Western Diet Modulates Insulin Signaling, c-Jun N-Terminal Kinase Activity, and Insulin Receptor Substrate-1ser307 Phosphorylation in a Tiss
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内分泌学杂志 2005年第3期
Departamento de Clínica Médica da Universidade Estadual de Campinas, Campinas, S?o Paulo 13083-970, Brazil
Address all correspondence and requests for reprints to: M. J. A. Saad, Departamento de Clínica Médica, FCM-UNICAMP, Cidade Universitária, Campinas, S?o Paulo 13083-970, Brazil. E-mail: msaad@fcm.unicamp.br.
Abstract
The mechanisms by which diet-induced obesity is associated with insulin resistance are not well established, and no study has until now integrated, in a temporal manner, functional insulin action data with insulin signaling in key insulin-sensitive tissues, including the hypothalamus. In this study, we evaluated the regulation of insulin sensitivity by hyperinsulinemic-euglycemic clamp procedures and insulin signaling, c-jun N-terminal kinase (JNK) activation and insulin receptor substrate (IRS)-1ser307 phosphorylation in liver, muscle, adipose tissue, and hypothalamus, by immunoprecipitation and immunoblotting, in rats fed on a Western diet (WD) or control diet for 10 or 30 d. WD increased visceral adiposity, serum triacylglycerol, and insulin levels and reduced whole-body glucose use. After 10 d of WD (WD10) there was a decrease in IRS-1/phosphatidylinositol 3-kinase/protein kinase B pathway in hypothalamus and muscle, associated with an attenuation of the anorexigenic effect of insulin in the former and reduced glucose transport in the latter. In WD10, there was an increased glucose transport in adipose tissue in parallel to increased insulin signaling in this tissue. After 30 d of WD, insulin was less effective in suppressing hepatic glucose production, and this was associated with a decrease in insulin signaling in the liver. JNK activity and IRS-1ser307 phosphorylation were higher in insulin-resistant tissues. In summary, the insulin resistance induced by WD is tissue specific and installs first in hypothalamus and muscle and later in liver, accompanied by activation of JNK and IRS-1ser307 phosphorylation. The impairment of the insulin signaling in these tissues, but not in adipose tissue, may lead to increased adiposity and insulin resistance in the WD rats.
Introduction
IN RODENTS AND HUMANS, dietary intake of high amounts of fat has been shown to have adverse effects on insulin sensitivity. Indeed, a more palatable cafeteria diet, which is closer to the Western diet (WD), has also been associated with increased adiposity and insulin resistance in humans (1, 2). However, the exact mechanism by which WD can induce obesity and insulin resistance is not yet known. In this context, insulin resistance could be tissue-specific to the muscle, liver, and hypothalamus, whereas adipose tissue remains sensitive to insulin.
At the molecular level, the insulin signaling begins when activation of insulin receptor (IR) results in tyrosine phosphorylation of several substrates, including the IR substrate 1 (IRS-1) and IRS-2 (3). After tyrosine phosphorylation, IRS-1 and IRS-2 bind and activate the enzyme phosphatidylinositol 3-kinase (PI3-K) (3, 4). The activation of PI3-K increases serine phosphorylation of protein kinase B (Akt), which in turn stimulates the glucose transport in the muscle and adipose tissue, stimulates glycogen synthesis in the liver and muscle, and stimulates lipogenesis in the adipose tissue. Therefore, the PI3-K/Akt pathway has an important role in the metabolic effects of insulin. In the hypothalamus, the activated PI3-K/Akt pathway suppresses feeding, and insulin resistance in this tissue has recently been described (5, 6).
Many mechanisms may contribute to the dysregulation of the insulin-signaling pathway, including serine phosphorylation of IRS proteins by protein kinases such as c-jun N-terminal kinase (JNK) (7). JNK is a member of the MAPK family (8, 9) and can be activated by TNF (10) and IL-1? (11, 12). In addition, JNK might serve as a feedback inhibitor during insulin stimulation (7). There are three JNK isoforms described, named JNK1, -2, and -3 (13), of which JNK1 is the most involved in the pathophysiology of obesity and insulin resistance (10). JNK activation induces inhibitory serine 307 (Ser307) phosphorylation of IRS-1, as shown in previous studies (7, 14). Ser307 is located next to the phosphotyrosine (pY)-binding domain in IRS-1 and its phosphorylation inhibits the interaction of the pY-binding domain with the phosphorylated NPEY motif in the activated IR, causing insulin resistance (14). Here, we investigate the effects of WD on insulin signaling in liver, skeletal muscle, hypothalamus, and adipose tissue of Wistar rats, given during 10 or 30 d, and the role of JNK activation and IRS-1ser307 in the dysregulation of the insulin-signaling pathway in these tissues. In parallel, we have also conducted functional studies of insulin sensitivity by hyperinsulinemic-euglycemic clamp procedures in combination with tracer infusions, which allowed us to isolate the hepatic, skeletal muscle, and adipose tissue components of insulin action and to associate these data with insulin signaling in each tissue.
Materials and Methods
Materials
Male Wistar rats were provided by the State University of Campinas Central Breeding Center (Campinas, Brazil). Anti-pY (-pY), anti-IR? (-IR), anti-IRS-1, anti-Akt1/2, anti-pJNK, and anti-JNK1 antibodies were from Santa Cruz Technology (Santa Cruz, CA). Anti-pAkt and anti-pFoxo1 were from Cell Signaling Technology (Beverly, MA). Anti-PI3-K, anti-IRS-2, and anti-phospho-IRS-1ser307 were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Human recombinant insulin was from Eli Lilly and Co. (Indianapolis, IN). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless specified elsewhere. [125I]Protein A, HPLC-purified [3-3H]glucose and 2-deoxy-D-[1-14C]glucose (2-[14C]DG1) were obtained from Amersham (Amersham Biosciences Group, Little Chalfont, UK). The Harvard apparatus (model 11) and Harvard compact infusion pumps (model 975) were obtained from South Natick, MA.
Animals
All experiments were approved by the Ethics Committee at the State University of Campinas.
Eight-week-old male Wistar rats were divided into two groups with similar body weights (255 ± 4 g) and assigned to receive two kinds of diet: a standard rodent chow and water ad libitum or WD for 10 and 30 d (C10, C30, WD10, and WD30, respectively). WD was adapted from a previous study (15) and included soft drinks ad libitum alternated daily (Coca-Cola and Guaraná Antarctica) and 37.5% standard rodent chow, 25% peanuts, 25% chocolate, and 12.5% cookies. In addition, these rats were offered specific amounts of palatable food items such as biscuits, cakes, and cookies totaling 5.4 kcal/g (carbohydrate, 38.5%; protein, 15%; fat, 46.5%) as opposed to the 3.8 kcal/g (carbohydrate, 70%; protein, 20%; fat, 10%) of the standard chow diet. The nutritional status of the rats was not affected by the WD regimen, as demonstrated by similar values of serum albumin, calcium, magnesium, phosphorus, iron, folate, and vitamin B12 in preliminary experiments (data not shown).
Each day, leftovers were collected and replaced with new items. This was a key point of our experiment that assured success of the hypercaloric regimen.
Animal characterization
At the end of the diet period, body weight, epididymal, retroperitoneal, and mesenteric fat pad were weighed. Food was withdrawn 12–14 h before the experiments and blood samples were taken for the determination of serum concentration of total cholesterol, high-density lipoprotein (HDL)-cholesterol, triacylglycerol (TG), and insulin. Lipids were measured in a routine diagnostic analyzer (Modular, Roche Diagnostics, Mannheim, Germany) using for cholesterol (CHOD-PAP, cholesterol oxidase-peroxidase enzymatic colorimetric assay) and for triacylglycerol (GPO-PAP, glycerol-3-phosphate oxidase enzymatic colorimetric assay). Blood glucose levels were measured by the glucose oxidase method (16). Serum insulin was determined by RIA.
Hyperinsulinemic-euglycemic clamp procedures
After 5 h fasting, animals were anesthetized ip with sodium pentobarbital (50 mg/kg body weight), and catheters were then inserted into the left jugular vein (for tracer infusions) and carotid artery (for blood sampling), as previously described (17). Each animal was monitored for food intake and weight gain for 5 d after surgery to ensure complete recovery. Food was removed for 12 h before the beginning of in vivo studies. A 120-min hyperinsulinemic-euglycemic clamp procedure was conducted in conscious, unrestrained, catheterized rats, as shown previously (18, 19), with a prime continuous infusion of human insulin at a rate of 3.6 mU/kg body weight per minute to raise the plasma insulin concentration to approximately 800–900 pmol/liter. Blood samples (20 μl) were collected at 5-min intervals for the immediate measurement of plasma glucose concentration, and 10% unlabeled glucose was infused at variable rates to maintain plasma glucose at fasting levels. Insulin-stimulated whole-body glucose flux was estimated using a prime continuous infusion of HPLC-purified [3-3H]glucose (10 μCi bolus, 0.1 μCi/min) throughout the clamp procedure (20). To estimate insulin-stimulated glucose-transport activity and metabolism in skeletal muscle and visceral fat, 2-[14C]DG1 was administered as a bolus (10 μCi) 45 min before the end of the clamp procedure. Blood samples (20 μl) were taken at 80, 90, 100, 110, and 120 min after the start of the clamp procedure to determine plasma [3H]glucose and 2-[14C]DG1 concentrations. Additional blood samples (10 μl) were collected before the start and at the end of the clamp procedure for measurement of plasma insulin concentrations. All infusions were performed using Harvard infusion pumps. At the end of the clamp procedure, animals were killed by a sodium pentobarbital iv injection. Within 2 min, two skeletal muscles (soleus and gastrocnemius) from both hind limbs and epididymal white adipose tissue were taken. Each tissue, once exposed, was dissected out within 2 sec, weighed, frozen with liquid N2, and stored at –70 C for later analysis. In separate experiments, the basal rates of glucose turnover were measured by continuously infusing [3-3H]glucose (0.02 μCi/min) for 120 min, and blood samples (20 μl) were taken at 100, 110, and 120 min after the start of the experiment for the determination of plasma [3H]glucose concentration.
