liver, the tyrosine phosphorylation of insulin receptor (IR) and IRS-1 is regulated by the nutritional state (Dupont et al. 1998a ). Compared to the fed state, prolonged starvation (48 h) decreased, whereas refeeding restored, their levels of phosphorylation. Surprisingly, in the same conditions, phosphorylation of muscle IR and IRS-1 was not altered, which remains unexplained. This demonstrates that the first steps of the insulin signaling cascade are present in chickens and are regulated, at least in the liver. During these studies, Src homology collagen protein (Shc) was also found to be phosphorylated in the liver on tyrosine residues, and the level of its phosphorylation was dependent on the animal's nutritional state (Dupont et al. 1998b ). Furthermore, the presence of a large complex involving IR, IRS-1, Shc (mostly the 52 kDa isoform) and a phosphatidyl inositol (PI) 3'-kinase activity was observed in chicken liver and muscle.

In the present study, we compared the phosphorylation of IRS-1 and Shc in the liver and muscle of genetically fat (FL) and lean (LL) chickens. In birds, the liver is the main organ of lipogenesis (Leveille et al. 1975 ). FL and LL chickens were obtained by divergent selection for abdominal fat pad size in males at 9 wk of age while maintaining similar body weight (Leclercq et al. 1980 ). Food intake, basal energy requirement, digestive use of dietary energy and body temperature are constant and cannot account for differences in fattening (Leclercq 1988 , Simon 1988 ). Therefore, a metabolic change leading to a different balance for nutrient use is involved. In the starved state (overnight, ~16 h) or the fed state, FL chickens exhibit lower plasma glucose and normal or slightly increased plasma insulin levels. During a glucose tolerance test, FL chickens exhibit higher plasma insulin levels that are not accounted for by insulin resistance as it is in obese mammals. Paradoxically, FL chickens that are starved overnight are in fact more sensitive to exogenous insulin when considering the hypoglycemic response (Saadoun et al. 1988 ). After ad libitum refeeding following overnight starvation, both plasma insulin and glucose levels are lower in FL chickens. In isolated perfused pancreas, the amount of insulin released during the first phase in response to glucose (42 mmol/L) is lower in FL chickens, which remains unexplained (Rideau et al. 1986 ). Plasma levels of counter-regulatory hormones (glucagon, growth hormone and corticosterone) as well as the insulin/glucagon ratio which varies between 1 (starved state) and 3 (fed state) are very similar in both lines (Leclercq et al. 1988b ). The tri- (T3) or tetraiodothyronine (T4) balance is also very close (Leclercq et al. 1988a ). As a whole, this suggests that a change in the glucose-insulin relationship is the most likely causal key mechanism accounting for the divergence between the two lines (Simon 1988 ). Presently, insulin signaling was compared in FL and LL chickens by studying liver and muscle tyrosine phosphorylation of IR, IRS-1 and Shc in three nutritional states: feeding, prolonged starvation and refeeding.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chemicals.

¹²⁵I was purchased from Amersham France (Les Ulis, France). (³²P) deoxy cytosine triphosphate (dCTP) and (³³P) ATP were obtained from Isotopchim (Peyruis, France). Bovine serum albumin (BSA, fraction V radioimmunoassay grade), phenylmethylsulfonylfluoride (PMSF), leupeptin, aprotinin, protein A agarose and phosphatidylinositol (PI) were purchased from Sigma Chemical (St Louis, MO). Triton X-100, SDS and nitrocellulose membrane were obtained from Bio-Rad Laboratories (Richmond, CA). Protogel was from National Diagnostic (Atlanta, GA). Silica TLC plates were obtained from Carlo Erba (Paris, France). Nylon membrane (Hybond-N⁺) was purchased from Amersham. Quick Spin purification columns sephadex G25 were obtained from Boehringer Mannheim (Mannheim, Germany). QuickPrep Total RNA Extraction Kit, Taq DNA polymerase and deoxyribonucleotides (dNTP) were purchased from Pharmacia (France). Avian Myeloblastosis Virus Reverse Transcriptase (AMV) and RNase ribonuclease inhibitor(rRNasin) were obtained from Oncor-Appligene (Illkirch, France). Random hexamer primers were purchased from Promega (France). Anti-rat IRS-1 (IRS-1), anti-mouse Shc (Shc) and monoclonal anti-phosphotyrosine (PY20) antibodies were obtained from Transduction Laboratories (Lexington, KY). Primers used for reverse transcription-polymerase chain reaction (RT-PCR) were provided by Genosys (Cambridge, UK).

