Both Saturated and n-6 Polyunsaturated Fat Diets Reduce Phosphorylation of Insulin Receptor Substrate-1 and Protein Kinase B in Muscle during the Init
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《内分泌学杂志》
Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital (G.F., J.-M.Y., G.J.C.), Darlinghurst, Sydney, 2010 New South Wales
Department of Medicine, St. Vincent’s Medical School, University of New South Wales (G.J.C.), Sydney, 2052 New South Wales, Australia
Abstract
Our aim was to determine the importance of changes in phosphorylation of key insulin signaling intermediates in the insulin resistance observed in skeletal muscle of rats fed diets high in saturated or n-6 polyunsaturated fat. We used phospho-specific antibodies to measure the time course of phosphorylation of key components of the insulin signaling pathway by immunoblotting during the initial stages of a physiological elevation in the circulating insulin concentration. The phosphorylation of insulin receptor at Tyr1162/1163 (IR Tyr1162/1163) increased over 20 min of insulin infusion, whereas the downstream phosphorylation of insulin receptor substrate-1 Tyr612 (IRS-1 Tyr612) peaked at 5 min and declined thereafter. Interestingly, phosphorylation of IRS-1 at Tyr895 continued to increase over the 20-min period, and protein kinase B (PKB) phosphorylation at Ser473 reached a plateau by 5 min, demonstrating that different profiles of phosphorylation are involved in transmission of the insulin signal despite a constant level of insulin stimulation. In muscle from rats fed high n-6 polyunsaturated or saturated fat diets, however, there was no insulin-stimulated increase in IRS-1 Tyr612 phosphorylation and a temporal difference in PKB Ser473 phosphorylation despite no difference in IR Tyr1162/1163 phosphorylation, IRS-1 Tyr895 phosphorylation, and ERK phosphorylation. These results demonstrate that under conditions of increased insulin, similar to those used to assess insulin action in vivo, chronic high-fat feeding impairs insulin signal transduction related to glucose metabolism at the level of IRS-1 Tyr612 and PKB Ser473 and that these effects are independent of the type of fat used in the high-fat diet.
Introduction
STUDIES IN CELL lines suggest that different fatty acid (FA) subtypes impact differently on insulin signal transduction to induce insulin resistance. For example, in C2C12 skeletal muscle cells, the monounsaturated FA oleate and the n-6 polyunsaturated FA linoleate impair insulin-stimulated phosphatidylinositol 3-kinase (PI3-K) activation without any significant effect on downstream protein kinase B (PKB) activation, whereas the saturated FA palmitate impairs insulin-stimulated PKB activation independent of any effect on upstream PI3-K activation (1). Evidence linking defects in insulin signaling to the insulin resistance produced by high-fat diets in rats, however, has not always been in agreement with findings from cultured cell systems. Kanoh et al. (2) reported no alterations in IRS-1 [insulin-stimulated insulin receptor (IR) substrate-1]-associated PI3-K activity and PKB phosphorylation, no alterations in total GLUT4 content, but reduced insulin-stimulated atypical protein kinase C/ activity in skeletal muscle of 4- to 5-wk high-fat-fed rats 15 min after a supraphysiological (1 U/kg) im injection of insulin. Conversely, Tremblay et al. (3) have shown reduced insulin-stimulated IRS-1-associated PI3-K activity, reduced PKB activity, and reduced total GLUT4 content, but no difference in insulin-stimulated atypical protein kinase C/ activity in skeletal muscle 4 min after an 8 U/kg iv injection of insulin in rats fed a high-fat diet for 4 wk. Some of the reported differences in the effects of high-fat diets on insulin signaling may be accounted for by different types and amounts of fat used in the diets, but the protocols employed for comparing insulin action with insulin signaling could also contribute to these inconsistencies.
The assessment of insulin action in vivo is normally determined using the euglycemic/hyperinsulinemic clamp technique. In rats, raising the circulating insulin level 3- to 5-fold to levels of 0.9–1.7 nM increases whole-body glucose turnover at least 10-fold, and maximum increases in glucose disposal occur at circulating insulin concentrations of approximately 7 nM (4). Most studies examining insulin signaling events in tissues (such as those described above), however, use bolus injections of insulin that result in pharmacological plasma concentrations of 10–20 nM. The subsequent insulin signaling events may therefore reflect maximal activation of the pathway, rather than changes that occur during physiological insulin action.
However, typical euglycemic-hyperinsulinemic clamp conditions are not necessarily ideal for assessing the phosphorylation status of insulin signaling pathway intermediates. The duration of insulin stimulation during a clamp study is usually 1.5–2 h. By the time tissues are taken for assay at the end of the clamp, the phosphorylation state of proteins may be down-regulated by feedback mechanisms and therefore not reflect changes that occur at the onset of insulin stimulation (5). Some studies have employed a short-term clamp of 30 min to examine insulin signaling after acute elevation of circulating lipids (6), but this approach has not been used to examine alterations in signaling in models of high-fat diet-induced insulin resistance.
The purpose of this study was 2-fold: to establish time-course relationships for insulin stimulation of important insulin signaling intermediates in vivo and to examine the effects that feeding high-fat diets enriched in particular fat subtypes have on these parameters. We describe methodology designed to measure initial, in vivo, insulin signaling in muscle of chow- and high fat-fed rats using conditions similar to those routinely employed to assess insulin action in muscle during a euglycemic/hyperinsulinemic clamp. Normal, chow-fed rats were compared with rats fed a high-fat diet enriched in either n-6 polyunsaturated or saturated fat for 3 wk to examine whether different fat diets produce different effects on site-specific phosphorylation of major insulin signaling components. We show that there are temporal differences in specific in vivo insulin-stimulated phosphorylation events in muscle after a physiological rise in insulin concentration and that both groups of high fat-fed rats exhibit similar impairments in signaling independent of the saturation status of the FAs used in the respective diets.
