Molecular and functional resistance to insulin in hypothalamus of rats exposed to cold
Márcio A. Torsoni,
José B. Carvalheira,
Márcio Pereira-Da-Silva,
Marco A. de Carvalho-Filho,
Mário J. A. Saad, and
Lício A. Velloso
Department of Internal Medicine, State University of Campinas, 13083-070 Campinas, Brazil
Submitted 21 January 2003
; accepted in final form 10 March 2003
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ABSTRACT
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Insulin and leptin act in the hypothalamus, providing robust anorexigenic signals. The exposure of homeothermic animals to a cold environment leads to increased feeding, accompanied by sustained low levels of insulin and leptin. In the present study, the initial and intermediate steps of the insulin-signaling cascade were evaluated in the hypothalamus of cold-exposed Wistar rats. By immunohistochemistry, most insulin receptor (IR) and insulin receptor substrate-2 (IRS-2) immunoreactivity localized to the arcuate nucleus. Basal levels of tyrosine phosphorylation of IR and IRS-2 were increased in cold-exposed rats compared with rats maintained at room temperature. However, after an acute, peripheral infusion of exogenous insulin, significantly lower increases of IR and IRS-2 tyrosine phosphorylation were detected in the hypothalamus of cold-exposed rats. Insulin-induced association of p85/phosphatidylinositol 3-kinase with IRS-2, Ser473 phosphorylation of Akt, and tyrosine phosphorylation of ERK was significantly reduced in the hypothalamus of cold-exposed rats. To test the hypothesis of functional impairment of insulin signaling in the hypothalamus, intracerebroventricularly cannulated rats were acutely treated with insulin, and food ingestion was measured over a period of 12 h. Cold-exposed animals presented a significantly lower insulin-induced reduction in food consumption compared with animals maintained at room temperature. Hence, the present studies reveal that animals exposed to cold are resistant, both at the molecular and the functional level, to the actions of insulin in the hypothalamus.
insulin receptor; insulin substrate receptor-2; food ingestion
EXPOSURE OF HOMEOTHERMIC ANIMALS to a cold environment has been extensively employed as a model for studying thermogenesis, stress, and regulation of several metabolic parameters (20, 38). To maintain body temperature, cold-exposed animals increase heat production and, in parallel, augment food ingestion. These physiological adaptations generate a unique situation characterized by increased feeding accompanied by weight loss. Characterization of hypothalamic mechanisms that integrate thermogenesis and feeding behavior may help in understanding some features of the pathogenesis of obesity and related disorders. In recent years, substantial advance was obtained by determining several characteristics of leptin and insulin signaling in the hypothalamus (17, 32, 44).
In the present study, the initial and intermediate steps of the insulin-signaling pathway were evaluated in the hypothalamus of rats exposed to cold. These are hyperphagic animals that possess low levels of circulating leptin and insulin and represent a unique model for evaluating the central effects of either hormone upon their respective signaling pathway. To our surprise, cold-exposed animals display both molecular and functional characteristics of resistance to insulin and may thus represent interesting tools for the study of insulin signal transduction in the hypothalamus.