Analytical procedures of hyperinsulinemic-euglycemic clamp
Plasma glucose was measured using a glucometer (Advantage, Roche Molecular Biochemicals). Plasma tracer samples were deproteinized with equal volumes of barium hydroxide and zinc sulfate (0.03 N) and stored overnight at 4 C. Radioactivity of [3-3H]glucose in plasma was measured from supernatants of Ba(OH)2/ZnSO4 precipitates, after each was evaporated to dryness for the removal of tritiated water. Rates of whole-body glucose uptake and basal glucose turnover were determined as the ratio of the [3-3H]glucose infusion rate (disintegrations per minute) to the specific activity of plasma glucose (disintegrations per minute per milligram glucose) during the final 30 min of respective experiments under steady-state conditions. The hepatic glucose output (HGO) during the clamp procedure was therefore obtained from the difference between the whole-body glucose uptake and the rate of unlabeled glucose infusion. Glucose transport activity in skeletal muscle and fat was calculated from the plasma 2-[14C]DG profile, as described before (21, 22).
Tissue extraction and immunoprecipitation
Rats were anesthetized with sodium thiopental and used 10–15 min later. As soon as anesthesia was assured by the loss of pedal and corneal reflexes, the abdominal cavity was opened, the portal vein was exposed, and 0.2 ml normal saline with or without insulin (2 μg) was injected. At 30 sec after the insulin injection, the liver was removed, and 90 sec later, muscle and adipose tissue were removed, minced coarsely, and homogenized immediately in extraction buffer, as described elsewhere (23). Extracts were then centrifuged at 15,000 rpm and 4 C for 40 min to remove insoluble material, and the supernatants were used for immunoprecipitation with -IR, -IRS-1 and -2, and protein A-Sepharose 6MB (Pharmacia, Uppsala, Sweden).
Protein analysis by immunoblotting
The precipitated proteins and/or whole-tissue extracts were treated with Laemmli sample buffer (24) containing 100 mM dithiothreitol and heated in a boiling water bath for 5 min, after which they were subjected to SDS-PAGE in a Bio-Rad miniature slab gel apparatus (Mini-Protean). For total extracts, 250 μg of proteins were subjected to SDS-PAGE. Electrotransfer of proteins from the gel to nitrocellulose was performed for 120 min at 120 V in a Bio-Rad Mini-Protean transfer apparatus (25). Nonspecific protein binding to the nitrocellulose was reduced by preincubating the filter for 2 h in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, 0.02% Tween 20). The nitrocellulose blot was incubated with specific antibodies overnight at 4 C and then incubated with 125I-labeled protein A. The results were visualized by autoradiography with preflashed Kodak XAR film. Band intensities were quantified by optical densitometry (Hoefer Scientific Instruments, San Francisco, CA; model GS300).
Hypothalamus study
Briefly, the animals were anesthetized with sodium thiopental, as described above, and 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 preestablished coordinates: anteroposterior, 0.2 mm from bregma; lateral, 1.5 mm; and vertical, 4.2 mm (23, 26). Rats were allowed 5 d of recovery before testing for cannula patency and position. Cannulas were considered patent and correctly positioned by dypsogenic response elicited after angiotensin II injection (27). For evaluation of the anorexigenic effect of insulin, we first measured the 12-h food intake before intracerebroventricular (ICV) insulin or vehicle (saline) infusion. Cannulated rats were then treated with an ICV infusion of 2 μl of 10–6 M insulin or 2 μl of saline. Treatment occurred invariably at 1800 h, and food intake was measured over the following 12 h (23). The following day, fasted cannulated rats from different groups received 2 μl of saline or 10–6 M insulin ICV and the hypothalamus was removed 15 min later (23). Tissue samples underwent the same procedures as the other tissues described above.
Statistical analysis
Data were expressed as means ± SEM accompanied by the indicated number of independent experiments. For statistical analysis, the groups were compared using a two-way ANOVA with the Bonferroni test for post hoc comparisons. The level of significance adopted was P < 0.05.
Results
Animal characteristics
Table 1 shows that the body weight was similar in the two groups after 10 d of WD. However, after 30 d, the body weight was higher in the WD group compared with the respective control group (P < 0.05). The animals fed with the WD displayed higher contents of fat mass in different sites. The epididymal, retroperitoneal, and mesenteric fat were heavier (P < 0.05) after 10 and 30 d of WD, compared with their control groups. There was no difference in fasting blood glucose and insulin levels after 10 d of WD compared with the control group. However, in WD30, the fasting insulin levels were higher (P < 0.05) than in C30, despite the normal blood glucose levels. The serum concentrations of TG were significantly higher in the WD10 and WD30 compared with their respective controls. The serum cholesterol levels were not different between the groups; neither were the serum HDL-cholesterol levels.
TABLE 1. Animal characteristics
Hyperinsulinemic-euglycemic clamp procedures
A hyperinsulinemic-euglycemic clamp procedure with tracer infusions was performed to examine the effects of WD on the metabolism of glucose in liver, skeletal muscle, and adipose tissue (Fig. 1). The glucose infusion rate needed to clamp glycemia at fasting levels in the presence of a constant infusion of insulin (3.6 mU/kg body weight·min) was more than 2-fold lower in rats fed on WD than in their respective controls (Fig. 1A). Accordingly, the insulin-stimulated whole-body glucose-disposal rates were also significantly decreased in rats fed on WD for 10 and 30 d compared with their respective controls, as depicted in Fig. 1B. However, insulin was not able to reduce the hepatic glucose output by the same amount in all groups of animals. In Fig. 1C, it may be observed that insulin was able to reduce the HGO in both controls (C10 and C30) and in WD10. However, its ability to suppress HGO was impaired in WD30 when compared with controls and WD10.
FIG. 1. A, Steady-state glucose infusion rates obtained from averaged rates of 90–120 min of 10% unlabeled glucose infusion during hyperinsulinemic-euglycemic clamp procedures in the control rats (C10 and C30) and in rats fed on WD for 10 (WD10) and 30 (WD30) d; means ± SEM, n = 4; *, WD10 vs. C10, P < 0.05; #, WD30 vs. C30, P < 0.01. B, Insulin-stimulated whole-body glucose transport during hyperinsulinemic-euglycemic clamp procedure in awake rats; means ± SEM, n = 4; *, WD10 vs. C10, P < 0.01; #, WD30 vs. C30, P < 0.01. C, Basal and insulin-stimulated rates of hepatic glucose production during the hyperinsulinemic-euglycemic clamp procedures in awake rats; means ± SEM, n = 4; *, C10+ vs. C10–, P < 0.05; #, WD10+ vs. WD10–, P < 0.01; , C30+ vs. C30–, P < 0.01. D, Glucose transport in skeletal muscle and in adipose tissue evaluated by 2DG uptake during the last 45 min of the hyperinsulinemic-euglycemic clamp studies; means ± SEM, n = 4; *, WD10 vs. C10, P < 0.001; #, WD30 vs. C30, P < 0.01.
Using 2DG uptake analysis, the insulin-stimulated glucose uptake in skeletal muscle and adipose tissue was quantified (Fig. 1D). As shown, WD10 and WD30 presented significant reductions in skeletal muscle glucose uptake, and a more severe reduction was observed in WD30 rats (80% reduction compared with C30). In contrast to the inhibition of glucose uptake in the muscle of WD rats, the adipose tissue showed a significantly higher glucose uptake (at least 2-fold) both in WD10 and WD30, when compared with their respective controls.
Insulin signaling in the liver of animals fed on a WD for 10 and 30 d
There were no differences in the IR protein expressions in the livers of control and WD rats after 10 and 30 d (Fig. 2A). After 10 d of WD, there was no difference in insulin-stimulated IR tyrosine phosphorylation in the livers of WD and control animals (Fig. 2B). Despite this observation, animals fed on WD for 30 d showed a significantly reduced insulin-stimulated IR tyrosine phosphorylation in liver (Fig. 2B) when compared with WD10 and with control animals.
FIG. 2. Insulin signaling in liver of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; B, IP with -IR and IB with -pY; C, IP with -IRS-1 and IB with -IRS-1; D, IP with -IRS-1 and IB with -pY; E, IP with -IRS-1 and IB with -PI3-K; F, IP with -IRS-2 and IB with -IRS-2; G, IP with -IRS-2 and IB with -pY; H, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments, i.e. six different cohorts of rats fed the control diet or WD for 10 or 30 d. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD30+ vs. WD10+, P < 0.001; &, WD10 vs. C10 and WD30, P < 0.001.
In parallel to an increase in IRS-1 protein expression in the liver of WD10 (Fig. 2C), there was an increase in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 2D) and in IRS-1/PI3-K association in WD10 when compared with C10 (Fig. 2E). Animals fed on WD for 30 d presented IRS-1 protein levels similar to those of the C30; however, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1/PI3-K association were significantly lower than those of the C30 (Fig. 2, C–E).
There were no differences in IRS-2 protein levels (Fig. 2F), in insulin-stimulated tyrosine phosphorylation of IRS-2 (Fig. 2G), and in its association with the p85 subunit of PI3-K in the liver of WD10 compared with C10 (Fig. 2H). However, in WD30, there was a significant reduction in both insulin-stimulated IRS-2 tyrosine phosphorylation and in IRS-2/PI3-K association when compared with the C30 group and with WD10 (Fig. 2, G and H).
Akt protein levels did not differ among the groups in this study (Fig. 3A). Insulin-stimulated Akt serine phosphorylation was similar in WD10 and C10 but showed a significant decrease in WD30 compared with WD10, C10, and C30 rats (Fig. 3B).