Materials and Methods
Experimental animals and dietary treatment
All procedures were approved by the Garvan Institute/St. Vincent’s Hospital animal experimentation ethics committee and were performed in accordance with the National Health and Medical Research Council of Australia Guidelines on Animal Experimentation. Male Wistar rats (250 g) supplied by the Animal Resources Center (Perth, Australia) were acclimatized in communal cages at 22 ± 1 C with a 12-h light, 12-h dark cycle (lights on at 0600 h) for 1 wk and had access to a standard chow diet (Gordon’s Specialty Stock Feed, Sydney, Australia) and water ad libitum. Rats were then randomly assigned to receive either the standard chow diet as the control group (CHOW) or an isocaloric high-fat (60% calories as fat) diet enriched in either n-6 polyunsaturated (PUFA) or saturated (SFA) fat for an additional 3 wk. The PUFA diet contained 79% linoleic acid, and the SFA diet contained 91% saturated fat, made up of myristic, palmitic, and stearic acids.
Animal preparation
One week before study, rats were chronically cannulated via the right jugular vein and left carotid artery under halothane (Fluothane, Cenvet Pty. Ltd., Adelaide, Australia) inhalation anesthesia (5% induction, 2% maintenance), and the cannulas were exteriorized via a small incision at the back of the neck. Postsurgery recovery over a 7-d period was closely monitored by measurement of food intake and body weight gain. Rats were handled daily to minimize stress, and only those with fully recovered body weight were used for the study.
Time course for plasma insulin elevation and signaling protein phosphorylation in insulin-infused rats
To establish optimum time points to study the onset of insulin signaling, a time course of plasma insulin elevation during insulin infusion was determined in normal rats. Experiments were performed in conscious rats after approximately 5 h of fasting. Cannulas were connected to the infusion apparatus, and the rats were allowed to rest for 50–60 min before a basal blood sample was taken. Blood glucose was determined using an automated glucose analyzer (YSI 2300, YSI, Inc., Yellow Springs, OH), and then plasma was separated and frozen in liquid nitrogen for subsequent metabolite measurements. Insulin was infused at a rate of 0.5 U/kg·h, and a 30% (wt/vol) variable glucose infusion was started at 4 min to maintain euglycemia. In these rats, plasma samples were taken at 0, 1, 2, 3, 4, 5, 6, 8, 10, and 20 min for the measurement of insulin and free FAs (FFA) concentrations only. To establish a time course for changes in the phosphorylation state of insulin signaling intermediates, insulin was infused at a rate of 0.5 U/kg·h for 5, 10, or 20 min, and a concurrent 30% (wt/vol) variable glucose infusion was started at 4 min to maintain euglycemia. At 5, 10, and 20 min, rats were killed with an overdose of sodium pentobarbitane (Nembutal, Cenvet Pty. Ltd.), and red quadriceps muscle was rapidly dissected, freeze-clamped with aluminum tongs precooled in liquid nitrogen, and stored at –80 C for subsequent analysis.
Insulin signaling in muscle from control and fat-fed rats
For subsequent experiments comparing the activation of insulin signaling in PUFA and SFA rats to CHOW, rats were infused with insulin at a rate of 0.5 U/kg·h for 5 and 10 min [with a concurrent 30% (wt/vol) variable glucose infusion started at 4 min to maintain euglycemia], because this was judged the most appropriate for measuring the initial events leading to the downstream effect of insulin from the time-course study. The animals were then killed, and red quadriceps muscles were collected as described above.
Metabolite measurements
Plasma FFA levels were determined spectrophotometrically using an enzymatic colorimetric method (NEFA-C kit, Wako Pure Chemical Industries, Osaka, Japan). Plasma triglycerides were also determined spectrophotometrically using an enzymatic colorimetric method (Triglycerides GPO-PAP reagent, Roche, Indianapolis, IN). Plasma insulin was determined by RIA using a rat-specific kit (Linco Research, Inc., St. Charles, MO). Red quadriceps skeletal muscle triglycerides were extracted using the method of Folch et al. (7) and quantified using an enzymatic colorimetric method (Triglycerides GPO-PAP reagent, Roche).
Protein extraction
Red quadriceps muscle was homogenized in an ice-cold solubilization buffer containing 65 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM sodium fluoride, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride. The homogenate was incubated for 2 h at 4 C and then centrifuged at 12,000 x g for 15 min to remove insoluble material. The protein concentration of the supernatants was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
Immunoblot analyses
Protein homogenates were prepared in Laemmli buffer (8) and subjected to SDS-PAGE. To allow for quantification between blots, an in-house standard was also run on each gel. Proteins were separated on 7.5% gels, transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences, Piscataway, NJ), and blocked in 1% BSA- or 5% skim milk-Tris-buffered saline containing 0.025% Tween 20. Membranes were probed with the following primary antibodies as required: anti-IR-pYpY1162/1163 (BioSource, Camarillo, CA), anti-IR (BD Transduction Laboratories, San Diego, CA), anti-IRS-1-pY612 (BioSource), anti-IRS-1 (Upstate Cell Signaling Solutions, Lake Placid, NY), anti-pY895-IRS-1 (Oncogene Research Products, Boston, MA), anti-IRS-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-phospho (Ser473)-Akt (Cell Signaling Technology, Beverly, MA), anti-Akt (Cell Signaling Technology), anti-phospho (Thr202/Tyr204)-p44/42 MAPK (Cell Signaling Technology), or anti-p44/42 MAPK (Cell Signaling Technology). Membranes were then incubated with donkey antirabbit horseradish peroxidase or protein A-horseradish peroxidase secondary antibody, and bands were detected by chemiluminescence (PerkinElmer Life Sciences, Boston, MA) after exposure to film (Fuji Photo Film Co., Tokyo, Japan). Membranes were probed with phosphorylated antibodies first, then stripped and reprobed with the corresponding total antibodies. Films were subsequently scanned, and the densities of bands of interest were quantified using IPLab Gel software (Signal Analytics Corp., Vienna, VA).
Statistics
Results are presented as the mean ± SE. Data were initially analyzed by two-way factorial ANOVA to establish an overall response to insulin stimulation and to examine the degree to which the response was modulated by diet. If a significant interaction was found, more detailed analyses within groups were subsequently performed by Fisher’s protected least significant difference post hoc tests. Statistical calculations were performed using a commercial software package (StatView 5.0; SAS Institute, Inc., Cary, NC). Differences were considered significant at P < 0.05.