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MATERIALS AND METHODS
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Animals and surgical procedures. Male Wistar-Hannover rats (12 wk old, 250280 g) from the State University of Campinas Animal Breeding Center were used in all experiments. For all experimental protocols, rats were randomly divided into two groups. In the control group, the rats were kept in individual cages at 25°C; in the cold-exposed group, the rats were kept in individual cages in a cold incubator at 4°C for 48 h. Light-dark cycles and feeding and drinking (ad libitum) conditions were similar for both experimental groups. For some experiments, the rats were chronically instrumented with an intracerebroventricular (ICV) cannula and kept under controlled temperature (25°C) and light-dark conditions (07001900) in individual metabolic cages, with free access to tap water and standard laboratory rodent chow. Seven days after ICV cannula installation, the rats were tested for cannula patency and position and thereafter randomly selected for one of the experimental groups. The general guidelines established by the Brazilian College of Animal Experimentation were followed throughout the study. The study was approved by the State University of Campinas Ethical Committee (protocol no. 51002). Briefly, the animals were anesthetized with amobarbital sodium (15 mg/kg body wt ip) and, after loss of corneal and pedal reflexes, were positioned on a Stoelting stereotaxic apparatus. A 23-gauge guide stainless steel cannula with indwelling 30-gauge obturator was stereotaxically implanted into the lateral cerebral ventricle by use of previously reported techniques and pre-established coordinates: anteroposterior, 0.2 mm from bregma; lateral, 1.5 mm; and vertical, 4.2 mm (26). Rats were allowed 1 wk of recovery before testing for cannula patency and position. Cannulas were considered patent and correctly positioned by dypsogenic response elicited after angiotensin II injection (21). Rectal temperature was measured with a Thermistor digital (HI-8753) high-precision thermometer (Hanna Instruments, Woonsocket, RI) inserted 1.5 cm from the anus. Biochemical and hormonal measurements. For determinations of plasma glucose and serum insulin, leptin, TSH, corticosterone, and nonesterified fatty acids (NEFA), blood samples were collected at the end of the experimental protocol. For that purpose, rats were anesthetized as described above, the abdominal cavity was opened, and a blood sample of 0.5 ml was collected from the cava vein. Glucose was measured by the glucose oxidase method [intra-assay coefficient of variation (CV) 0.7; interassay CV 2.2] (37); insulin was measured by RIA, as previously described (intra-assay CV 1.9) (33); leptin (intra-assay CV 1.8), TSH (intra-assay CV 2.2), and corticosterone (intra-assay CV 2.3) were measured by RIA with commercial kits (Amersham Biosciences, Biotrak, Aylesbury, UK); and NEFA were determined by ELISA with a commercial kit (intra-assay CV 1.1, inter-assay CV 1.9) from Wako Chemicals (Richmond, VA).
Protocols for insulin treatment. For both groups (control and cold exposed), food was withdrawn 12 h before insulin treatment. Two protocols of insulin administration were employed. For evaluation of the molecular events occurring at the insulin signal transduction pathway, 0.1 ml of saline (0.9% NaCl), with or without insulin (10-6 M), was injected through the cava vein and, after a 10-min time course of insulin-induced insulin receptor (IR) tyrosine phosphorylation was performed, the hypothalamus was obtained and homogenized as described below. Peripheral injection of insulin for the study of signal transduction events in the hypothalamus was previously optimized and shown to produce similar effects to ICV-injected insulin (6). For evaluation of the anorexigenic effect of insulin, ICV-cannulated rats were ICV treated with 0.002 ml of 10-10 M, 10-8 M, 10-6 M, 10-4 M insulin, or a similar volume of saline. Treatment occurred invariably at 1800, and food intake was measured over the following 12 h.
Tissue extraction, immunoprecipitation, and immunoblotting. Tissue extraction, immunoprecipitation, and immunoblotting were performed as previously described (11, 40, 41). Briefly, rats were treated with insulin or saline, according to the protocols described in the preceding section. They were then anesthetized and subjected to craniotomy. Hypothalami were obtained and homogenized in freshly prepared ice-cold buffer (1% Triton X-100, 100 mM Tris, pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PMSF, and 0.01 mg aprotinin/ml). Insoluble material was removed by centrifugation (10,000 g) for 25 min at 4°C. Aliquots of the resulting supernatants containing 2.0 mg of total protein were used for immunoprecipitation with specific antibodies [anti-IR (rabbit; sc-711), anti-IR substrate-1 (IRS-1; rabbit; sc-559), or anti-IRS-2 (goat; sc-1555)], all from Santa Cruz Biotechnology (Santa Cruz, CA), at 4°C overnight, followed by addition of protein A-Sepharose 6MB (Pharmacia, Uppsala, Sweden) for 2 h. The pellets were washed three times in ice-cold buffer (0.5% Triton X-100, 100 mM Tris, pH 7.4, 10 mM EDTA, and 2 mM sodium vanadate) and then resuspended in Laemmli sample buffer (23) and boiled for 5 min before SDS-PAGE in a miniature slab gel apparatus (Bio-Rad, Richmond, CA). Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant). The nitrocellulose transfers were probed with specific antibodies [polyclonal anti-phosphotyrosine antibodies raised in rabbit and affinitypurified on phosphotyramine columns, or anti-rat-p85/phosphatidylinositol (PI) 3-kinase (rabbit; no. 06195) antiserum from UBI (Lake Placid, NY)]. Reliability of the method was further evaluated by performing immunoprecipitation with anti-phosphotyrosine antibodies and blotting with anti-IR, -IRS-1, or -IRS-2 antibodies as previously described (31). The blots were subsequently incubated with 125I-labeled protein A (Amersham, Aylesbury, UK). For direct immunoblot analyses, 0.2 mg protein from hypothalamus extracts was separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with specific antibodies [anti-IR, anti-IRS-1, anti-IRS-2, anti-phospho-Ser473-Akt (goat; sc-1618), or anti-phospho-ERK (mouse; sc-7383), all from Santa Cruz]. Results were visualized by autoradiography with preflashed Kodak XAR film. Band intensities were quantified by optical densitometry of developed autoradiographs (Scion Image software, ScionCorp).