Animals.

Nine-week-old male FL and LL chickens were subjected to nutritional conditions that produce marked changes in the plasma insulin and glucose levels [fed, starved (48 h) and refed for 30 min following 48 h starvation]. The chickens were killed by cervical dislocation; livers and thigh muscles were removed, quickly frozen and powdered in liquid nitrogen, then stored at -80°C.

Animals were treated according to French legislation.

Determination of plasma glucose and insulin levels.

Plasma glucose levels were measured by the glucose oxidase method (Glucose Beckman Analyser 2, Palo Alto, CA) and plasma insulin levels by a radioimmunoassay with a guinea pig anti-porcine insulin antibody (Ab 27–6, generously provided by Dr. G. Rosselin, Hopital Saint-Antoine, Paris) using chicken insulin as the standard (Simon et al. 1974 ).

Insulin binding to liver membranes.

Crude liver membranes were prepared by differential centrifugation as previously described (Havrankova et al. 1978 ). Insulin binding to liver membranes was measured in 0.15 mL of Krebs-Ringer phosphate buffer containing 10 g/L BSA and 1 g bacitracin/L using 0.1 µg ¹²⁵I-porcine insulin/L and 0.4 g membrane protein/L (final concentration) (Lowry et al. 1951 ). Tracer binding was inhibited by increasing concentrations of unlabeled monocomponent porcine insulin (Novo, Copenhagen, Denmark). After 16 h incubation at 4°C, the reaction was stopped by centrifugation at 12000 x g for 3 min. The resulting pellets were washed and incorporated radioactivity was counted. Nonspecific binding was determined in the presence of an excess of unlabeled insulin (13.3 g/L) and was found to be 18–25% of total binding.

Determination of insulin receptor substrate-1 or Src homolgy and collagen protein.

The amount of IRS-1 or Shc protein was determined by immunoblotting with an IRS-1 or Shc antibody, respectively. Liver or muscle protein lysates were prepared from samples of frozen powdered tissues. As previously described (Taouis et al. 1994 ), tissues (1 g) were homogenized on ice with an ultraturax homogeneizer in buffer A containing 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 0.5% NP 40, protease inhibitors (2 mmol/L PMSF, 10 mg leupeptin/mL, 10 mg aprotinin/mL) and phosphatase inhibitors (100 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 2 mmol/L sodium orthovanadate). Homogenates were centrifuged at 600 x g for 20 min at 4°C, then the supernatants were ultracentrifuged for 45 min at 150,000 x g. The protein concentration of the supernatants was determined by using the Bradford dye binding assay kit (Bio-Rad). Aliquots of liver supernatants (125 µg protein) were separated by SDS-PAGE (7.5%), followed by electrotransfer to a nitrocellulose membrane. Membranes were blocked in Buffer B (Tris 10 mmol/L, pH 7.5, containing 100 mmol/L NaCl and 0.01% Tween 20) plus 5% nonfat dried milk for 1 h at room temperature followed by the addition of IRS-1 or Shc (1:200) overnight at 4°C. After removal of unbound antibody, the membranes were incubated with horse-radish peroxidase conjugated anti-rabbit IgG for 1 h followed by washing for 6 x 5 min in blocking buffer without milk. Detection was accomplished by using enhanced chemiluminescence (Levy-Toledano et al. 1994 , Taouis et al. 1994 ). Band densities were estimated by using the NIH Image Software version 1.6 (Chow 1994, Division of computer research and technology, NIH, Bethesda MD).

Measurement of IRS-1 mRNA expression by quantitative RT-PCR.