Results
Insulin time-course metabolic parameters
Infusion of insulin at a rate of 0.5 U/kg·h resulted in significant increases in circulating insulin after 2 min (P < 0.01), and by 5 min the plasma insulin concentration had reached a plateau of approximately 1.5 nM, indicating that steady-state insulin levels were achieved within 5 min of the start of the insulin infusion (Fig. 1). These levels are within the high physiological range for rats (9). The plasma FFA concentration was significantly decreased after 5 min of insulin infusion (P < 0.05; Fig. 1), demonstrating an insulin-mediated inhibition of lipolysis. There was also a significant effect of insulin on whole body glucose metabolism, because a glucose infusion of 34 ± 1.0 mg/kg·min was required to maintain plasma euglycemia at 7 mM over the 20 min of the study.
Time course of signaling protein phosphorylation
The time course of activation of insulin signaling intermediates was established in CHOW rats by examining phosphorylation of IR, IRS-1, and PKB at specific phosphorylation sites. Using a phospho-specific antibody directed at Tyr1162/Tyr1163 of the IR (sites critical for full tyrosine kinase activity of IR) (10), it was observed that insulin infusion resulted in a significant elevation in IR phosphorylation that increased throughout the 20-min infusion period (Fig. 2A). Phospho-specific antibodies directed at Tyr612 of IRS-1 (a putative binding domain for the p85 subunit of PI3-K) (11, 12) and Tyr895 of IRS-1 [a growth receptor-bound-2 (Grb-2) binding site] (12, 13, 14) were used to examine the insulin-stimulated activation of IRS-1. We detected a 2-fold increase in phosphorylation of IRS-1 Tyr612 at 5 min (P < 0.01 vs. basal), which was similar at 10 min, but had returned to basal levels by 20 min (Fig. 2B). Interestingly, phosphorylation of IRS-1 at Tyr895 followed a different time course from that of Tyr612, reaching a plateau at 10 min. This suggested that different functions of the IRS-1 scaffolding protein could be dependent on differential phosphorylation of specific sites. Phosphorylation of PKB at Ser473, a critical phosphorylation site required for full activation of PKB (15, 16), was increased 3-fold 5 min after the beginning of the insulin infusion, and this degree of phosphorylation was maintained for the remaining insulin infusion time (Fig. 2C). The results in Figs. 1 and 2 demonstrate that a 5-min insulin infusion at a rate of 0.5 U/kg·h is enough to produce a significant effect on whole body insulin action and phosphorylation of signaling intermediates. This protocol is therefore suitable for assessment of the effect of high-fat diets on initial insulin signaling events.
Effects of fat feeding and insulin infusion on plasma and tissue metabolic parameters
Infusion of insulin at a rate of 0.5 U/kg·h for 5 min resulted in similar significant 4- to 5-fold increases in circulating insulin levels in all groups of rats (Table 1). Glucose infusion was required to maintain euglycemia in all groups of rats, but this was significantly lower in both the PUFA and SFA rats, which is an indication of whole body insulin resistance (CHOW, 35 ± 1.3 mg/kg· min; PUFA, 25 ± 1.7 mg/kg·min; SFA, 28 ± 0.4 mg/kg· min; P < 0.001, PUFA and SFA vs. CHOW). The high-fat diet enriched in SFA resulted in higher plasma triglycerides (at least 1.7-fold) and higher circulating FFA (at least 1.5-fold) compared with both levels in both CHOW and PUFA rats (Table 1). There was an effect of insulin stimulation to reduce plasma FFA in the CHOW and PUFA rats only (Table 1). The tissue triglyceride content in red quadriceps muscle was elevated 1.3-fold in both PUFA and SFA rats compared with CHOW rats (CHOW, 2.8 ± 0.2 μmol/g; PUFA, 3.6 ± 0.3 μmol/g; SFA, 3.7 ± 0.4 μmol/g; P < 0.05, PUFA and SFA vs. CHOW).
IR tyrosine phosphorylation
Basal levels of IR Tyr1162/1163 phosphorylation were equivalent in all three groups of rats (Fig. 3). In CHOW rats, there were approximately 2- to 3-fold increases in IR Tyr1162/1163 phosphorylation over basal with insulin stimulation (Fig. 3). A similar increase in phosphorylation at these sites was observed in both PUFA and SFA rats with insulin stimulation for 5 or 10 min (Fig. 3). Although the responses in both high-fat-fed groups of rats appeared marginally lower than the corresponding CHOW values, the differences were not statistically significant. The total IR protein content was not affected by diet or insulin stimulation (Fig. 3B). Overall, there was a significant effect of insulin stimulation (P < 0.0001), but no effect of diet (P = 0.2528), on muscle IR tyrosine phosphorylation at the Tyr1162/1163 residues.
IRS-1 tyrosine phosphorylation
Phosphorylation at the PI3-K binding Tyr612 site of IRS-1 was similar in all groups of rats in the basal state (Fig. 4). In CHOW rats, insulin infusion produced a 2-fold increase in IRS-1 Tyr612 phosphorylation at both 5 and 10 min (Fig. 4). However, there was no effect of insulin infusion on IRS-1 Tyr612 phosphorylation in either PUFA or SFA rats at 5 or 10 min (Fig. 4). Total IRS-1 protein expression was similar in all groups, at all time points (Fig. 4B). The effect of diet treatment on insulin-stimulated IRS-1 Tyr612 phosphorylation in muscle was highly significant (P < 0.0001). Phosphorylation of IRS-1 at Tyr895 (the Grb-2-binding site) by insulin was not affected by either fat diet (Fig. 5), suggesting that the effects of a high-fat diet on insulin-stimulated IRS-1 tyrosine phosphorylation were specific for certain IRS-1 mediated functions.
PKB phosphorylation
Basal phosphorylation of PKB at the Ser473 site was not different from that in CHOW animals with either high-fat diet treatment (Fig. 6). Insulin stimulation for 5 min resulted in significant increases in Ser473-PKB phosphorylation in all groups (CHOW, P < 0.001 vs. basal; PUFA, P < 0.001 vs. basal; SFA, P < 0.01 vs. basal; Fig. 6). However, this insulin-stimulated increase in phosphorylation was significantly blunted in the PUFA and SFA rats compared with CHOW rats (P < 0.001, PUFA vs. CHOW, 5 min insulin; P < 0.001, SFA vs. CHOW, 5 min insulin). After 10 min of insulin infusion, Ser473-PKB phosphorylation remained significantly elevated over basal levels in all groups of rats. At this time point, the phosphorylation of PKB in the fat-fed rats was still lower than that in the CHOW rats (P = 0.06, PUFA vs. CHOW, 10 min insulin; P < 0.05, SFA vs. CHOW, 10 min insulin; Fig. 6). Total PKB protein was unaffected by diet or insulin treatment (Fig. 6B).