Immunohistochemistry. Rat hypothalami were fixed in 4% paraformaldehyde-0.2 M phosphate-buffered saline (PBS, pH 7.4) for 24 h and embedded in paraffin, and 5-µm sections were obtained. The glass-mounted sections were cleared from paraffin with xylene and rehydrated by sequential washings with graded ethanol solutions (70100%). After permeabilization with 0.1% Triton X-100 in PBS, pH 7.4, for 10 min at room temperature, the sections were incubated in 1% H2O2 in PBS for 30 min to quench the endogenous peroxidase activity. The sections were pretreated in a microwave oven in sodium citrate buffer (pH 7.4) for 10 min. After being washed in PBS, the sections were blocked by 3% nonfat dry milk in PBS for 1 h at 37°C, followed by overnight incubation with the primary antibody (rabbit anti-IR, rabbit anti-IRS-1, or goat anti-IRS-2) in 1% BSA in PBS at 4°C in a moisture chamber. After incubation with the primary antibody, sections were washed and incubated with a specific biotinylated anti-rabbit secondary antibody (1:150 dilution) for 2 h at room temperature, followed by incubation with Streptavidin reagent (containing avidin-conjugated peroxidase) and color reaction with the DAB substrate kit (Vector Laboratories, Burlingame, CA) according to recommendations of the manufacturer. After the color reaction, sections were counterstained with Harris hematoxylin, dehydrated through an ethanol series into xylene, and mounted using Entellan mounting media (Merck, Darmstadt, Germany). Secondary antibody specificity was tested in a series of positive and negative control measurements. In the absence of primary antibodies, application of secondary antibodies (negative controls) failed to produce any significant staining. The images were obtained using an optical microscope (Leica, Wetzlar, Germany) and a Focus Imagecorder Plus System. For single immunofluorescence staining, FITC-conjugated secondary antibodies were employed, and analysis and photo documentation were performed using an Olympus BX60 Microscope and a Zeiss LSM 510 Laser Scanning Confocal Microscope, according to a previously described technique (2).
Data presentation and statistical analysis. All numerical results are expressed as means ± SE of the indicated number of experiments. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry by use of the Scion Image software (ScionCorp); when appropriate, comparisons were performed of the variation of means in band intensity between saline and insulintreated conditions. For comparison of food intake, means of ingested chow and variation in food consumption induced by insulin treatment were statistically analyzed. Student's t-test for unpaired samples was used for statistical analysis. The level of significance was set at P < 0.05.
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RESULTS
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Hypothalamic distribution of proteins that participate in insulin signaling. A thorough immunohistochemical evaluation of IR, IRS-1, and IRS-2 expression was performed in the hypothalamus of Wistar rats to characterize the presence and tissue distribution of elements that participate in insulin signaling in hypothalamic areas known to control feeding behavior. Most immunoreactivity to IR was detected in the arcuate nucleus (Fig. 1C). Moreover, neurons apparently expressing high levels of IR were also detected in the paraventricular nucleus (PVN, Fig. 1A). IRS-1 was not detected in the same areas expressing IR. Only occasional neurons expressing IRS-1 were encountered in hypothalamic areas involved with feeding behavior. Considerable amounts of IRS-1 were encountered in the posterior hypothalamus (Fig. 1B), a region known to participate in the control of sleep and wakefulness. Finally, IRS-2 immunoreactivity was mostly detected in the arcuate nucleus (Fig. 1D), thus coinciding with IR expression in a region primarily involved in the control of feeding.