Total RNAs were extracted from the liver or muscle with the Quick Prep Total RNA extraction kit by following the manufacturer's instructions. Samples were subjected to RT-PCR as previously described (Taouis et al. 1996 ). Briefly, 1 µg total RNA was reverse transcribed by AMV reverse transcriptase (15 U) in the presence of random hexamers (1 g/L) as primer. RT was carried out in the presence of MgCl₂ (25 mmol/L), dNTP mixture (10 mmol/L) and rRNasin. The RT reaction was assessed at 42°C for 60 min followed by an incubation at 95°C for 5 min. RT products were then subjected to PCR in standard conditions (35 cycles), in the presence of two pairs of primers specific to chicken IRS-1: sense, ^5'GCCCGGCCCACGAGGCTG^3' (2630–2648), and antisense, ^3'GTACGCTTGTCCGTAACG^5' (3120–3102), flanking a 490 bp region. As an internal control, the same RT products were also subjected to PCR in the presence of a second pair of primers specific to mouse 18S RNA: sense, ^5'CTGCCCTATCAACTTTCG^3', and antisense, ^5'CATTATTCCTAGCTGCGG^3', flanking a 515 bp fragment. RT-PCR products were analyzed by an agarose gel (1%) electrophoresis, stained with ethidium bromide (1 g/L), then transferred to a nylon membrane by capillarity overnight and immobilized by exposure to UV light. Briefly, the nylon membrane was prehybridized for 16 h at 55°C in a buffer containing 50% (v/v) formamide, 5XSSC (1XSSC = 0.15 mol/L sodium chloride, 0.015 mol/L sodium citrate), 50% 10X Denhardts, 1% SDS and 50 mmol/L Tris HCl (pH 7.5). The cDNA probes coding for either chicken IRS-1 (2630–3120) or mouse 18S RNA (515 bp) (25–50 ng) were labeled with (³²P) dCTP by a rediprime labeling kit (Amersham) and used for Southern blotting. Hybridized radioactivity was measured by using a Storm apparatus (Molecular Dynamic, Paris, France). Autoradiography was also carried out at -70°C for 12 h. The integrity and the quantification of the IRS-1 RNA transcript were assessed by using the 18S RNA transcript. The ratio of amplified IRS-1/18S cDNA was then determined.

Tyrosyl phosphorylation of insulin receptor, insulin receptor substrate-1 and Src homolgy and collagen protein.

Liver and muscle lysates were prepared as described earlier. Solubilized proteins (5 mg) were immunoprecipitated with PY20 at 1:200 dilution for 16 h at 4°C. The immune complexes were precipitated by the addition of protein A agarose beads for 1 h at 4°C, as previously described (Levy-Toledano et al. 1994 , Taouis et al. 1994 ). After two sequential washes with diluted buffer A (1/2), the resulting pellets were resuspended in Laemmli buffer containing 80 mmol/L dithiothreitol. Following SDS-PAGE and electrotransfer, to assess the phosphorylation of IR ß-subunit, blots were incubated for 16 h at 4°C in buffer B plus 5% BSA with PY20 (1:1000), as previously described (Dupont et al. 1998a ). To detect phosphotyrosine IRS-1 and Shc, other blots were incubated for 16 h at 4°C in buffer B plus 5% nonfat, dried milk with IRS-1 and Shc, respectively. After several washings, membranes were incubated with horse-radish peroxidase conjugated anti-rabbit or anti-mouse IgG for 1 h. Membranes were washed again and after detection by using enhanced chemilumuniscence (ECL), bands corresponding to phosphorylated IR ß-subunit, IRS-1 and Shc were quantitated by using NIH Image Software.

Phosphatidyl inositol 3'-kinase assay.

PI 3'-kinase was determined as previously described (Taouis et al. 1994 ). Briefly, livers were homogenized on ice in extraction buffer B composed of 20 mmol/L Tris (pH 7.5), 137 mmol/L NaCl, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 150 mmol/L Na₃V0₄, 1% Nonidet P-40, 10% glycerol, 2 mmol/L PMSF, 10 mg aprotinin/mL in PBS. After centrifugation for 35 min at 40,000 x g at 4°C, equal protein amounts (5 mg) from supernatants were immunoprecipitated overnight at 4°C with PY20 (1/200). Immunoprecipitates were collected with protein A-agarose beads and washed successively once in PBS containing 1% Nonidet P-40 and 100 µmol/L Na₃VO₄; twice in 100 mmol/L Tris-HCl (pH 7.5), 500 mmol/L LiCl₂, 100 µmol/L Na₃V0₄; and once in 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 100 µmol/L Na₃V0₄. The pellet was resuspended in 40 µL of a buffer containing 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA. To each tube was added 10 µL 100 mmol/L MnCl₂ and 20 µg phosphatidylinositol. The reaction was started by the addition of 10 µL ATP (440 µmol/L) containing 10⁶ Bq of [-³³P]ATP, as previously described. After 10 min at room temperature, the reaction was stopped by the addition of 20 µL HCl (8 N) and 160 µL of chloroform: methanol (1:1). After centrifugation (3000 x g, 4 min at 4°C), the organic phase was extracted and applied to a silica gel thin layer chromatography plate. Thin layer chromatography plates were developed in CHCl₃/CH₃OH/H₂O/NH₄OH (120:94:22.6:4), dried and visualized by autoradiography. The radioactivity was quantitated with a Storm apparatus (Molecular Dynamics).