FIG. 3. Insulin signaling in liver of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pJNK; D, immunoprecipitation (IP) with -IRS-1 and IB with -JNK1; E, IB with -IRS-1ser307. Data are means ± SEM of six independent experiments. , WD30+ vs. C30+ and WD10+, P < 0.001; #, WD30 vs. C30 and WD10, P < 0.001.
Liver from animals fed on WD for 10 d showed similar JNK phosphorylation/activation to C10 animals (Fig. 3C), accompanied by a similar degree of association between IRS-1/JNK1 (Fig. 3D), which probably led to similar IRS-1ser307 phosphorylation in WD10 and C10 (Fig. 3E). Animals who received WD for 30 d, however, demonstrated increased JNK activation, increased IRS-1/JNK1 association, and increased IRS-1ser307 phosphorylation in liver when compared with WD10, C10, and C30 animals (Fig. 3, C–E).
Insulin signaling in the muscle of animals fed on a WD for 10 and 30 d
There were no differences in the IR protein expression in the muscles of control and WD rats after 10 and 30 d (Fig. 4A). After 10 d of WD, there was no difference in insulin-stimulated IR tyrosine phosphorylation in the muscle of WD10 and C10 rats (Fig. 4B). However, animals fed with WD for 30 d showed a significantly reduced insulin-stimulated IR tyrosine phosphorylation in muscle (Fig. 4B) compared with C10, C30, and WD30 animals.
FIG. 4. Insulin signaling in muscle of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; B, IP with -IR and IB with -pY; C, IP with -IRS-1 and IB with -IRS-1; D, IP with -IRS-1 and IB with -pY; E, IP with -IRS-1 and IB with -PI3-K; F, IP with -IRS-2 and IB with -IRS-2; G, IP with -IRS-2 and IB with -pY; H, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+ and WD10+, P < 0.001; , WD30 vs. C30 and WD10, P < 0.001.
Similar tissue levels of IRS-1 were found in muscle from each group of animals studied (Fig. 4C). There was a progressive reduction in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 4D) and in IRS-1/PI3-K association (Fig. 4E) in muscle from animals fed with WD for 10 and 30 d when compared with their respective controls.
There were no differences in IRS-2 protein levels in the muscle of WD10 compared with C10 (Fig. 4F). In addition, there were no differences in insulin-stimulated tyrosine phosphorylation of IRS-2 (Fig. 4G) and in its association with the p85 subunit of PI3-K in muscle of WD10 compared with C10 (Fig. 4H). In WD30, however, there was a significant reduction in IRS-2 protein expression, in insulin-stimulated IRS-2 tyrosine phosphorylation, and in IRS-2/PI3-K association when compared with C10, C30, and WD10 rats (Fig. 4, G and H).
Akt protein levels did not differ among the groups studied (Fig. 5A). However, similar to observations made with IRS-1, there was also a progressive reduction in insulin-stimulated Akt serine phosphorylation in animals fed with WD for 10 and 30 d when compared with controls (Fig. 5B).
FIG. 5. Insulin signaling in muscle of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pJNK; D, immunoprecipitation (IP) with -IRS-1 and IB with -JNK1; E, IB with anti-IRS-1ser307. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; , WD10 vs. C10, P < 0.001; #, WD30+ vs. C30+ and WD10+, P < 0.001; &, WD30 vs. C30, P < 0.001; %, WD30 vs. C30 and WD10, P < 0.001.
Muscle from animals fed with WD for 10 and 30 d showed higher JNK activation than C10 and C30 rats, respectively (Fig. 5C), accompanied by significantly higher degrees of association between IRS-1/JNK1 (Fig. 5D) and by increased IRS-1ser307 phosphorylation (Fig. 5E).
Insulin signaling in adipose tissue of animals fed on a WD for 10 and 30 d
There were no differences in IR protein levels (Fig. 6A) or in insulin-induced IR tyrosine phosphorylation in adipose tissue of WD and control animals (Fig. 6B).
FIG. 6. Insulin signaling in adipose tissue of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; B, IP with -IR and IB with -pY; C, IP with -IRS-1 and IB with -IRS-1; D, IP with -IRS-1 and IB with -pY; E, IP with -IRS-1 and IB with -PI3-K; F, IP with -IRS-2 and IB with -IRS-2; G, IP with -IRS-2 and IB with -pY; H, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD10+ vs. C10+ and WD30+, P < 0.001.
Despite similar levels of IRS-1 in adipose tissue of animals fed with WD and control diet (Fig. 6C), there was a decrease in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 6D) and in IRS-1/PI3-K association (Fig. 6E) in WD10 and WD30 when compared with their respective control groups.
There were no differences in IRS-2 protein levels in adipose tissue of WD rats, compared with controls (Fig. 6F). However, there was an increase in insulin-stimulated IRS-2 tyrosine phosphorylation (Fig. 6G) and in IRS-2/PI3-K association (Fig. 6H) in WD10 when compared with C10. Conversely, animals fed with WD for 30 d presented insulin-stimulated IRS-2 tyrosine phosphorylation and IRS-2/PI3-K association similar to those of C30 animals (Fig. 6, G and H).
Akt protein levels showed no differences among the groups in this study (Fig. 7A). Insulin-stimulated Akt serine phosphorylation was increased in WD10 compared with C10 rats. However, Akt phosphorylation in adipose tissue from WD30 was similar to C30 animals (Fig. 7B). Basal forkhead transcription factor 1 (Foxo1) phosphorylation was increased in WD10 compared with the other groups (Fig. 7C). Insulin-induced Foxo1 phosphorylation was not evaluated because its maximal effect occurs later than Akt activation at a time when hypoglycemia occurs in the animal, which could introduce a bias in analysis. Adipose tissue from animals fed with WD for 10 and 30 d demonstrated similar JNK activation to those of control rats (Fig. 7D), accompanied by a similar degree of association between IRS-1/JNK1 (Fig. 7E), which probably led to similar IRS-1ser307 phosphorylation in control and WD groups (Fig. 7F).
FIG. 7. Insulin signaling in adipose tissue of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pFoxo1; D, IB with -pJNK; E, IP with -IRS-1 and IB with -JNK1; F, IB with -IRS-1ser307. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+ and WD30+, P < 0.001; #, WD10 vs. C10 and WD30, P < 0.001.
Insulin action in hypothalamus of animals fed on a WD for 10 and 30 d
The anorexigenic effect of insulin was measured in cannulated rats by recording both the 12-h food intake before and after ICV insulin or saline infusion. Before ICV insulin or saline infusion, the food intake was similar among the control animals, WD10, and WD30 (C10, 17.6 ± 2.5 g; WD10, 16.8 ± 2.2 g; C30, 17.5 ± 5.3 g; WD30, 17 ± 4.8 g; n = 8 for each group). However, after ICV insulin infusion, the food intake was different among the groups (C10, 7.4 ± 3.2 g; WD10, 13.6 ± 2.7 g; C30, 7.5 ± 3.5 g; WD30, 14.7 ± 3 g; n = 8 each group). As shown in Fig. 8A, the reduction in food intake after ICV insulin infusion occurred to a much lesser degree in rats fed on WD compared with control rats (Fig. 8A: 57.8 ± 7% for C10 vs. 18.8 ± 6.8% for WD10, P < 0.01; 55 ± 13% for C30 vs. 11.2 ± 4.2% for WD30, P < 0.01). After saline infusion, the food intake was similar among control animals, WD10, and WD30 (C10, 17 ± 1.8 g; WD10, 17.3 ± 2.0 g; C30, 16.8 ± 3.5 g; WD30, 16.4 ± 2.7 g; n = 8 each group).
FIG. 8. A, Insulin action in hypothalamus represented by percent reduction of food intake after ICV insulin injection (n = 8 rats in each group). B–I, Insulin signaling in hypothalamus of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. B, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; C, IP with -IR and IB with -pY; D, IP with -IRS-1 and IB with -IRS-1; E, IP with -IRS-1 and IB with -pY; F, IP with -IRS-1 and IB with -PI3-K; G, IP with -IRS-2 and IB with -IRS-2; H, IP with -IRS-2 and IB with -pY; I, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments (B–I). . WD10 vs. C10, P < 0.01; &, WD30 vs. C30, P < 0.01; *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD30+ vs. C30+ and WD10+, P < 0.001.
There were no differences in the IR protein expression in hypothalamus of controls and WD rats after 10 and 30 d (Fig. 8B). After 10 and 30 d of WD, there was a decrease in insulin-stimulated IR tyrosine phosphorylation in hypothalamus compared with control animals (Fig. 8C). Similar tissue levels of IRS-1 were found in hypothalamus of each group of animals studied (Fig. 8D). There was a progressive and significant reduction in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 8E) and in IRS-1/PI3-K association (Fig. 8F) in hypothalamus from animals fed with WD for 10 and 30 d when compared with their respective control groups.
There were no differences in IRS-2 protein levels in hypothalamus of rats fed with WD compared with controls (Fig. 8G). However, there was also a significant reduction in insulin-stimulated IRS-2 tyrosine phosphorylation (Fig. 8H) and in IRS-2/PI3-K association (Fig. 8I) in the hypothalamus of animals fed on WD for 10 and 30 d when compared with their controls.
Akt protein levels did not differ among the groups studied (Fig. 9A). Similar to observations with IRS-1 and -2, there was also a significant reduction in insulin-stimulated Akt serine phosphorylation in animals fed with WD for 10 and 30 d when compared with their controls (Fig. 9B). There was a decrease in insulin-induced Foxo1 phosphorylation in WD10 and WD30 compared with controls (Fig. 9C).