ERK phosphorylation
ERK has a role in cell growth and differentiation and is activated downstream of IRS-1 Tyr895 phosphorylation (17). A phospho-specific antibody directed at Thr202/Tyr204 of ERK (key regulatory phosphorylation sites in this kinase) (18, 19) was used to examine the activation of this protein. ERK Thr202/Tyr204 phosphorylation in PUFA and SFA rats followed a pattern similar to that in CHOW rats (Fig. 7). In all rats, there was no significant increase in insulin-stimulated ERK Thr202/Tyr204 phosphorylation at 5 min, but with 10-min insulin infusion, ERK Thr202/Tyr204 phosphorylation was similarly elevated approximately 50% over basal (CHOW, P < 0.05 vs. basal; PUFA, P = 0.08 vs. basal; SFA, P = 0.1 vs. basal; Fig. 7).
Discussion
In the present study we examined the effects of time and different high-fat diets on the phosphorylation of intermediates of the insulin signaling pathway in skeletal muscle in vivo using experimental conditions similar to those routinely employed to assess insulin action during a euglycemic/hyperinsulinemic clamp. After 5 min of insulin infusion, plasma insulin levels reached a steady-state concentration (1.5 nM) and produced clear downstream metabolic effects and a significant and coordinated increase in the phosphorylation state of IR Tyr1162/1163, IRS-1 Tyr612, and PKB Ser473 in skeletal muscle. One interesting observation from this study was the difference in the time course of phosphorylation of IRS-1 on two specific sites associated with distinct physiological processes. The phosphorylation of Tyr612 on IRS-1 is involved in insulin signaling via the PI3-K pathway to PKB and the stimulation of glucose uptake and glycogen synthesis (11, 12), whereas IRS-1 phosphorylation at Tyr895 is thought to be linked to insulin stimulation of mitogenic pathways via Grb-2 (12, 13, 14). The rapid onset of IR Tyr1162/1163, IRS-1 Tyr612, and PKB Ser473 phosphorylation suggests an immediate transduction of the insulin signal for glucose uptake in muscle, whereas the insulin stimulation of mitogenic pathways (via IRS-1 Tyr895 and downstream ERK Thr202/Tyr204) may involve a slower onset and more sustained signal. The decrease in phosphorylation at Tyr612 of IRS-1 after 10 min, whereas the phosphorylation of IR and PKB was further increased or remained unchanged, highlights the possibility that the initial rate of phosphorylation of the signaling intermediates, rather than a sustained level of phosphorylation, is more important for transduction of the metabolic effects of insulin (20).
The effects of different dietary fat subtypes on the development of insulin resistance assessed by whole-body and tissue-specific glucose uptake have been studied previously (21). This study did not, however, extend to the effect of fat subtypes on in vivo insulin signaling in muscle or other insulin-sensitive tissues. In the studies reported here we show that a high-fat diet enriched in either n-6 polyunsaturated or saturated fats had different effects on circulating FAs and triglycerides, but produced similar increases in muscle triglyceride content, similar reductions in insulin action, and similar deleterious effects on skeletal muscle insulin signaling compared with those in control rats. Although in PUFA and SFA rats there was no significant difference in insulin-stimulated IR Tyr1162/1163 phosphorylation compared with CHOW rats, there was an absence of insulin-stimulated IRS-1 Tyr612 phosphorylation in PUFA and SFA rats and a decreased insulin-stimulated PKB Ser473 phosphorylation effect in PUFA and SFA rats at 5 min. The magnitude of the reduction in PKB phosphorylation in muscle of fat-fed rats was less than the reduction seen upstream at Tyr612-IRS-1. One explanation for this is that other PI3-K binding sites on IRS-1 are not affected or are less affected by the dietary treatment, thereby producing some downstream transmission of the insulin signal to PKB. Both fat diets also had no effect on insulin-stimulated IRS-1 Tyr895 phosphorylation and downstream ERK Thr202/Tyr204 phosphorylation, suggesting that the diets had site-specific effects on phosphorylation that could be relevant to specific signaling pathways, but might not be detected if activation of IRS-1 was assessed using antibodies that detect global IRS-1 tyrosine phosphorylation. At least 30 tyrosine phosphorylation sites exist on IRS-1, but only six are known PI3-K binding sites (22). Therefore, the use of phospho-specific antibodies may be more appropriate than assessment of global tyrosine phosphorylation when investigating the role of changes in IRS-1 phosphorylation relevant to stimulating glucose metabolism or other insulin-stimulated processes.
The changes in phosphorylation observed in the current study show similarity to some reports on the effect of a high-fat diet on insulin signaling (3, 23, 24), but not others (2, 25). All these previous studies were conducted using pharmacological insulin stimulation (which produces plasma insulin concentrations of 10–20 nM) and were thus more likely to reflect the maximal capacity of the muscle to respond to insulin stimulation rather than the level of signaling required to stimulate glucose transport in vivo. Our results suggest that the total amount of fat provided in the high-fat diets, rather than the particular FA subtype provided, has a major impact on insulin action in vivo. Studies using cell culture systems have shown distinct effects of different FAs on impairing insulin signaling in skeletal muscle cells in vitro (1). The current results appear contrary to these observations; however, the presentation of excess lipids to cells or tissue is very different in the two experimental situations. In culture systems, cells are exposed to only one FA, whereas in vivo all FA species are still present in the circulation even if there is enrichment of one subtype of FA because of the composition of the diet. It would seem likely that the in vivo effects of increased SFA or PUFA will always result in a combination of any individual effects of specific FAs because it is not possible to produce an experimental situation in vivo that generates exposure of muscle tissue to only one FA, and there can be interconversion of FA species in vivo (26, 27).
The results of the current study show a major defect in insulin signaling with chronic high-fat diet-induced insulin resistance at the level of IRS-1 and a lower Ser473-PKB phosphorylation downstream. It is difficult to be definitive, however, about whether one defect is primary to the other. It is possible that the decrease in PKB phosphorylation is secondary to the reduced phosphorylation of IRS-1, but it is also possible that the high-fat diets have direct effects on both signaling molecules or that a decrease in the phosphorylation and activity of PKB reduces the phosphorylation of specific serine residues on IRS-1 that normally protects it from protein-tyrosine phosphatases (28).