Statistical analysis.

Differences between the LL and FL lines were determined within in each nutritional state by using a Student's t-test. Data are presented as mean ± SEM and P < 0.05 was considered significant. For Table 1 , a two-way ANOVA test was used (Super ANOVA Software, Abacus Concepts, Berkeley, CA).

	RESULTS

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Table 1 shows that body weight, insulinemia and glycemia were significantly affected by the genotype and nutritional status. In addition, the ANOVA showed a clear interaction of the genotype and the nutritional status for body weight (P < 0.01), glycemia (P < 0.05) and insulinemia (P < 0.01). This indicates that FL and LL chickens reacted differently to nutritional status changes (Table 1) .

Insulin signaling in liver. Effect of nutritional states on liver IR number and tyrosine phosphorylation in LL and FL chickens.

Specific tracer insulin binding, as a percentage of total radioactivity, did not differ between genotypes and significantly increased in the starved state in both lines (P < 0.05, Fig. 1 ). Figure 1 shows that neither nutritional state nor genotypes affected IR affinity, as estimated by the concentration of unlabeled insulin that was required to inhibit tracer binding by 50%. Liver membrane insulin receptor number did not differ between genotypes and was similarly enhanced after 48 h starvation (Fig. 1) .

View larger version (28K): [in this window] [in a new window]   Figure 1. Competitive inhibition of 125I-labeled porcine insulin binding to liver membranes from chickens subjected to various nutritional states. Liver membranes were prepared from chickens that were starved for 48 h, fed or refed for 30 min after 48-h starvation. (A) Crude membranes were incubated in presence of labeled insulin (0.1 µg/L) for 16 h at 4°C in the absence or presence of increasing concentrations of unlabeled porcine insulin. (B) Percentage of maximal specific binding of 125I-insulin was determined and plotted against different unlabelled insulin concentrations. Upper panel: specific binding of labeled insulin (B) to lean or to fat line was expressed as percentage of total radioactivity added (T). Data were presented as means ± SEM; n = 4.

View larger version (30K): [in this window] [in a new window]   Figure 2. Liver insulin receptor (IR) tyrosine phosphorylation in genetically lean or fat chickens. Liver from fed, refed and starved LL or FL chickens were solubilized and immunoprecipitated using anti-phosphotyrosine antibody (PY20). (A) Immunoprecipitates were resolved on SDS-PAGE (10%) followed by electrotransfer. Blots were probed with PY20. (B) Bands were quantified with NIH-image software and are presented as arbitrary units. Results were presented as mean ± SEM, n = 4, * indicates P < 0.05.

View larger version (26K): [in this window] [in a new window]   Figure 3. Liver insulin receptor substrate 1 (IRS-1) and Src homology and collagen protein (Shc) protein amounts in lean and fat chickens. Liver from ad libitum fed, starved, and refed (for 30 min after 48-h starvation) lean or fat chickens were isolated and solubilized as described in Materials and Methods. Aliquots were resolved on 7.5% SDS-PAGE (120 µg of proteins) and transferred to nitrocellulose membrane by electroblotting. Blots were probed with either an (A) anti-IRS-1 antibody or (B) an anti-Shc antibody, and bands were revealed by ECL.