FIG. 9. Insulin signaling in hypothalamus of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pFoxo1, D, IB with anti-pJNK; E, immunoprecipitation (IP) with anti-IRS-1 and IB with anti-JNK1; F, IB with anti-IRS-1ser307. Data are expressed as means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD30+ vs. C30+ and WD10+, P < 0.001; , WD10 vs. C10, P < 0.001; &, WD30 vs. C30 and WD10, P < 0.001.
Hypothalamus from animals fed with WD showed higher JNK activation than control rats (Fig. 9D), accompanied by higher degrees of association between IRS-1/JNK1 (Fig. 9E) and by increased IRS-1ser307 phosphorylation (Fig. 9F).
Table 2 summarizes the main results of insulin signaling in the tissues studied.
TABLE 2. Insulin signaling in tissues of WD-fed rats
Discussion
In the present study, we have associated functional studies of insulin sensitivity with immunoblotting to investigate insulin action and insulin signaling in rats that received a (WD) for 10 or 30 d to investigate the temporal response to the diet in key insulin-sensitive tissues. To our knowledge, this is the first study that integrates functional insulin action data with molecular insulin signaling, as well as its modulation, in liver, muscle, adipose tissues, and hypothalamus, allowing us to determine the tissue-specific installation of insulin resistance in a model of diet-induced obesity.
Animals fed on WD for 10 d showed a 50% increase in their central fat depot, which was associated with insulin resistance, as manifested by a significant decrease in whole-body glucose disposal, with a decrease in insulin-stimulated skeletal muscle glucose uptake without any significant effect on hepatic glucose output. There was also an increase in plasma triacylglycerol levels, whereas fasting blood glucose and insulin levels did not change. These metabolic characteristics were accompanied by impairment in insulin signaling in muscle and hypothalamus. Conversely, 30 d of WD induced a more severe insulin resistance than 10 d of WD, characterized by a more pronounced decrease in whole-body glucose disposal and insulin-stimulated skeletal muscle glucose uptake, which were accompanied by a less effective action of insulin in suppressing the hepatic glucose output. In addition, there was an increase in body weight, hyperinsulinemia, and reduced insulin signaling in liver.
In WD10 rats, there was a substantial decrease in insulin-stimulated IRS-1/PI3-K/Akt pathway in muscle, which might have an important role in the insulin resistance of these animals, because this pathway has been implicated in glucose transport and in glycogen synthesis (28). Accordingly, in these animals, there was a decrease in insulin-stimulated skeletal muscle glucose uptake. Interestingly, a reduction in IRS-1 tyrosine phosphorylation, without changes in IRS-2, was sufficient to reduce Akt activation, reinforcing that the former is more important than the latter in modulation of Akt and insulin signaling in muscle (29).
WD for 10 d induced differential modulation of IRSs in liver, without affecting Akt activation. Although insulin-stimulated IRS-1 phosphorylation was higher in WD10 rats, higher contents of IRS-1 protein levels were observed in this tissue. However, the IRS-2/PI3-K/Akt pathway did not change, suggesting that insulin signaling is preserved in the liver of these animals, in parallel with a preserved insulin effect on the suppression of hepatic glucose output. In contrast, after 30 d, the IRS-1 and IRS-2 phosphorylation and the Akt activation were reduced in WD rats, suggesting impairment in insulin signaling in liver after 30 d of WD, accompanied by a less effective action of insulin on hepatic glucose output. In both cases, the Akt activation followed the same profile of IRS-2 phosphorylation. Previous data have shown that IRS-2 is more important than IRS-1 in liver for mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in this tissue (29).
Interestingly, despite the early insulin resistance in muscle and hypothalamus in rats fed with WD, the compensatory hyperinsulinemia was evident only when altered insulin signaling was installed in liver. This finding is in accordance with previous studies using the Cre-loxP system to disrupt the IR gene in specific tissues. The muscle-specific IR knockout mice exhibit a muscle-specific, greater-than-95% reduction in receptor content and early signaling events. These mice display elevated serum TG but normal serum insulin levels (30). Male mice with a neuron-specific disruption of the IR gene do not show a significant increase in insulin levels compared with controls (31). Conversely, the liver-specific IR knockout mice demonstrated blunted insulin signaling in liver as well as compensatory hyperinsulinemia (32). Taking these previous data together with our results, it may be suggested that altered insulin signaling in muscle and hypothalamus is not sufficient to induce compensatory hyperinsulinemia, but insulin resistance in liver is accompanied by hyperinsulinemia.
Despite the reduced glucose uptake in the muscle of WD10 rats, in the adipose tissue of these animals there was a significant increase in insulin-induced glucose transport. With regard to the insulin signaling in the adipose tissue of these animals, there was a differential modulation of IRS activation, with a decrease in IRS-1 and an increase in IRS-2 tyrosine phosphorylation, resulting in an increase in insulin-induced Akt activation. Previous studies point out that the IRS-2 regulation may predominate over IRS-1 in downstream insulin signaling in adipose tissue (33). In another model of insulin resistance, the monoglutamate-insulin-resistant rats, an increase in the IRS-2/PI3-K/Akt pathway in adipose tissue and increased adiposity was demonstrated (34). In addition, muscle-specific IR knockout mice display increased fat pad mass and increased insulin-stimulated glucose transport activity in epididymal white adipose tissue, indicating that the decrease in insulin-stimulated muscle glucose transport may be partially compensated for increases in insulin-stimulated glucose transport activity in adipose tissue (35). Taken together with the present data, it may be suggested that the increase in the IRS/PI3-K/Akt pathway may play an important role in the increased visceral adiposity described in WD10 rats. Recently, it has been demonstrated that Akt phosphorylates the Foxos (36) and inhibits their transcriptional activity (37). Foxo1 is the most abundant Foxo isoform in several insulin-responsive tissues, such as liver, adipose tissue, and pancreatic ?-cells. It was also demonstrated that Foxo1 plays an important role in coupling insulin signaling to adipocyte differentiation (38). The increased basal phosphorylation and inactivation of Foxo1 described in our study might have a role in the enhanced adiposity in the WD group.
Interestingly, in adipose tissue of WD30 animals, an increase in insulin-induced glucose transport in fat still occurred. However, early steps of insulin signaling were not increased. This result suggests that the molecular mechanism that accounts for this increase in glucose transport in the fat of WD30 rats is probably downstream from Akt, because there was no alteration in the IRS/PI3-K/Akt pathway. The association of the results obtained from muscle, liver, and adipose tissue suggest that, in the presence of reduced insulin action in muscle, and later in liver and muscle, glucose was partly shunted to white adipose tissue.
In this study, ICV insulin infusion significantly reduced food intake in controls (59%). In contrast, ICV insulin administration was less effective in reducing food intake in rats fed with WD10 (19%), and this effect was more intensive after 30 d of WD (11%). Thus, WD led to a significant impairment of the insulin-induced anorexigenic effect, which may characterize central resistance to insulin action. In parallel, we demonstrated that the sites of insulin signaling impairment appear to be at the IR and postreceptor levels in the hypothalamus from animals fed with WD.
Neuron-specific IR knockout mice (31) or rats with a selective decrease in IR expression in hypothalamic nuclei (achieved using an antisense oligodeoxynucleotide directed against the IR precursor protein) (39) demonstrated increased food intake and increased fat mass. The same result was observed in female IRS-2 knockout mice. These animals presented increased daily food intake, increased body weight, and adiposity. In addition, they demonstrated a disrupted control of hypothalamic neuropeptide level linkage to feeding (40). Our data suggest that the antiobesity actions induced by insulin in hypothalamus could be blunted because of partial inhibition of the PI3-K pathway, suggesting that neuronal PI3-K is important for the effects of insulin on food intake. If the mechanism used by insulin to reduce food intake is PI3-K dependent, as has been previously demonstrated (6, 41, 42, 43), the defective activation of PI3-K/Akt/Foxo1 in hypothalamic neurons could attenuate the ability of insulin to reduce food intake in WD rats.
There are probably a number of mechanisms that may lead to an impairment of the insulin-signaling pathway in muscle, liver, and hypothalamus of rats fed with WD. Previous studies demonstrated that an increase in IRS serine phosphorylation could induce marked insulin resistance, pointing to this as an important mechanism in the control of insulin signaling (44, 45, 46). It has been reported that activation of JNK induces serine 307 phosphorylation of IRS-1 (14), leading to a decrease in insulin-stimulated PI3-K activity. Our data showed an increase in JNK phosphorylation/activation, in agreement with altered insulin signaling in muscle and hypothalamus of WD10 and also in liver of WD30, suggesting that this serine kinase may have an important role in down-regulating insulin signaling in these tissues. In addition, we demonstrated an increase in IRS-1/JNK1 association in parallel with IRS-1ser307 phosphorylation with the same time course and tissue distribution of JNK activation. These data suggest that JNK may have an important role in the altered insulin signaling in tissues of rats fed with WD.
In summary, our data provide direct evidence that insulin resistance, induced by WD, is tissue specific. The findings also support the hypothesis that insulin can activate the PI3-K/Akt/Foxo1 pathway in the hypothalamus, which can be modulated by WD. In addition, the insulin resistance induced by WD seems to install first in hypothalamus and skeletal muscle, characterized by attenuation of the anorexigenic effect of insulin and reduced muscle glucose transport, respectively, and later in liver, with less suppressibility of hepatic glucose production. These temporal alterations in insulin action and signaling are accompanied by activation of JNK and IRS-1ser307 phosphorylation. This situation of an impaired PI3-K/Akt signaling pathway in the hypothalamus, muscle, and liver but not in adipose tissue may lead to the development of increased adiposity and insulin resistance in the WD rats.
Acknowledgments
We thank Mr. Luis Janieri, Mr. Márcio Alves da Cruz, and Mr. Jósimo Pinheiro for their technical assistance.
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Address all correspondence and requests for reprints to: M. J. A. Saad, Departamento de Clínica Médica, FCM-UNICAMP, Cidade Universitária, Campinas, S?o Paulo 13083-970, Brazil. E-mail: msaad@fcm.unicamp.br.