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Fig. 1. Immunohistochemical evaluation of insulin receptor (IR), IR substrate-1 (IRS-1, IRS1) and IRS-2 (IRS2) distribution in hypothalamus of rats. Paraformaldehyde-fixed rat hypothalamic sections (5 µm) were primarily incubated with anti-IR (A and C), anti-IRS1 (B), or anti-IRS2 (D) specific antibodies (as stated in MATERIALS AND METHODS). Visualization of immunoreactivity was obtained by using secondary antibodies conjugated with FITC (A, C, and D) and photographed under a fluorescence microscope, or secondary biotinylated antibodies, recognized by avidin-conjugated peroxidase and photographed under an optical microscope (B). Approximate anatomic sites of staining for each protein are depicted in the schemes and are indicated with arrows. Three different sets of experiments were performed and presented the same histological characteristics. Arc, arcuate nucleus; PaV, paraventricular nucleus; PH, posterior hypothalamic area; 3V, third ventricle.
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Characterization of rats exposed to cold. The exposure of homeothermic animals to cold provides a physiological model of increased thermogenesis accompanied by increased food intake and sustained hypoinsulinemia. To evaluate the characteristics of the animal model and compare them with previously reported data, a series of measurements was performed (Table 1). Two-day exposure to cold led to loss of body weight, accompanied by a significant increase in daily food ingestion and a modest but significant decrease in body temperature. The levels of insulin and leptin were significantly reduced, whereas a tendency for an increase was detected for blood NEFA. Finally, the blood levels of glucose, TSH, and corticosterone were unaffected by 2-day exposure to cold. As a whole, the animal model utilized in the present study is in agreement with previous metabolic characterizations (10, 39) and completely fulfilled the expectations of an animal model with hypoinsulinemia and increased food intake.
Effect of cold exposure on initial and intermediate steps of insulin signal transduction in the hypothalamus. To investigate the molecular mechanisms of insulin signal transduction in the hypothalamus of coldexposed rats, a protocol of peripheral insulin infusion (100 µl of 10-6 M insulin through the cava vein) that has been extensively characterized (11, 13, 29, 31, 36) was employed. This protocol is known to promote a reproducible molecular response to the hormone in the central nervous system, muscle, liver, and adipose tissue. Moreover, with use of this protocol, the lowest blood glucose levels during the first 15 min after insulin infusion were 61.09 ± 1.34 mg/dl in control and 59.98 ± 1.40 mg/dl in cold-exposed rats, which means that hypoglycemia and activation of counterregulatory factors do not participate in the induction of the molecular events herein described.
In preliminary experiments, the method was optimized by performing a time course evaluation of insulin-induced IR tyrosine phosphorylation, which is presented in Fig. 2. When the protein amounts of IR, IRS-1, and IRS-2 in hypothalamic protein extracts of rats maintained at room temperature or exposed to cold during 2, 24, and 48 h were compared, no significant variation was observed (Figs. 2B and 3, A and D). However, concerning the molecular phenomena related to protein functional activation or signaling engagement, significant modulations induced by cold exposure were detected. Thus the exposure to cold led to increased (
40%, P < 0.05) steady-state (non-insulinstimulated) tyrosine phosphorylation of the IR and to a significant reduction (
65%, P < 0.05) of the insulininduced increase in the phosphorylation of IR (Fig. 1B). Concerning IRS values, no significant differences were detected in steady-state or insulin-stimulated tyrosine phosphorylation of IRS-1, and the increase in tyrosine phosphorylation of IRS-1 was not significantly influenced by cold exposure. Nevertheless, both basal (reduction of
50%, P < 0.05) and insulin-stimulated (increase of
25%, P < 0.05) associations of IRS-1 with p85/PI 3-kinase were different between control and cold-exposed rats. Because immunohistochemical studies evidenced that neuronal coexpression of IR and IRS-1 was a rare event in the hypothalamus of rats, a question concerning the possible mechanisms involved in insulin-induced tyrosine phosphorylation of IRS-1 in this anatomic site was raised. No IRS-1/IRS-2 crossreactivity was observed with the antibodies employed (sc-559 and sc-1555). However, the affinity of sc-559 (anti-IRS-1) toward IRS-1 was 3.5-fold higher than the affinity of sc-1555 (anti-IRS-2) toward IRS-2, which suggests that even discrete increase of IRS-1 tyrosine phosphorylation occurring in a minority of neurons of the hypothalamus might be detected by the method employed.