View larger version (22K): [in this window] [in a new window]   Figure 4. Liver insulin receptor substrate 1 (IRS-1) mRNA expression in lean and fat chickens. Total RNA from the liver of fed, refed or starved fat and lean chickens were prepared from 100 mg of tissue as indicated in Materials and Methods. RNAs were subjected to reverse transcription and PCR in the presence of two sets of primers: primers specific to IRS-1 and others specific to 18S rRNA. (A) Amplified products were run on a 1.5% agarose gel and visualized by staining with ethidium bromide. Expected size of amplified products is 490 bp and 515 for IRS-1 and 18S, respectively. Following transfer to nylon membranes, RT-PCR products were subjected to Southern blotting by using radiolabeled cDNA probes directed toward either IRS-1 or 18S. (B) Incorporated radioactivity was measured by using STORM Apparatus and expressed as the ratio IRS-1/18S, n = 4, * indicates P < 0.05.

View larger version (25K): [in this window] [in a new window]   Figure 5. Liver insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation in lean and fat chickens. Liver was extracted from fed, refed and starved lean or fat chickens, solubilized and immunoprecipitated by using anti-phosphotyrosine antibody (PY20). Immunoprecipitates were resolved on SDS-PAGE (10%) followed by electrotransfer. (A) Blots were probed with IRS-1. (B) Bands were quantified with NIH-image software and are presented as arbitrary units. Results were presented as mean ± SEM, n = 4, * indicates P < 0.05.

View larger version (28K): [in this window] [in a new window]   Figure 6. Liver Src homology and collagen protein (Shc) tyrosine phosphorylation in lean and fat chickens. Liver was extracted from fed, refed and starved lean or fat chickens, solubilized and immunoprecipitated by using anti-phosphotyrosine antibody (PY20). Immunoprecipitates were resolved on SDS-PAGE (10%) followed by electrotransfer. (A) Blots were probed with Shc. (B) Bands were quantified with NIH image software and are presented as arbitrary units. Results were presented as mean ± SEM, n = 4, * indicates P < 0.05.

View larger version (42K): [in this window] [in a new window]   Figure 7. Phosphatidyl Inositol 3'-kinase activity in lean and fat chickens. Liver from ad libitum fed, starved for 48 h and refed for 30 min following 48-h starvation were solubilized as described in Materials and Methods. Solubilized materials were subjected to immunoprecipitation with PY20 and PI 3'-kinase activity was measured in immunoprecipitates in the presence of (33P)ATP and phosphatidylinositol (PI). After separation onto TLC silica plates, phosphorylated PI was measured using by STORM apparatus and expressed as arbitrary units, n = 6.

The first elements of the insulin signaling cascade (IR, IRS-1 and Shc) are present in chicken muscle, however as previously described (Dupont et al. 1998 ), only the tyrosine phosphorylation of Shc is altered by the same nutritional states (fed, refed and food deprived). Despite this feature, it was of interest to look for the presence of a possible change at this level between the two genotypes. No major differences were observed in insulin signaling in muscle between the two genotypes. Data are summarized in Table 2 . In the starved state, IR phosphorylation did not differ between genotypes, but was higher (P < 0.05) in FL compared to LL chickens in the fed and refed states. Amounts of IRS-1 protein and mRNA and Shc protein were similar for the two genotypes. Tyrosine phosphorylation of IRS-1 and Shc was higher in FL than in LL chickens (P < 0.05) only in the fed state. Finally, PI 3'-kinase activity was not affected by genotype (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Early components of insulin signaling were not altered in the muscles of FL chickens (tyrosine phosphorylation of IR, IRS-1 and Shc was even higher in FL than in LL chickens in the fed state). Therefore, the lower amount of body nitrogen found in FL chickens (Leclercq 1988 ) and, to a lesser extent, the decrease in breast muscle (as a percentage of body weight, Alleman et al. 1999 and unpublished data) are not associated with changes in the early steps of insulin signaling in muscle.

Previous studies showed that the liver membrane insulin receptor number and the intrinsic tyrosine kinase activity of solubilized and lectin purified receptors were similar in FL and LL chickens (Simon et al. 1991 ). In the present study, irrespective of the nutritional status, liver membrane IR number did not differ between the two genotypes.