Abstract
The mechanisms by which diet-induced obesity is associated with insulin resistance are not well established, and no study has until now integrated, in a temporal manner, functional insulin action data with insulin signaling in key insulin-sensitive tissues, including the hypothalamus. In this study, we evaluated the regulation of insulin sensitivity by hyperinsulinemic-euglycemic clamp procedures and insulin signaling, c-jun N-terminal kinase (JNK) activation and insulin receptor substrate (IRS)-1ser307 phosphorylation in liver, muscle, adipose tissue, and hypothalamus, by immunoprecipitation and immunoblotting, in rats fed on a Western diet (WD) or control diet for 10 or 30 d. WD increased visceral adiposity, serum triacylglycerol, and insulin levels and reduced whole-body glucose use. After 10 d of WD (WD10) there was a decrease in IRS-1/phosphatidylinositol 3-kinase/protein kinase B pathway in hypothalamus and muscle, associated with an attenuation of the anorexigenic effect of insulin in the former and reduced glucose transport in the latter. In WD10, there was an increased glucose transport in adipose tissue in parallel to increased insulin signaling in this tissue. After 30 d of WD, insulin was less effective in suppressing hepatic glucose production, and this was associated with a decrease in insulin signaling in the liver. JNK activity and IRS-1ser307 phosphorylation were higher in insulin-resistant tissues. In summary, the insulin resistance induced by WD is tissue specific and installs first in hypothalamus and muscle and later in liver, accompanied by activation of JNK and IRS-1ser307 phosphorylation. The impairment of the insulin signaling in these tissues, but not in adipose tissue, may lead to increased adiposity and insulin resistance in the WD rats.
Introduction
IN RODENTS AND HUMANS, dietary intake of high amounts of fat has been shown to have adverse effects on insulin sensitivity. Indeed, a more palatable cafeteria diet, which is closer to the Western diet (WD), has also been associated with increased adiposity and insulin resistance in humans (1, 2). However, the exact mechanism by which WD can induce obesity and insulin resistance is not yet known. In this context, insulin resistance could be tissue-specific to the muscle, liver, and hypothalamus, whereas adipose tissue remains sensitive to insulin.
At the molecular level, the insulin signaling begins when activation of insulin receptor (IR) results in tyrosine phosphorylation of several substrates, including the IR substrate 1 (IRS-1) and IRS-2 (3). After tyrosine phosphorylation, IRS-1 and IRS-2 bind and activate the enzyme phosphatidylinositol 3-kinase (PI3-K) (3, 4). The activation of PI3-K increases serine phosphorylation of protein kinase B (Akt), which in turn stimulates the glucose transport in the muscle and adipose tissue, stimulates glycogen synthesis in the liver and muscle, and stimulates lipogenesis in the adipose tissue. Therefore, the PI3-K/Akt pathway has an important role in the metabolic effects of insulin. In the hypothalamus, the activated PI3-K/Akt pathway suppresses feeding, and insulin resistance in this tissue has recently been described (5, 6).
Many mechanisms may contribute to the dysregulation of the insulin-signaling pathway, including serine phosphorylation of IRS proteins by protein kinases such as c-jun N-terminal kinase (JNK) (7). JNK is a member of the MAPK family (8, 9) and can be activated by TNF (10) and IL-1? (11, 12). In addition, JNK might serve as a feedback inhibitor during insulin stimulation (7). There are three JNK isoforms described, named JNK1, -2, and -3 (13), of which JNK1 is the most involved in the pathophysiology of obesity and insulin resistance (10). JNK activation induces inhibitory serine 307 (Ser307) phosphorylation of IRS-1, as shown in previous studies (7, 14). Ser307 is located next to the phosphotyrosine (pY)-binding domain in IRS-1 and its phosphorylation inhibits the interaction of the pY-binding domain with the phosphorylated NPEY motif in the activated IR, causing insulin resistance (14). Here, we investigate the effects of WD on insulin signaling in liver, skeletal muscle, hypothalamus, and adipose tissue of Wistar rats, given during 10 or 30 d, and the role of JNK activation and IRS-1ser307 in the dysregulation of the insulin-signaling pathway in these tissues. In parallel, we have also conducted functional studies of insulin sensitivity by hyperinsulinemic-euglycemic clamp procedures in combination with tracer infusions, which allowed us to isolate the hepatic, skeletal muscle, and adipose tissue components of insulin action and to associate these data with insulin signaling in each tissue.
Materials and Methods
Materials
Male Wistar rats were provided by the State University of Campinas Central Breeding Center (Campinas, Brazil). Anti-pY (-pY), anti-IR? (-IR), anti-IRS-1, anti-Akt1/2, anti-pJNK, and anti-JNK1 antibodies were from Santa Cruz Technology (Santa Cruz, CA). Anti-pAkt and anti-pFoxo1 were from Cell Signaling Technology (Beverly, MA). Anti-PI3-K, anti-IRS-2, and anti-phospho-IRS-1ser307 were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Human recombinant insulin was from Eli Lilly and Co. (Indianapolis, IN). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless specified elsewhere. [125I]Protein A, HPLC-purified [3-3H]glucose and 2-deoxy-D-[1-14C]glucose (2-[14C]DG1) were obtained from Amersham (Amersham Biosciences Group, Little Chalfont, UK). The Harvard apparatus (model 11) and Harvard compact infusion pumps (model 975) were obtained from South Natick, MA.
Animals
All experiments were approved by the Ethics Committee at the State University of Campinas.
Eight-week-old male Wistar rats were divided into two groups with similar body weights (255 ± 4 g) and assigned to receive two kinds of diet: a standard rodent chow and water ad libitum or WD for 10 and 30 d (C10, C30, WD10, and WD30, respectively). WD was adapted from a previous study (15) and included soft drinks ad libitum alternated daily (Coca-Cola and Guaraná Antarctica) and 37.5% standard rodent chow, 25% peanuts, 25% chocolate, and 12.5% cookies. In addition, these rats were offered specific amounts of palatable food items such as biscuits, cakes, and cookies totaling 5.4 kcal/g (carbohydrate, 38.5%; protein, 15%; fat, 46.5%) as opposed to the 3.8 kcal/g (carbohydrate, 70%; protein, 20%; fat, 10%) of the standard chow diet. The nutritional status of the rats was not affected by the WD regimen, as demonstrated by similar values of serum albumin, calcium, magnesium, phosphorus, iron, folate, and vitamin B12 in preliminary experiments (data not shown).
Each day, leftovers were collected and replaced with new items. This was a key point of our experiment that assured success of the hypercaloric regimen.
Animal characterization
At the end of the diet period, body weight, epididymal, retroperitoneal, and mesenteric fat pad were weighed. Food was withdrawn 12–14 h before the experiments and blood samples were taken for the determination of serum concentration of total cholesterol, high-density lipoprotein (HDL)-cholesterol, triacylglycerol (TG), and insulin. Lipids were measured in a routine diagnostic analyzer (Modular, Roche Diagnostics, Mannheim, Germany) using for cholesterol (CHOD-PAP, cholesterol oxidase-peroxidase enzymatic colorimetric assay) and for triacylglycerol (GPO-PAP, glycerol-3-phosphate oxidase enzymatic colorimetric assay). Blood glucose levels were measured by the glucose oxidase method (16). Serum insulin was determined by RIA.
Hyperinsulinemic-euglycemic clamp procedures
After 5 h fasting, animals were anesthetized ip with sodium pentobarbital (50 mg/kg body weight), and catheters were then inserted into the left jugular vein (for tracer infusions) and carotid artery (for blood sampling), as previously described (17). Each animal was monitored for food intake and weight gain for 5 d after surgery to ensure complete recovery. Food was removed for 12 h before the beginning of in vivo studies. A 120-min hyperinsulinemic-euglycemic clamp procedure was conducted in conscious, unrestrained, catheterized rats, as shown previously (18, 19), with a prime continuous infusion of human insulin at a rate of 3.6 mU/kg body weight per minute to raise the plasma insulin concentration to approximately 800–900 pmol/liter. Blood samples (20 μl) were collected at 5-min intervals for the immediate measurement of plasma glucose concentration, and 10% unlabeled glucose was infused at variable rates to maintain plasma glucose at fasting levels. Insulin-stimulated whole-body glucose flux was estimated using a prime continuous infusion of HPLC-purified [3-3H]glucose (10 μCi bolus, 0.1 μCi/min) throughout the clamp procedure (20). To estimate insulin-stimulated glucose-transport activity and metabolism in skeletal muscle and visceral fat, 2-[14C]DG1 was administered as a bolus (10 μCi) 45 min before the end of the clamp procedure. Blood samples (20 μl) were taken at 80, 90, 100, 110, and 120 min after the start of the clamp procedure to determine plasma [3H]glucose and 2-[14C]DG1 concentrations. Additional blood samples (10 μl) were collected before the start and at the end of the clamp procedure for measurement of plasma insulin concentrations. All infusions were performed using Harvard infusion pumps. At the end of the clamp procedure, animals were killed by a sodium pentobarbital iv injection. Within 2 min, two skeletal muscles (soleus and gastrocnemius) from both hind limbs and epididymal white adipose tissue were taken. Each tissue, once exposed, was dissected out within 2 sec, weighed, frozen with liquid N2, and stored at –70 C for later analysis. In separate experiments, the basal rates of glucose turnover were measured by continuously infusing [3-3H]glucose (0.02 μCi/min) for 120 min, and blood samples (20 μl) were taken at 100, 110, and 120 min after the start of the experiment for the determination of plasma [3H]glucose concentration.