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Fig. 2. Effects of cold exposure on IR protein expression and insulininduced IR tyrosine phosphorylation. A: anesthetized rats were infused with 100 µl of 10-6 M insulin (Ins) through the cava vein, and after elapsed time shown, hypothalami were obtained and homogenized; 2.0 mg of total protein were used in immunoprecipitation (IP) experiments with anti-IR antibodies. Immunocomplexed proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots (IB) were performed with anti-phosphotyrosine (pY) antibodies. B: 0.2 mg of total protein obtained from hypothalami of control rats (C) or rats exposed during 2, 24, or 48 h to cold were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-IR antibodies. C: control rats (C) or 48-h cold-exposed rats (4°C) were acutely injected with 100 µl of 10-6 M insulin (Ins+) or saline (Ins-) through the cava vein; after 10 min, hypothalami were obtained and homogenized, and 2.0 mg total protein were utilized in IP experiments with anti-IR antibodies. Immunocomplexed proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-pY antibodies. Bars, relative tyrosine phosphorylation of IR; C+ corresponds to 100%; in all experiments n = 4; *P < 0.05 vs. C-; #P < 0.05 vs. difference between C- and C+.
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Fig. 3. Effects of cold exposure on hypothalamic IRS1 and IRS2 protein expression (A and D), insulin-induced tyrosine phosphorylation (B and E), and p85/phosphatidylinositol (PI) 3-kinase association (C and F). A and D: 0.2 mg of total protein obtained from hypothalami of control rats (C) or rats exposed for 2, 24, or 48 h to cold (4°C) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with anti-IRS1 (A) or anti-IRS2 (D) antibodies. B and E: control rats (C) or 48-h cold-exposed rats (4°C) were acutely injected with 100 µl of 10-6 M insulin (Ins+) or saline (Ins-) through the cava vein; after 10 min, hypothalami were obtained and homogenized, and 2.0 mg of total protein were utilized in IP experiments with anti-IRS1 (B) or anti-IRS2 (E) antibodies. Immunocomplexed proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-phosphotyrosine (pY) antibodies. C and F: control rats (C) or 48-h cold-exposed rats (4°C) were acutely injected with 100 µl of 10-6 M insulin (Ins+) or saline (Ins-) through the cava vein; after 10 min, hypothalami were obtained and homogenized, and 2.0 mg of total protein were utilized in IP experiments with anti-IRS1 (B) or anti-IRS2 (E) antibodies. Immunocomplexed proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-p85/PI 3-kinase antibodies. Bars, relative tyrosine phosphorylation of IRSs (B and E) or relative association of p85/PI 3-kinase (C and F), C+ corresponds to 100%; in all experiments n = 4; *P < 0.05 vs. C-; #P < 0.05 vs. difference between C- and C+; P < 0.05 vs. C+.
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Exposure to cold promoted a significant increase (
45%, P < 0.05) in steady-state IRS-2 tyrosine phosphorylation (Fig. 3E) and a significant reduction in insulin-induced IRS-2 tyrosine phosphorylation (
25%, P < 0.05) and p85/PI 3-kinase association (
35%, P < 0.05) (Fig. 3, D and E). PI-(3,4)P2 and PI-(3,4,5)P, which are formed as a result of PI 3-kinase activation, serve as docking sites for the enzymes Akt and phosphoinositide-dependent kinase-1. Activated Akt may participate in events that control glucose uptake, apoptosis, membrane ruffling, and vesicular trafficking. In the present study, it was observed that cold exposure induced a highly significant increase (
150%, P < 0.05) in steady-state Akt serine phosphorylation. However, the treatment with insulin promoted almost no further increase in serine phosphorylation levels in the cold-exposed animals, whereas in control rats, this effect was significant (Fig. 4A).