In conclusion, we have used a method of physiological insulin stimulation designed to study the onset of insulin signaling events in vivo to show that rats fed a high-fat diet enriched in either PUFA or SFA developed similar impairments in the phosphorylation of insulin signaling intermediates in skeletal muscle. There was no defect at the level of IR, reduced insulin-stimulated tyrosine phosphorylation at IRS-1 Tyr612 (but not at IRS-1 Tyr895 or ERK), and reduced insulin-stimulated PKB Ser473 phosphorylation in both PUFA and SFA rats. This suggests that impairments in skeletal muscle insulin signal transduction in vivo after physiological insulin stimulation are a consequence of chronic high-fat treatment, independent of fat subtype, and that decreased IRS-1 Tyr612 and PKB Ser473 phosphorylations are key components of the reduced insulin action observed in situations of chronic lipid oversupply.
Acknowledgments
We thank Elaine Preston, Donna Wilks, Joanna Edema, and the staff of Garvan Institute’s Biological Testing Facility for technical assistance with this study.
Footnotes
This work was supported by the National Health and Medical Research Council of Australia and the Diabetes Australia Research Trust. G.F. was the recipient of an Australian Postgraduate Research Award.
First Published Online September 8, 2005
Abbreviations: FA, Fatty acid; Grb-2, growth receptor-bound-2; IR, insulin receptor; IRS-1, IR substrate-1; PI3-K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PUFA, n-6 polyunsaturated fatty acid; SFA, saturated fatty acid.
Accepted for publication September 1, 2005.
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Department of Medicine, St. Vincent’s Medical School, University of New South Wales (G.J.C.), Sydney, 2052 New South Wales, Australia
Abstract
Our aim was to determine the importance of changes in phosphorylation of key insulin signaling intermediates in the insulin resistance observed in skeletal muscle of rats fed diets high in saturated or n-6 polyunsaturated fat. We used phospho-specific antibodies to measure the time course of phosphorylation of key components of the insulin signaling pathway by immunoblotting during the initial stages of a physiological elevation in the circulating insulin concentration. The phosphorylation of insulin receptor at Tyr1162/1163 (IR Tyr1162/1163) increased over 20 min of insulin infusion, whereas the downstream phosphorylation of insulin receptor substrate-1 Tyr612 (IRS-1 Tyr612) peaked at 5 min and declined thereafter. Interestingly, phosphorylation of IRS-1 at Tyr895 continued to increase over the 20-min period, and protein kinase B (PKB) phosphorylation at Ser473 reached a plateau by 5 min, demonstrating that different profiles of phosphorylation are involved in transmission of the insulin signal despite a constant level of insulin stimulation. In muscle from rats fed high n-6 polyunsaturated or saturated fat diets, however, there was no insulin-stimulated increase in IRS-1 Tyr612 phosphorylation and a temporal difference in PKB Ser473 phosphorylation despite no difference in IR Tyr1162/1163 phosphorylation, IRS-1 Tyr895 phosphorylation, and ERK phosphorylation. These results demonstrate that under conditions of increased insulin, similar to those used to assess insulin action in vivo, chronic high-fat feeding impairs insulin signal transduction related to glucose metabolism at the level of IRS-1 Tyr612 and PKB Ser473 and that these effects are independent of the type of fat used in the high-fat diet.
Introduction
STUDIES IN CELL lines suggest that different fatty acid (FA) subtypes impact differently on insulin signal transduction to induce insulin resistance. For example, in C2C12 skeletal muscle cells, the monounsaturated FA oleate and the n-6 polyunsaturated FA linoleate impair insulin-stimulated phosphatidylinositol 3-kinase (PI3-K) activation without any significant effect on downstream protein kinase B (PKB) activation, whereas the saturated FA palmitate impairs insulin-stimulated PKB activation independent of any effect on upstream PI3-K activation (1). Evidence linking defects in insulin signaling to the insulin resistance produced by high-fat diets in rats, however, has not always been in agreement with findings from cultured cell systems. Kanoh et al. (2) reported no alterations in IRS-1 [insulin-stimulated insulin receptor (IR) substrate-1]-associated PI3-K activity and PKB phosphorylation, no alterations in total GLUT4 content, but reduced insulin-stimulated atypical protein kinase C/ activity in skeletal muscle of 4- to 5-wk high-fat-fed rats 15 min after a supraphysiological (1 U/kg) im injection of insulin. Conversely, Tremblay et al. (3) have shown reduced insulin-stimulated IRS-1-associated PI3-K activity, reduced PKB activity, and reduced total GLUT4 content, but no difference in insulin-stimulated atypical protein kinase C/ activity in skeletal muscle 4 min after an 8 U/kg iv injection of insulin in rats fed a high-fat diet for 4 wk. Some of the reported differences in the effects of high-fat diets on insulin signaling may be accounted for by different types and amounts of fat used in the diets, but the protocols employed for comparing insulin action with insulin signaling could also contribute to these inconsistencies.
The assessment of insulin action in vivo is normally determined using the euglycemic/hyperinsulinemic clamp technique. In rats, raising the circulating insulin level 3- to 5-fold to levels of 0.9–1.7 nM increases whole-body glucose turnover at least 10-fold, and maximum increases in glucose disposal occur at circulating insulin concentrations of approximately 7 nM (4). Most studies examining insulin signaling events in tissues (such as those described above), however, use bolus injections of insulin that result in pharmacological plasma concentrations of 10–20 nM. The subsequent insulin signaling events may therefore reflect maximal activation of the pathway, rather than changes that occur during physiological insulin action.
However, typical euglycemic-hyperinsulinemic clamp conditions are not necessarily ideal for assessing the phosphorylation status of insulin signaling pathway intermediates. The duration of insulin stimulation during a clamp study is usually 1.5–2 h. By the time tissues are taken for assay at the end of the clamp, the phosphorylation state of proteins may be down-regulated by feedback mechanisms and therefore not reflect changes that occur at the onset of insulin stimulation (5). Some studies have employed a short-term clamp of 30 min to examine insulin signaling after acute elevation of circulating lipids (6), but this approach has not been used to examine alterations in signaling in models of high-fat diet-induced insulin resistance.