Analytical procedures of hyperinsulinemic-euglycemic clamp
Plasma glucose was measured using a glucometer (Advantage, Roche Molecular Biochemicals). Plasma tracer samples were deproteinized with equal volumes of barium hydroxide and zinc sulfate (0.03 N) and stored overnight at 4 C. Radioactivity of [3-3H]glucose in plasma was measured from supernatants of Ba(OH)2/ZnSO4 precipitates, after each was evaporated to dryness for the removal of tritiated water. Rates of whole-body glucose uptake and basal glucose turnover were determined as the ratio of the [3-3H]glucose infusion rate (disintegrations per minute) to the specific activity of plasma glucose (disintegrations per minute per milligram glucose) during the final 30 min of respective experiments under steady-state conditions. The hepatic glucose output (HGO) during the clamp procedure was therefore obtained from the difference between the whole-body glucose uptake and the rate of unlabeled glucose infusion. Glucose transport activity in skeletal muscle and fat was calculated from the plasma 2-[14C]DG profile, as described before (21, 22).
Tissue extraction and immunoprecipitation
Rats were anesthetized with sodium thiopental and used 10–15 min later. As soon as anesthesia was assured by the loss of pedal and corneal reflexes, the abdominal cavity was opened, the portal vein was exposed, and 0.2 ml normal saline with or without insulin (2 μg) was injected. At 30 sec after the insulin injection, the liver was removed, and 90 sec later, muscle and adipose tissue were removed, minced coarsely, and homogenized immediately in extraction buffer, as described elsewhere (23). Extracts were then centrifuged at 15,000 rpm and 4 C for 40 min to remove insoluble material, and the supernatants were used for immunoprecipitation with -IR, -IRS-1 and -2, and protein A-Sepharose 6MB (Pharmacia, Uppsala, Sweden).
Protein analysis by immunoblotting
The precipitated proteins and/or whole-tissue extracts were treated with Laemmli sample buffer (24) containing 100 mM dithiothreitol and heated in a boiling water bath for 5 min, after which they were subjected to SDS-PAGE in a Bio-Rad miniature slab gel apparatus (Mini-Protean). For total extracts, 250 μg of proteins were subjected to SDS-PAGE. Electrotransfer of proteins from the gel to nitrocellulose was performed for 120 min at 120 V in a Bio-Rad Mini-Protean transfer apparatus (25). Nonspecific protein binding to the nitrocellulose was reduced by preincubating the filter for 2 h in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, 0.02% Tween 20). The nitrocellulose blot was incubated with specific antibodies overnight at 4 C and then incubated with 125I-labeled protein A. The results were visualized by autoradiography with preflashed Kodak XAR film. Band intensities were quantified by optical densitometry (Hoefer Scientific Instruments, San Francisco, CA; model GS300).
Hypothalamus study
Briefly, the animals were anesthetized with sodium thiopental, as described above, and 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 preestablished coordinates: anteroposterior, 0.2 mm from bregma; lateral, 1.5 mm; and vertical, 4.2 mm (23, 26). Rats were allowed 5 d of recovery before testing for cannula patency and position. Cannulas were considered patent and correctly positioned by dypsogenic response elicited after angiotensin II injection (27). For evaluation of the anorexigenic effect of insulin, we first measured the 12-h food intake before intracerebroventricular (ICV) insulin or vehicle (saline) infusion. Cannulated rats were then treated with an ICV infusion of 2 μl of 10–6 M insulin or 2 μl of saline. Treatment occurred invariably at 1800 h, and food intake was measured over the following 12 h (23). The following day, fasted cannulated rats from different groups received 2 μl of saline or 10–6 M insulin ICV and the hypothalamus was removed 15 min later (23). Tissue samples underwent the same procedures as the other tissues described above.
Statistical analysis
Data were expressed as means ± SEM accompanied by the indicated number of independent experiments. For statistical analysis, the groups were compared using a two-way ANOVA with the Bonferroni test for post hoc comparisons. The level of significance adopted was P < 0.05.
Results
Animal characteristics
Table 1 shows that the body weight was similar in the two groups after 10 d of WD. However, after 30 d, the body weight was higher in the WD group compared with the respective control group (P < 0.05). The animals fed with the WD displayed higher contents of fat mass in different sites. The epididymal, retroperitoneal, and mesenteric fat were heavier (P < 0.05) after 10 and 30 d of WD, compared with their control groups. There was no difference in fasting blood glucose and insulin levels after 10 d of WD compared with the control group. However, in WD30, the fasting insulin levels were higher (P < 0.05) than in C30, despite the normal blood glucose levels. The serum concentrations of TG were significantly higher in the WD10 and WD30 compared with their respective controls. The serum cholesterol levels were not different between the groups; neither were the serum HDL-cholesterol levels.
TABLE 1. Animal characteristics
Hyperinsulinemic-euglycemic clamp procedures
A hyperinsulinemic-euglycemic clamp procedure with tracer infusions was performed to examine the effects of WD on the metabolism of glucose in liver, skeletal muscle, and adipose tissue (Fig. 1). The glucose infusion rate needed to clamp glycemia at fasting levels in the presence of a constant infusion of insulin (3.6 mU/kg body weight·min) was more than 2-fold lower in rats fed on WD than in their respective controls (Fig. 1A). Accordingly, the insulin-stimulated whole-body glucose-disposal rates were also significantly decreased in rats fed on WD for 10 and 30 d compared with their respective controls, as depicted in Fig. 1B. However, insulin was not able to reduce the hepatic glucose output by the same amount in all groups of animals. In Fig. 1C, it may be observed that insulin was able to reduce the HGO in both controls (C10 and C30) and in WD10. However, its ability to suppress HGO was impaired in WD30 when compared with controls and WD10.
FIG. 1. A, Steady-state glucose infusion rates obtained from averaged rates of 90–120 min of 10% unlabeled glucose infusion during hyperinsulinemic-euglycemic clamp procedures in the control rats (C10 and C30) and in rats fed on WD for 10 (WD10) and 30 (WD30) d; means ± SEM, n = 4; *, WD10 vs. C10, P < 0.05; #, WD30 vs. C30, P < 0.01. B, Insulin-stimulated whole-body glucose transport during hyperinsulinemic-euglycemic clamp procedure in awake rats; means ± SEM, n = 4; *, WD10 vs. C10, P < 0.01; #, WD30 vs. C30, P < 0.01. C, Basal and insulin-stimulated rates of hepatic glucose production during the hyperinsulinemic-euglycemic clamp procedures in awake rats; means ± SEM, n = 4; *, C10+ vs. C10–, P < 0.05; #, WD10+ vs. WD10–, P < 0.01; , C30+ vs. C30–, P < 0.01. D, Glucose transport in skeletal muscle and in adipose tissue evaluated by 2DG uptake during the last 45 min of the hyperinsulinemic-euglycemic clamp studies; means ± SEM, n = 4; *, WD10 vs. C10, P < 0.001; #, WD30 vs. C30, P < 0.01.
Using 2DG uptake analysis, the insulin-stimulated glucose uptake in skeletal muscle and adipose tissue was quantified (Fig. 1D). As shown, WD10 and WD30 presented significant reductions in skeletal muscle glucose uptake, and a more severe reduction was observed in WD30 rats (80% reduction compared with C30). In contrast to the inhibition of glucose uptake in the muscle of WD rats, the adipose tissue showed a significantly higher glucose uptake (at least 2-fold) both in WD10 and WD30, when compared with their respective controls.
Insulin signaling in the liver of animals fed on a WD for 10 and 30 d
There were no differences in the IR protein expressions in the livers of control and WD rats after 10 and 30 d (Fig. 2A). After 10 d of WD, there was no difference in insulin-stimulated IR tyrosine phosphorylation in the livers of WD and control animals (Fig. 2B). Despite this observation, animals fed on WD for 30 d showed a significantly reduced insulin-stimulated IR tyrosine phosphorylation in liver (Fig. 2B) when compared with WD10 and with control animals.
FIG. 2. Insulin signaling in liver of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; B, IP with -IR and IB with -pY; C, IP with -IRS-1 and IB with -IRS-1; D, IP with -IRS-1 and IB with -pY; E, IP with -IRS-1 and IB with -PI3-K; F, IP with -IRS-2 and IB with -IRS-2; G, IP with -IRS-2 and IB with -pY; H, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments, i.e. six different cohorts of rats fed the control diet or WD for 10 or 30 d. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD30+ vs. WD10+, P < 0.001; &, WD10 vs. C10 and WD30, P < 0.001.
In parallel to an increase in IRS-1 protein expression in the liver of WD10 (Fig. 2C), there was an increase in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 2D) and in IRS-1/PI3-K association in WD10 when compared with C10 (Fig. 2E). Animals fed on WD for 30 d presented IRS-1 protein levels similar to those of the C30; however, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1/PI3-K association were significantly lower than those of the C30 (Fig. 2, C–E).
There were no differences in IRS-2 protein levels (Fig. 2F), in insulin-stimulated tyrosine phosphorylation of IRS-2 (Fig. 2G), and in its association with the p85 subunit of PI3-K in the liver of WD10 compared with C10 (Fig. 2H). However, in WD30, there was a significant reduction in both insulin-stimulated IRS-2 tyrosine phosphorylation and in IRS-2/PI3-K association when compared with the C30 group and with WD10 (Fig. 2, G and H).
Akt protein levels did not differ among the groups in this study (Fig. 3A). Insulin-stimulated Akt serine phosphorylation was similar in WD10 and C10 but showed a significant decrease in WD30 compared with WD10, C10, and C30 rats (Fig. 3B).
FIG. 3. Insulin signaling in liver of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pJNK; D, immunoprecipitation (IP) with -IRS-1 and IB with -JNK1; E, IB with -IRS-1ser307. Data are means ± SEM of six independent experiments. , WD30+ vs. C30+ and WD10+, P < 0.001; #, WD30 vs. C30 and WD10, P < 0.001.