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Fig. 4. Effects of cold exposure on insulin-induced serine phosphorylation of Akt or tyrosine phosphorylation of Erk. A and B: 0.2 mg of total protein obtained from hypothalami of control rats (C) or rats exposed to cold for 48 h (4°C), treated with 100 µl of 10-6 M insulin (Ins+) or saline (Ins-) through the cava vein, were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-pSer473 Akt (A) or anti-pErk (B) antibodies. Bars, relative serine (A) or tyrosine (B) phosphorylation of Akt (A) and Erk (B), respectively; C+ corresponds to 100%; in all experiments n = 4; #P < 0.05 vs. difference between C- and C+.
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ERK proteins are downstream effectors of the Raf/MEK-signaling pathway, which links membrane receptor-derived signals to intranuclear control of gene expression (5, 8). In control rats, the peripheral treatment with insulin induced a significant increase in tyrosine phosphorylation of ERK (Fig. 4B). However, after 2-day cold exposure, a significantly lower (
60%, P < 0.05) effect of insulin to promote tyrosine phosphorylation of ERK was detected (Fig. 4B).
Effect of cold exposure on insulin-induced satiety. To investigate whether the apparent disturbance of the molecular events involved in insulin signaling detected in the hypothalamus of cold-exposed rats would coincide with the modulation of a functional event controlled, at least in part, by insulin, experiments were performed for determining the anorexigenic effect of ICV-injected insulin in control and cold-exposed rats. Insulin promoted, after a dose-dependent pattern, ≤50% decrease in 12-h food intake in control rats, whereas, surprisingly, during the same period it reduced by only 25% the amount of food ingested in cold-exposed rats (Fig. 5) (n = 5, P < 0.05). Thus cold exposure led to a significant impairment of the insulininduced anorexigenic effect, which may characterize central resistance to insulin action.

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Fig. 5. Effects of intracerebroventricular (ICV)-infused insulin on 12-h food intake of rats maintained at thermoneutrality or exposed to cold temperature. ICV-instrumented rats maintained in metabolic cages and exposed (4°C) or not (C) to cold were fasted for 12 h and at 1800 were injected with 0.002 ml of insulin at concentrations given in A or 10-6 Min B. Food intake was measured over the next 12 h. Bars, food intake in g. In A, n = 4; in B, n = 6; #P < 0.05 vs. difference between C- and C+.
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DISCUSSION
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Peripheral resistance to insulin action is one of the most important features of type 2 diabetes mellitus and obesity (9, 22). By definition, insulin resistance refers to a loss of efficiency of the hormone to promote its physiological effects (14, 22). It may be clinically measured by different methods (14) at the molecular level by evaluating the response of IR and its substrates to a given dose of insulin. Several molecular and functional defects are correlated with clinical insulin resistance. That is the case for decreases in receptor or substrate concentration (25), impairment of kinase activity (42), enhanced or reduced phosphatase function (3, 12), and defects of the glucose transport system (34). Because the most remarkable physiological functions of insulin are the control of glucose uptake by fat and muscle and the inhibition of glucose output by the liver, clinical and experimental evaluation of insulin action is based on tests that measure peripheral glucose mobilization, such as the clamp study that serves as standard for all of the remaining methods (15).
After the initial descriptions of the presence of insulin (44) and its receptor (19) in the central nervous system, many efforts have been devoted to characterize its functional role in this area of the body. Suggestively, insulin might participate in cognitive functions such as learning and memory (4, 35), in the control of apoptotic events (24), or as a growth stimulus during brain development (1, 13). Nevertheless, the participation of insulin in the control of food ingestion was first suggested by Woods et al. (44) and is, perhaps, the most concrete and well-characterized physiological role of this hormone in the brain.