In the fed state, the degree of activation of the early steps of the insulin signaling pathways was increased in the liver of FL chickens. This was associated with a lower content of IRS-1 in this genotype, but a similar content of Shc in the two genotypes. Therefore, the enhanced phosphorylation of IR and the two substrates in fed FL chickens is most likely the direct consequence of the fact that plasma insulin levels were also higher in the fat genotype in the fed state. In these conditions, liver PI 3'-kinase should also have been higher in FL than in LL chickens. This was not the case. This enzyme exerts a critical role in the insulin signaling pathway. However, evidence is accumulating to suggest that other, subsequent or parallel steps are also crucial for the control of metabolism by insulin. For instance, this is the case for glucose transport in rat adipocytes (Guilherme and Czech 1998 ). Furthermore, in chicken liver, both IRS-1 and Shc are components of a large signaling complex involving IR and a PI 3'-kinase activity (Dupont et al. 1998b ). This suggest that the two substrates (IRS-1 and Shc) share or compete for the same pathways. The physiological implications of this are still unknown. However, we speculate that the higher degree of activation that is found in the liver of fed FL chickens (higher tyrosine phosphorylation of IR, IRS-1 and Shc) has important consequences for the metabolism of this organ, which is the lipogenic site in birds. Among these potential consequences are a higher in vivo lipogenesis (73% as a mean of three experiments; Saadoun et al. 1988 ) and a higher plasma VLDL concentration (Hermier et al. 1984 ). As a whole, these changes may appear minor when considering the extent of the divergency of the two lines, however as a cumulative and long-term response, the changes may account for the divergency in body composition.

Results obtained in the two other nutritional states extended the comparison of insulin signaling in the two genotypes. Starvation for 48 h decreased liver IR tyrosine phosphorylation, which was expected from previous experiments (Dupont et al. 1998a ), however liver IR and IRS-1 phosphorylation were higher in LL chickens. This feature is most likely because in this state, both plasma glucose and insulin levels were much higher in the FL than in LL chickens. The variation of glycemia and insulinemia is in contrast with what is observed after an overnight fast and remains to be explored (Saadoun et al. 1988 ). In the refed state, a similar degree of activation of the signaling cascade was obtained in the liver of the two genotypes (except for the phosphorylation of Shc), with much lower insulin levels in the fat genotype. This clearly indicates that FL chickens are not insulin resistant, a fact that was previously evidenced during the measurement of the hypoglycemic effect of exogenous insulin (Saadoun et al. 1988 ). After an overnight food withdrawal exogenous insulin is slightly but consistently more hypoglycemic in FL chickens.

Taken together, the data obtained in fat chickens largely contrast with those observed in obese mammals, which are insulin resistant. For instance, in the db/db and ob/ob mice and the Zucker fatty rat (Heydrick et al. 1993 ; Kerouz et al. 1997 ; King et al. 1992 ; Soll et al. 1975 ), the IR number and autophosphorylation are decreased, as well as IRS-1 phosphorylation and PI 3'-kinase activity in both the liver and muscle (Goodyear et al. 1995 ; Heydrick et al. 1993 ; Kerouz et al. 1997 ; King et al. 1992 ).

In conclusion, given the present knowledge of chicken physiology, the changes we observed in the liver would at least partly account for the metabolic differences that exist between FL and LL chickens. In the absence of insulin resistance, higher plasma insulin levels stimulate tyrosine phosphorylation of IR, IRS-1 and Shc in the liver of fed-state, FL chickens. This would favor liver lipogenesis and, as a consequence, body fat deposition in the fat genotype.

	ACKNOWLEDGMENTS

 
We thank Joël Michel for the maintenance of experimental FL and LL chickens and C. Moisy and J. M. Brigand for animal care.

	FOOTNOTES

 
¹ This work was financially supported by AIP- GENOME (INRA) and AFM (Association Française contre les Myopathies).

³ Abbreviations used: IRS-1, anti-rat IRS-1; PY20, monoclonal anti-phosphotyrosine; Shc, anti-mouse Shc; AMV, avian mylenoma virus; BSA, bovine serum albumin; dCTP, deoxy cytosine triphosphate; dNTP, deoxyribonucleotides; FL, genetically fat line; IR, insulin receptor; IRS-1, insulin receptor substrate 1; LL, genetically lean line; PI, phosphatidyl inositol; PI 3'-kinase, phosphatidyl inositol 3'-kinase; PMSF, phenylmethylsulfonylfluride; rRNasin, RNase ribonuclease inhibitor; RT-PCR, reverse transcriptase-polymerase chain reaction; Shc, Src homolgy and collagen protein; Tris, tri(hydroxymethyl)aminomethane hydrochloride.

Manuscript received March 30, 1999. Initial review completed June 3, 1999. Revision accepted July 13, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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