Liver from animals fed on WD for 10 d showed similar JNK phosphorylation/activation to C10 animals (Fig. 3C), accompanied by a similar degree of association between IRS-1/JNK1 (Fig. 3D), which probably led to similar IRS-1ser307 phosphorylation in WD10 and C10 (Fig. 3E). Animals who received WD for 30 d, however, demonstrated increased JNK activation, increased IRS-1/JNK1 association, and increased IRS-1ser307 phosphorylation in liver when compared with WD10, C10, and C30 animals (Fig. 3, C–E).
Insulin signaling in the muscle of animals fed on a WD for 10 and 30 d
There were no differences in the IR protein expression in the muscles of control and WD rats after 10 and 30 d (Fig. 4A). After 10 d of WD, there was no difference in insulin-stimulated IR tyrosine phosphorylation in the muscle of WD10 and C10 rats (Fig. 4B). However, animals fed with WD for 30 d showed a significantly reduced insulin-stimulated IR tyrosine phosphorylation in muscle (Fig. 4B) compared with C10, C30, and WD30 animals.
FIG. 4. Insulin signaling in muscle of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; B, IP with -IR and IB with -pY; C, IP with -IRS-1 and IB with -IRS-1; D, IP with -IRS-1 and IB with -pY; E, IP with -IRS-1 and IB with -PI3-K; F, IP with -IRS-2 and IB with -IRS-2; G, IP with -IRS-2 and IB with -pY; H, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+ and WD10+, P < 0.001; , WD30 vs. C30 and WD10, P < 0.001.
Similar tissue levels of IRS-1 were found in muscle from each group of animals studied (Fig. 4C). There was a progressive reduction in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 4D) and in IRS-1/PI3-K association (Fig. 4E) in muscle from animals fed with WD for 10 and 30 d when compared with their respective controls.
There were no differences in IRS-2 protein levels in the muscle of WD10 compared with C10 (Fig. 4F). In addition, there were no differences in insulin-stimulated tyrosine phosphorylation of IRS-2 (Fig. 4G) and in its association with the p85 subunit of PI3-K in muscle of WD10 compared with C10 (Fig. 4H). In WD30, however, there was a significant reduction in IRS-2 protein expression, in insulin-stimulated IRS-2 tyrosine phosphorylation, and in IRS-2/PI3-K association when compared with C10, C30, and WD10 rats (Fig. 4, G and H).
Akt protein levels did not differ among the groups studied (Fig. 5A). However, similar to observations made with IRS-1, there was also a progressive reduction in insulin-stimulated Akt serine phosphorylation in animals fed with WD for 10 and 30 d when compared with controls (Fig. 5B).
FIG. 5. Insulin signaling in muscle of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pJNK; D, immunoprecipitation (IP) with -IRS-1 and IB with -JNK1; E, IB with anti-IRS-1ser307. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; , WD10 vs. C10, P < 0.001; #, WD30+ vs. C30+ and WD10+, P < 0.001; &, WD30 vs. C30, P < 0.001; %, WD30 vs. C30 and WD10, P < 0.001.
Muscle from animals fed with WD for 10 and 30 d showed higher JNK activation than C10 and C30 rats, respectively (Fig. 5C), accompanied by significantly higher degrees of association between IRS-1/JNK1 (Fig. 5D) and by increased IRS-1ser307 phosphorylation (Fig. 5E).
Insulin signaling in adipose tissue of animals fed on a WD for 10 and 30 d
There were no differences in IR protein levels (Fig. 6A) or in insulin-induced IR tyrosine phosphorylation in adipose tissue of WD and control animals (Fig. 6B).
FIG. 6. Insulin signaling in adipose tissue of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; B, IP with -IR and IB with -pY; C, IP with -IRS-1 and IB with -IRS-1; D, IP with -IRS-1 and IB with -pY; E, IP with -IRS-1 and IB with -PI3-K; F, IP with -IRS-2 and IB with -IRS-2; G, IP with -IRS-2 and IB with -pY; H, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD10+ vs. C10+ and WD30+, P < 0.001.
Despite similar levels of IRS-1 in adipose tissue of animals fed with WD and control diet (Fig. 6C), there was a decrease in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 6D) and in IRS-1/PI3-K association (Fig. 6E) in WD10 and WD30 when compared with their respective control groups.
There were no differences in IRS-2 protein levels in adipose tissue of WD rats, compared with controls (Fig. 6F). However, there was an increase in insulin-stimulated IRS-2 tyrosine phosphorylation (Fig. 6G) and in IRS-2/PI3-K association (Fig. 6H) in WD10 when compared with C10. Conversely, animals fed with WD for 30 d presented insulin-stimulated IRS-2 tyrosine phosphorylation and IRS-2/PI3-K association similar to those of C30 animals (Fig. 6, G and H).
Akt protein levels showed no differences among the groups in this study (Fig. 7A). Insulin-stimulated Akt serine phosphorylation was increased in WD10 compared with C10 rats. However, Akt phosphorylation in adipose tissue from WD30 was similar to C30 animals (Fig. 7B). Basal forkhead transcription factor 1 (Foxo1) phosphorylation was increased in WD10 compared with the other groups (Fig. 7C). Insulin-induced Foxo1 phosphorylation was not evaluated because its maximal effect occurs later than Akt activation at a time when hypoglycemia occurs in the animal, which could introduce a bias in analysis. Adipose tissue from animals fed with WD for 10 and 30 d demonstrated similar JNK activation to those of control rats (Fig. 7D), accompanied by a similar degree of association between IRS-1/JNK1 (Fig. 7E), which probably led to similar IRS-1ser307 phosphorylation in control and WD groups (Fig. 7F).
FIG. 7. Insulin signaling in adipose tissue of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pFoxo1; D, IB with -pJNK; E, IP with -IRS-1 and IB with -JNK1; F, IB with -IRS-1ser307. Data are means ± SEM of six independent experiments. *, WD10+ vs. C10+ and WD30+, P < 0.001; #, WD10 vs. C10 and WD30, P < 0.001.
Insulin action in hypothalamus of animals fed on a WD for 10 and 30 d
The anorexigenic effect of insulin was measured in cannulated rats by recording both the 12-h food intake before and after ICV insulin or saline infusion. Before ICV insulin or saline infusion, the food intake was similar among the control animals, WD10, and WD30 (C10, 17.6 ± 2.5 g; WD10, 16.8 ± 2.2 g; C30, 17.5 ± 5.3 g; WD30, 17 ± 4.8 g; n = 8 for each group). However, after ICV insulin infusion, the food intake was different among the groups (C10, 7.4 ± 3.2 g; WD10, 13.6 ± 2.7 g; C30, 7.5 ± 3.5 g; WD30, 14.7 ± 3 g; n = 8 each group). As shown in Fig. 8A, the reduction in food intake after ICV insulin infusion occurred to a much lesser degree in rats fed on WD compared with control rats (Fig. 8A: 57.8 ± 7% for C10 vs. 18.8 ± 6.8% for WD10, P < 0.01; 55 ± 13% for C30 vs. 11.2 ± 4.2% for WD30, P < 0.01). After saline infusion, the food intake was similar among control animals, WD10, and WD30 (C10, 17 ± 1.8 g; WD10, 17.3 ± 2.0 g; C30, 16.8 ± 3.5 g; WD30, 16.4 ± 2.7 g; n = 8 each group).
FIG. 8. A, Insulin action in hypothalamus represented by percent reduction of food intake after ICV insulin injection (n = 8 rats in each group). B–I, Insulin signaling in hypothalamus of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. B, Immunoprecipitation (IP) with -IR and immunoblotting (IB) with -IR antibodies; C, IP with -IR and IB with -pY; D, IP with -IRS-1 and IB with -IRS-1; E, IP with -IRS-1 and IB with -pY; F, IP with -IRS-1 and IB with -PI3-K; G, IP with -IRS-2 and IB with -IRS-2; H, IP with -IRS-2 and IB with -pY; I, IP with -IRS-2 and IB with -PI3-K. Data are means ± SEM of six independent experiments (B–I). . WD10 vs. C10, P < 0.01; &, WD30 vs. C30, P < 0.01; *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD30+ vs. C30+ and WD10+, P < 0.001.
There were no differences in the IR protein expression in hypothalamus of controls and WD rats after 10 and 30 d (Fig. 8B). After 10 and 30 d of WD, there was a decrease in insulin-stimulated IR tyrosine phosphorylation in hypothalamus compared with control animals (Fig. 8C). Similar tissue levels of IRS-1 were found in hypothalamus of each group of animals studied (Fig. 8D). There was a progressive and significant reduction in insulin-stimulated IRS-1 tyrosine phosphorylation (Fig. 8E) and in IRS-1/PI3-K association (Fig. 8F) in hypothalamus from animals fed with WD for 10 and 30 d when compared with their respective control groups.
There were no differences in IRS-2 protein levels in hypothalamus of rats fed with WD compared with controls (Fig. 8G). However, there was also a significant reduction in insulin-stimulated IRS-2 tyrosine phosphorylation (Fig. 8H) and in IRS-2/PI3-K association (Fig. 8I) in the hypothalamus of animals fed on WD for 10 and 30 d when compared with their controls.
Akt protein levels did not differ among the groups studied (Fig. 9A). Similar to observations with IRS-1 and -2, there was also a significant reduction in insulin-stimulated Akt serine phosphorylation in animals fed with WD for 10 and 30 d when compared with their controls (Fig. 9B). There was a decrease in insulin-induced Foxo1 phosphorylation in WD10 and WD30 compared with controls (Fig. 9C).