Exposure of homeothermic animals to cold has been adopted as a useful model for studying thermogenesis and stressful events (38). Previous reports have shown that higher glucose mobilization is a consequence of cold exposure (39). During the first few hours of cold stimulus, the activation of a stress response may directly influence whole body metabolism; however, after adaptation, central inputs coordinate responses to warrant optimization of energy consumption for longer survival under the adversity of cold. Some of these adaptations include higher substrate disposal for optimal thermogenesis and increased food consumption. Thus, because insulin participates in the central control of feeding behavior, under cold exposure a modulation of insulin signaling in hypothalamus would be expected. As a result of the present study, it was demonstrated that reduced anorexigenic response to insulin infused in the central nervous system is paralleled by molecular phenomena characteristic of impaired insulin signal transduction. Two-day exposure of rats to cold promoted no changes in IR, IRS-1, and IRS-2 protein expression in the hypothalamus. Nevertheless, significant reductions in insulin-induced tyrosine phosphorylation of IR and IRS-2 were detected. Moreover, significant reductions in insulin-induced serine phosphorylation of Akt and tyrosine phosphorylation of ERK were observed as well, and these may reflect the defective transmission of the insulin signal toward more distal steps of two different branches of this signaling pathway. As stated above, the physiological roles of insulin in the hypothalamus are only beginning to be elucidated. Several studies have reported modulation of hypothalamic neuropeptide expression as a consequence of insulin infusion or insulin deficiency (7, 18, 30). Thus molecular impairment of insulin signal transduction in the hypothalamus may lead to disturbed production and secretion of neuropeptides primarily involved in control of appetite and energy expenditure. Those neuropeptides may be fundamental intermediaries in the control exerted by insulin on feeding and energy homeostasis. In which hypothalamic areas, and by which subcellular pathways insulin might act to promote those actions in the hypothalamus are still debated questions.
Other than evaluating the peripheral action of insulin, there are no clinical or experimental means of measuring insulin activity in the hypothalamus. By utilizing an animal model that presents sustained low blood levels of insulin and leptin associated with hyperphagia, we hypothesized that it would serve well to investigate separately the effects of either hormone on the molecular steps of their signaling pathways in the hypothalamus. By utilizing a method optimized for evaluating molecular response to acutely injected insulin in classic target tissues (6, 31), we could evaluate the effects of cold exposure on the protein expression and functional status of the IR and some of the proximal and distal participants of the insulin-signaling pathway in the hypothalamus of rats.
By immunohistochemistry, the presence of IR in the arcuate nucleus was detected. This is a site known for its involvement with the control of food ingestion and has previously been demonstrated as a region with high expression of both insulin and leptin receptors (28, 32). Besides that, IR was also detected in the PVN and to a lesser extent in other scattered neurons of the hypothalamus. To transduce the signal delivered by insulin, the IR must coexist with its substrates in the same cells. Performing a thorough immunohistochemical evaluation of the brain, Folli et al. (16) found that IRS-1 was widely distributed in several areas of the central nervous system, but in none of those areas did its expression coincide with the presence of high levels of IR. Similarly, in the present study, IRS-1 expression was detected mostly in the posterior hypothalamus, an area not related to feeding behavior and almost devoid of IR expression. Thus, on the basis of tissue distribution analyses, it seems that IRS-1 is not an important substrate of the IR in the brain, unlike most peripheral tissues. This fact was recently confirmed by Niswender et al. (27). On the other hand, IRS-2 expression was localized predominantly at the arcuate nucleus, coinciding with the expression of IR, and at the main site of feeding behavior control. It is of major importance to notice that IRS-2, but not IRS-1, knockout mice present some of the same characteristics of IR brain knockout mice concerning weight gain and reproductive disability (43), providing strong support for the hypothesis of IRS-2 preferential usage in hypothalamic insulin signaling.
In conclusion, during cold exposure, rats become hyperphagic and display both functional and molecular impairment of insulin action in the hypothalamus. Characterization of the molecular mechanisms involved in hypothalamic resistance to insulin during cold exposure may provide useful information for understanding the integration between central control of food ingestion and thermogenesis.
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ACKNOWLEDGMENTS
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We are indebted to L. Janeri for technical assistance.
These studies were supported by a grant of Fundação de Amparo à Pesquisa do Estado de São Paulo (São Paulo State Agency for Research Support).
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FOOTNOTES
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Address for reprint requests and other correspondence: L. A. Velloso, Departamento de Clínica Médica, FCM-UNICAMP, 13083-970 Campinas, Brazil (E-mail: lavelloso{at}fcm.unicamp.br).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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