The purpose of this study was 2-fold: to establish time-course relationships for insulin stimulation of important insulin signaling intermediates in vivo and to examine the effects that feeding high-fat diets enriched in particular fat subtypes have on these parameters. We describe methodology designed to measure initial, in vivo, insulin signaling in muscle of chow- and high fat-fed rats using conditions similar to those routinely employed to assess insulin action in muscle during a euglycemic/hyperinsulinemic clamp. Normal, chow-fed rats were compared with rats fed a high-fat diet enriched in either n-6 polyunsaturated or saturated fat for 3 wk to examine whether different fat diets produce different effects on site-specific phosphorylation of major insulin signaling components. We show that there are temporal differences in specific in vivo insulin-stimulated phosphorylation events in muscle after a physiological rise in insulin concentration and that both groups of high fat-fed rats exhibit similar impairments in signaling independent of the saturation status of the FAs used in the respective diets.
Materials and Methods
Experimental animals and dietary treatment
All procedures were approved by the Garvan Institute/St. Vincent’s Hospital animal experimentation ethics committee and were performed in accordance with the National Health and Medical Research Council of Australia Guidelines on Animal Experimentation. Male Wistar rats (250 g) supplied by the Animal Resources Center (Perth, Australia) were acclimatized in communal cages at 22 ± 1 C with a 12-h light, 12-h dark cycle (lights on at 0600 h) for 1 wk and had access to a standard chow diet (Gordon’s Specialty Stock Feed, Sydney, Australia) and water ad libitum. Rats were then randomly assigned to receive either the standard chow diet as the control group (CHOW) or an isocaloric high-fat (60% calories as fat) diet enriched in either n-6 polyunsaturated (PUFA) or saturated (SFA) fat for an additional 3 wk. The PUFA diet contained 79% linoleic acid, and the SFA diet contained 91% saturated fat, made up of myristic, palmitic, and stearic acids.
Animal preparation
One week before study, rats were chronically cannulated via the right jugular vein and left carotid artery under halothane (Fluothane, Cenvet Pty. Ltd., Adelaide, Australia) inhalation anesthesia (5% induction, 2% maintenance), and the cannulas were exteriorized via a small incision at the back of the neck. Postsurgery recovery over a 7-d period was closely monitored by measurement of food intake and body weight gain. Rats were handled daily to minimize stress, and only those with fully recovered body weight were used for the study.
Time course for plasma insulin elevation and signaling protein phosphorylation in insulin-infused rats
To establish optimum time points to study the onset of insulin signaling, a time course of plasma insulin elevation during insulin infusion was determined in normal rats. Experiments were performed in conscious rats after approximately 5 h of fasting. Cannulas were connected to the infusion apparatus, and the rats were allowed to rest for 50–60 min before a basal blood sample was taken. Blood glucose was determined using an automated glucose analyzer (YSI 2300, YSI, Inc., Yellow Springs, OH), and then plasma was separated and frozen in liquid nitrogen for subsequent metabolite measurements. Insulin was infused at a rate of 0.5 U/kg·h, and a 30% (wt/vol) variable glucose infusion was started at 4 min to maintain euglycemia. In these rats, plasma samples were taken at 0, 1, 2, 3, 4, 5, 6, 8, 10, and 20 min for the measurement of insulin and free FAs (FFA) concentrations only. To establish a time course for changes in the phosphorylation state of insulin signaling intermediates, insulin was infused at a rate of 0.5 U/kg·h for 5, 10, or 20 min, and a concurrent 30% (wt/vol) variable glucose infusion was started at 4 min to maintain euglycemia. At 5, 10, and 20 min, rats were killed with an overdose of sodium pentobarbitane (Nembutal, Cenvet Pty. Ltd.), and red quadriceps muscle was rapidly dissected, freeze-clamped with aluminum tongs precooled in liquid nitrogen, and stored at –80 C for subsequent analysis.
Insulin signaling in muscle from control and fat-fed rats
For subsequent experiments comparing the activation of insulin signaling in PUFA and SFA rats to CHOW, rats were infused with insulin at a rate of 0.5 U/kg·h for 5 and 10 min [with a concurrent 30% (wt/vol) variable glucose infusion started at 4 min to maintain euglycemia], because this was judged the most appropriate for measuring the initial events leading to the downstream effect of insulin from the time-course study. The animals were then killed, and red quadriceps muscles were collected as described above.
Metabolite measurements
Plasma FFA levels were determined spectrophotometrically using an enzymatic colorimetric method (NEFA-C kit, Wako Pure Chemical Industries, Osaka, Japan). Plasma triglycerides were also determined spectrophotometrically using an enzymatic colorimetric method (Triglycerides GPO-PAP reagent, Roche, Indianapolis, IN). Plasma insulin was determined by RIA using a rat-specific kit (Linco Research, Inc., St. Charles, MO). Red quadriceps skeletal muscle triglycerides were extracted using the method of Folch et al. (7) and quantified using an enzymatic colorimetric method (Triglycerides GPO-PAP reagent, Roche).
Protein extraction
Red quadriceps muscle was homogenized in an ice-cold solubilization buffer containing 65 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM sodium fluoride, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride. The homogenate was incubated for 2 h at 4 C and then centrifuged at 12,000 x g for 15 min to remove insoluble material. The protein concentration of the supernatants was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
Immunoblot analyses
Protein homogenates were prepared in Laemmli buffer (8) and subjected to SDS-PAGE. To allow for quantification between blots, an in-house standard was also run on each gel. Proteins were separated on 7.5% gels, transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences, Piscataway, NJ), and blocked in 1% BSA- or 5% skim milk-Tris-buffered saline containing 0.025% Tween 20. Membranes were probed with the following primary antibodies as required: anti-IR-pYpY1162/1163 (BioSource, Camarillo, CA), anti-IR (BD Transduction Laboratories, San Diego, CA), anti-IRS-1-pY612 (BioSource), anti-IRS-1 (Upstate Cell Signaling Solutions, Lake Placid, NY), anti-pY895-IRS-1 (Oncogene Research Products, Boston, MA), anti-IRS-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-phospho (Ser473)-Akt (Cell Signaling Technology, Beverly, MA), anti-Akt (Cell Signaling Technology), anti-phospho (Thr202/Tyr204)-p44/42 MAPK (Cell Signaling Technology), or anti-p44/42 MAPK (Cell Signaling Technology). Membranes were then incubated with donkey antirabbit horseradish peroxidase or protein A-horseradish peroxidase secondary antibody, and bands were detected by chemiluminescence (PerkinElmer Life Sciences, Boston, MA) after exposure to film (Fuji Photo Film Co., Tokyo, Japan). Membranes were probed with phosphorylated antibodies first, then stripped and reprobed with the corresponding total antibodies. Films were subsequently scanned, and the densities of bands of interest were quantified using IPLab Gel software (Signal Analytics Corp., Vienna, VA).