FIG. 9. Insulin signaling in hypothalamus of control animals (C10 and C30) and rats fed on WD for 10 (WD10) and 30 (WD30) d. A, Immunoblotting (IB) with -Akt1/2 antibodies; B, IB with -pAkt; C, IB with -pFoxo1, D, IB with anti-pJNK; E, immunoprecipitation (IP) with anti-IRS-1 and IB with anti-JNK1; F, IB with anti-IRS-1ser307. Data are expressed as means ± SEM of six independent experiments. *, WD10+ vs. C10+, P < 0.001; #, WD30+ vs. C30+, P < 0.001; %, WD30+ vs. C30+ and WD10+, P < 0.001; , WD10 vs. C10, P < 0.001; &, WD30 vs. C30 and WD10, P < 0.001.
Hypothalamus from animals fed with WD showed higher JNK activation than control rats (Fig. 9D), accompanied by higher degrees of association between IRS-1/JNK1 (Fig. 9E) and by increased IRS-1ser307 phosphorylation (Fig. 9F).
Table 2 summarizes the main results of insulin signaling in the tissues studied.
TABLE 2. Insulin signaling in tissues of WD-fed rats
Discussion
In the present study, we have associated functional studies of insulin sensitivity with immunoblotting to investigate insulin action and insulin signaling in rats that received a (WD) for 10 or 30 d to investigate the temporal response to the diet in key insulin-sensitive tissues. To our knowledge, this is the first study that integrates functional insulin action data with molecular insulin signaling, as well as its modulation, in liver, muscle, adipose tissues, and hypothalamus, allowing us to determine the tissue-specific installation of insulin resistance in a model of diet-induced obesity.
Animals fed on WD for 10 d showed a 50% increase in their central fat depot, which was associated with insulin resistance, as manifested by a significant decrease in whole-body glucose disposal, with a decrease in insulin-stimulated skeletal muscle glucose uptake without any significant effect on hepatic glucose output. There was also an increase in plasma triacylglycerol levels, whereas fasting blood glucose and insulin levels did not change. These metabolic characteristics were accompanied by impairment in insulin signaling in muscle and hypothalamus. Conversely, 30 d of WD induced a more severe insulin resistance than 10 d of WD, characterized by a more pronounced decrease in whole-body glucose disposal and insulin-stimulated skeletal muscle glucose uptake, which were accompanied by a less effective action of insulin in suppressing the hepatic glucose output. In addition, there was an increase in body weight, hyperinsulinemia, and reduced insulin signaling in liver.
In WD10 rats, there was a substantial decrease in insulin-stimulated IRS-1/PI3-K/Akt pathway in muscle, which might have an important role in the insulin resistance of these animals, because this pathway has been implicated in glucose transport and in glycogen synthesis (28). Accordingly, in these animals, there was a decrease in insulin-stimulated skeletal muscle glucose uptake. Interestingly, a reduction in IRS-1 tyrosine phosphorylation, without changes in IRS-2, was sufficient to reduce Akt activation, reinforcing that the former is more important than the latter in modulation of Akt and insulin signaling in muscle (29).
WD for 10 d induced differential modulation of IRSs in liver, without affecting Akt activation. Although insulin-stimulated IRS-1 phosphorylation was higher in WD10 rats, higher contents of IRS-1 protein levels were observed in this tissue. However, the IRS-2/PI3-K/Akt pathway did not change, suggesting that insulin signaling is preserved in the liver of these animals, in parallel with a preserved insulin effect on the suppression of hepatic glucose output. In contrast, after 30 d, the IRS-1 and IRS-2 phosphorylation and the Akt activation were reduced in WD rats, suggesting impairment in insulin signaling in liver after 30 d of WD, accompanied by a less effective action of insulin on hepatic glucose output. In both cases, the Akt activation followed the same profile of IRS-2 phosphorylation. Previous data have shown that IRS-2 is more important than IRS-1 in liver for mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in this tissue (29).
Interestingly, despite the early insulin resistance in muscle and hypothalamus in rats fed with WD, the compensatory hyperinsulinemia was evident only when altered insulin signaling was installed in liver. This finding is in accordance with previous studies using the Cre-loxP system to disrupt the IR gene in specific tissues. The muscle-specific IR knockout mice exhibit a muscle-specific, greater-than-95% reduction in receptor content and early signaling events. These mice display elevated serum TG but normal serum insulin levels (30). Male mice with a neuron-specific disruption of the IR gene do not show a significant increase in insulin levels compared with controls (31). Conversely, the liver-specific IR knockout mice demonstrated blunted insulin signaling in liver as well as compensatory hyperinsulinemia (32). Taking these previous data together with our results, it may be suggested that altered insulin signaling in muscle and hypothalamus is not sufficient to induce compensatory hyperinsulinemia, but insulin resistance in liver is accompanied by hyperinsulinemia.
Despite the reduced glucose uptake in the muscle of WD10 rats, in the adipose tissue of these animals there was a significant increase in insulin-induced glucose transport. With regard to the insulin signaling in the adipose tissue of these animals, there was a differential modulation of IRS activation, with a decrease in IRS-1 and an increase in IRS-2 tyrosine phosphorylation, resulting in an increase in insulin-induced Akt activation. Previous studies point out that the IRS-2 regulation may predominate over IRS-1 in downstream insulin signaling in adipose tissue (33). In another model of insulin resistance, the monoglutamate-insulin-resistant rats, an increase in the IRS-2/PI3-K/Akt pathway in adipose tissue and increased adiposity was demonstrated (34). In addition, muscle-specific IR knockout mice display increased fat pad mass and increased insulin-stimulated glucose transport activity in epididymal white adipose tissue, indicating that the decrease in insulin-stimulated muscle glucose transport may be partially compensated for increases in insulin-stimulated glucose transport activity in adipose tissue (35). Taken together with the present data, it may be suggested that the increase in the IRS/PI3-K/Akt pathway may play an important role in the increased visceral adiposity described in WD10 rats. Recently, it has been demonstrated that Akt phosphorylates the Foxos (36) and inhibits their transcriptional activity (37). Foxo1 is the most abundant Foxo isoform in several insulin-responsive tissues, such as liver, adipose tissue, and pancreatic ?-cells. It was also demonstrated that Foxo1 plays an important role in coupling insulin signaling to adipocyte differentiation (38). The increased basal phosphorylation and inactivation of Foxo1 described in our study might have a role in the enhanced adiposity in the WD group.
Interestingly, in adipose tissue of WD30 animals, an increase in insulin-induced glucose transport in fat still occurred. However, early steps of insulin signaling were not increased. This result suggests that the molecular mechanism that accounts for this increase in glucose transport in the fat of WD30 rats is probably downstream from Akt, because there was no alteration in the IRS/PI3-K/Akt pathway. The association of the results obtained from muscle, liver, and adipose tissue suggest that, in the presence of reduced insulin action in muscle, and later in liver and muscle, glucose was partly shunted to white adipose tissue.
In this study, ICV insulin infusion significantly reduced food intake in controls (59%). In contrast, ICV insulin administration was less effective in reducing food intake in rats fed with WD10 (19%), and this effect was more intensive after 30 d of WD (11%). Thus, WD led to a significant impairment of the insulin-induced anorexigenic effect, which may characterize central resistance to insulin action. In parallel, we demonstrated that the sites of insulin signaling impairment appear to be at the IR and postreceptor levels in the hypothalamus from animals fed with WD.
Neuron-specific IR knockout mice (31) or rats with a selective decrease in IR expression in hypothalamic nuclei (achieved using an antisense oligodeoxynucleotide directed against the IR precursor protein) (39) demonstrated increased food intake and increased fat mass. The same result was observed in female IRS-2 knockout mice. These animals presented increased daily food intake, increased body weight, and adiposity. In addition, they demonstrated a disrupted control of hypothalamic neuropeptide level linkage to feeding (40). Our data suggest that the antiobesity actions induced by insulin in hypothalamus could be blunted because of partial inhibition of the PI3-K pathway, suggesting that neuronal PI3-K is important for the effects of insulin on food intake. If the mechanism used by insulin to reduce food intake is PI3-K dependent, as has been previously demonstrated (6, 41, 42, 43), the defective activation of PI3-K/Akt/Foxo1 in hypothalamic neurons could attenuate the ability of insulin to reduce food intake in WD rats.
There are probably a number of mechanisms that may lead to an impairment of the insulin-signaling pathway in muscle, liver, and hypothalamus of rats fed with WD. Previous studies demonstrated that an increase in IRS serine phosphorylation could induce marked insulin resistance, pointing to this as an important mechanism in the control of insulin signaling (44, 45, 46). It has been reported that activation of JNK induces serine 307 phosphorylation of IRS-1 (14), leading to a decrease in insulin-stimulated PI3-K activity. Our data showed an increase in JNK phosphorylation/activation, in agreement with altered insulin signaling in muscle and hypothalamus of WD10 and also in liver of WD30, suggesting that this serine kinase may have an important role in down-regulating insulin signaling in these tissues. In addition, we demonstrated an increase in IRS-1/JNK1 association in parallel with IRS-1ser307 phosphorylation with the same time course and tissue distribution of JNK activation. These data suggest that JNK may have an important role in the altered insulin signaling in tissues of rats fed with WD.
In summary, our data provide direct evidence that insulin resistance, induced by WD, is tissue specific. The findings also support the hypothesis that insulin can activate the PI3-K/Akt/Foxo1 pathway in the hypothalamus, which can be modulated by WD. In addition, the insulin resistance induced by WD seems to install first in hypothalamus and skeletal muscle, characterized by attenuation of the anorexigenic effect of insulin and reduced muscle glucose transport, respectively, and later in liver, with less suppressibility of hepatic glucose production. These temporal alterations in insulin action and signaling are accompanied by activation of JNK and IRS-1ser307 phosphorylation. This situation of an impaired PI3-K/Akt signaling pathway in the hypothalamus, muscle, and liver but not in adipose tissue may lead to the development of increased adiposity and insulin resistance in the WD rats.
Acknowledgments
We thank Mr. Luis Janieri, Mr. Márcio Alves da Cruz, and Mr. Jósimo Pinheiro for their technical assistance.
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