Statistics
Results are presented as the mean ± SE. Data were initially analyzed by two-way factorial ANOVA to establish an overall response to insulin stimulation and to examine the degree to which the response was modulated by diet. If a significant interaction was found, more detailed analyses within groups were subsequently performed by Fisher’s protected least significant difference post hoc tests. Statistical calculations were performed using a commercial software package (StatView 5.0; SAS Institute, Inc., Cary, NC). Differences were considered significant at P < 0.05.
Results
Insulin time-course metabolic parameters
Infusion of insulin at a rate of 0.5 U/kg·h resulted in significant increases in circulating insulin after 2 min (P < 0.01), and by 5 min the plasma insulin concentration had reached a plateau of approximately 1.5 nM, indicating that steady-state insulin levels were achieved within 5 min of the start of the insulin infusion (Fig. 1). These levels are within the high physiological range for rats (9). The plasma FFA concentration was significantly decreased after 5 min of insulin infusion (P < 0.05; Fig. 1), demonstrating an insulin-mediated inhibition of lipolysis. There was also a significant effect of insulin on whole body glucose metabolism, because a glucose infusion of 34 ± 1.0 mg/kg·min was required to maintain plasma euglycemia at 7 mM over the 20 min of the study.
Time course of signaling protein phosphorylation
The time course of activation of insulin signaling intermediates was established in CHOW rats by examining phosphorylation of IR, IRS-1, and PKB at specific phosphorylation sites. Using a phospho-specific antibody directed at Tyr1162/Tyr1163 of the IR (sites critical for full tyrosine kinase activity of IR) (10), it was observed that insulin infusion resulted in a significant elevation in IR phosphorylation that increased throughout the 20-min infusion period (Fig. 2A). Phospho-specific antibodies directed at Tyr612 of IRS-1 (a putative binding domain for the p85 subunit of PI3-K) (11, 12) and Tyr895 of IRS-1 [a growth receptor-bound-2 (Grb-2) binding site] (12, 13, 14) were used to examine the insulin-stimulated activation of IRS-1. We detected a 2-fold increase in phosphorylation of IRS-1 Tyr612 at 5 min (P < 0.01 vs. basal), which was similar at 10 min, but had returned to basal levels by 20 min (Fig. 2B). Interestingly, phosphorylation of IRS-1 at Tyr895 followed a different time course from that of Tyr612, reaching a plateau at 10 min. This suggested that different functions of the IRS-1 scaffolding protein could be dependent on differential phosphorylation of specific sites. Phosphorylation of PKB at Ser473, a critical phosphorylation site required for full activation of PKB (15, 16), was increased 3-fold 5 min after the beginning of the insulin infusion, and this degree of phosphorylation was maintained for the remaining insulin infusion time (Fig. 2C). The results in Figs. 1 and 2 demonstrate that a 5-min insulin infusion at a rate of 0.5 U/kg·h is enough to produce a significant effect on whole body insulin action and phosphorylation of signaling intermediates. This protocol is therefore suitable for assessment of the effect of high-fat diets on initial insulin signaling events.
Effects of fat feeding and insulin infusion on plasma and tissue metabolic parameters
Infusion of insulin at a rate of 0.5 U/kg·h for 5 min resulted in similar significant 4- to 5-fold increases in circulating insulin levels in all groups of rats (Table 1). Glucose infusion was required to maintain euglycemia in all groups of rats, but this was significantly lower in both the PUFA and SFA rats, which is an indication of whole body insulin resistance (CHOW, 35 ± 1.3 mg/kg· min; PUFA, 25 ± 1.7 mg/kg·min; SFA, 28 ± 0.4 mg/kg· min; P < 0.001, PUFA and SFA vs. CHOW). The high-fat diet enriched in SFA resulted in higher plasma triglycerides (at least 1.7-fold) and higher circulating FFA (at least 1.5-fold) compared with both levels in both CHOW and PUFA rats (Table 1). There was an effect of insulin stimulation to reduce plasma FFA in the CHOW and PUFA rats only (Table 1). The tissue triglyceride content in red quadriceps muscle was elevated 1.3-fold in both PUFA and SFA rats compared with CHOW rats (CHOW, 2.8 ± 0.2 μmol/g; PUFA, 3.6 ± 0.3 μmol/g; SFA, 3.7 ± 0.4 μmol/g; P < 0.05, PUFA and SFA vs. CHOW).
IR tyrosine phosphorylation
Basal levels of IR Tyr1162/1163 phosphorylation were equivalent in all three groups of rats (Fig. 3). In CHOW rats, there were approximately 2- to 3-fold increases in IR Tyr1162/1163 phosphorylation over basal with insulin stimulation (Fig. 3). A similar increase in phosphorylation at these sites was observed in both PUFA and SFA rats with insulin stimulation for 5 or 10 min (Fig. 3). Although the responses in both high-fat-fed groups of rats appeared marginally lower than the corresponding CHOW values, the differences were not statistically significant. The total IR protein content was not affected by diet or insulin stimulation (Fig. 3B). Overall, there was a significant effect of insulin stimulation (P < 0.0001), but no effect of diet (P = 0.2528), on muscle IR tyrosine phosphorylation at the Tyr1162/1163 residues.
IRS-1 tyrosine phosphorylation
Phosphorylation at the PI3-K binding Tyr612 site of IRS-1 was similar in all groups of rats in the basal state (Fig. 4). In CHOW rats, insulin infusion produced a 2-fold increase in IRS-1 Tyr612 phosphorylation at both 5 and 10 min (Fig. 4). However, there was no effect of insulin infusion on IRS-1 Tyr612 phosphorylation in either PUFA or SFA rats at 5 or 10 min (Fig. 4). Total IRS-1 protein expression was similar in all groups, at all time points (Fig. 4B). The effect of diet treatment on insulin-stimulated IRS-1 Tyr612 phosphorylation in muscle was highly significant (P < 0.0001). Phosphorylation of IRS-1 at Tyr895 (the Grb-2-binding site) by insulin was not affected by either fat diet (Fig. 5), suggesting that the effects of a high-fat diet on insulin-stimulated IRS-1 tyrosine phosphorylation were specific for certain IRS-1 mediated functions.
PKB phosphorylation
Basal phosphorylation of PKB at the Ser473 site was not different from that in CHOW animals with either high-fat diet treatment (Fig. 6). Insulin stimulation for 5 min resulted in significant increases in Ser473-PKB phosphorylation in all groups (CHOW, P < 0.001 vs. basal; PUFA, P < 0.001 vs. basal; SFA, P < 0.01 vs. basal; Fig. 6). However, this insulin-stimulated increase in phosphorylation was significantly blunted in the PUFA and SFA rats compared with CHOW rats (P < 0.001, PUFA vs. CHOW, 5 min insulin; P < 0.001, SFA vs. CHOW, 5 min insulin). After 10 min of insulin infusion, Ser473-PKB phosphorylation remained significantly elevated over basal levels in all groups of rats. At this time point, the phosphorylation of PKB in the fat-fed rats was still lower than that in the CHOW rats (P = 0.06, PUFA vs. CHOW, 10 min insulin; P < 0.05, SFA vs. CHOW, 10 min insulin; Fig. 6). Total PKB protein was unaffected by diet or insulin treatment (Fig. 6B).
ERK phosphorylation
ERK has a role in cell growth and differentiation and is activated downstream of IRS-1 Tyr895 phosphorylation (17). A phospho-specific antibody directed at Thr202/Tyr204 of ERK (key regulatory phosphorylation sites in this kinase) (18, 19) was used to examine the activation of this protein. ERK Thr202/Tyr204 phosphorylation in PUFA and SFA rats followed a pattern similar to that in CHOW rats (Fig. 7). In all rats, there was no significant increase in insulin-stimulated ERK Thr202/Tyr204 phosphorylation at 5 min, but with 10-min insulin infusion, ERK Thr202/Tyr204 phosphorylation was similarly elevated approximately 50% over basal (CHOW, P < 0.05 vs. basal; PUFA, P = 0.08 vs. basal; SFA, P = 0.1 vs. basal; Fig. 7).
Discussion
In the present study we examined the effects of time and different high-fat diets on the phosphorylation of intermediates of the insulin signaling pathway in skeletal muscle in vivo using experimental conditions similar to those routinely employed to assess insulin action during a euglycemic/hyperinsulinemic clamp. After 5 min of insulin infusion, plasma insulin levels reached a steady-state concentration (1.5 nM) and produced clear downstream metabolic effects and a significant and coordinated increase in the phosphorylation state of IR Tyr1162/1163, IRS-1 Tyr612, and PKB Ser473 in skeletal muscle. One interesting observation from this study was the difference in the time course of phosphorylation of IRS-1 on two specific sites associated with distinct physiological processes. The phosphorylation of Tyr612 on IRS-1 is involved in insulin signaling via the PI3-K pathway to PKB and the stimulation of glucose uptake and glycogen synthesis (11, 12), whereas IRS-1 phosphorylation at Tyr895 is thought to be linked to insulin stimulation of mitogenic pathways via Grb-2 (12, 13, 14). The rapid onset of IR Tyr1162/1163, IRS-1 Tyr612, and PKB Ser473 phosphorylation suggests an immediate transduction of the insulin signal for glucose uptake in muscle, whereas the insulin stimulation of mitogenic pathways (via IRS-1 Tyr895 and downstream ERK Thr202/Tyr204) may involve a slower onset and more sustained signal. The decrease in phosphorylation at Tyr612 of IRS-1 after 10 min, whereas the phosphorylation of IR and PKB was further increased or remained unchanged, highlights the possibility that the initial rate of phosphorylation of the signaling intermediates, rather than a sustained level of phosphorylation, is more important for transduction of the metabolic effects of insulin (20).
The effects of different dietary fat subtypes on the development of insulin resistance assessed by whole-body and tissue-specific glucose uptake have been studied previously (21). This study did not, however, extend to the effect of fat subtypes on in vivo insulin signaling in muscle or other insulin-sensitive tissues. In the studies reported here we show that a high-fat diet enriched in either n-6 polyunsaturated or saturated fats had different effects on circulating FAs and triglycerides, but produced similar increases in muscle triglyceride content, similar reductions in insulin action, and similar deleterious effects on skeletal muscle insulin signaling compared with those in control rats. Although in PUFA and SFA rats there was no significant difference in insulin-stimulated IR Tyr1162/1163 phosphorylation compared with CHOW rats, there was an absence of insulin-stimulated IRS-1 Tyr612 phosphorylation in PUFA and SFA rats and a decreased insulin-stimulated PKB Ser473 phosphorylation effect in PUFA and SFA rats at 5 min. The magnitude of the reduction in PKB phosphorylation in muscle of fat-fed rats was less than the reduction seen upstream at Tyr612-IRS-1. One explanation for this is that other PI3-K binding sites on IRS-1 are not affected or are less affected by the dietary treatment, thereby producing some downstream transmission of the insulin signal to PKB. Both fat diets also had no effect on insulin-stimulated IRS-1 Tyr895 phosphorylation and downstream ERK Thr202/Tyr204 phosphorylation, suggesting that the diets had site-specific effects on phosphorylation that could be relevant to specific signaling pathways, but might not be detected if activation of IRS-1 was assessed using antibodies that detect global IRS-1 tyrosine phosphorylation. At least 30 tyrosine phosphorylation sites exist on IRS-1, but only six are known PI3-K binding sites (22). Therefore, the use of phospho-specific antibodies may be more appropriate than assessment of global tyrosine phosphorylation when investigating the role of changes in IRS-1 phosphorylation relevant to stimulating glucose metabolism or other insulin-stimulated processes.
The changes in phosphorylation observed in the current study show similarity to some reports on the effect of a high-fat diet on insulin signaling (3, 23, 24), but not others (2, 25). All these previous studies were conducted using pharmacological insulin stimulation (which produces plasma insulin concentrations of 10–20 nM) and were thus more likely to reflect the maximal capacity of the muscle to respond to insulin stimulation rather than the level of signaling required to stimulate glucose transport in vivo. Our results suggest that the total amount of fat provided in the high-fat diets, rather than the particular FA subtype provided, has a major impact on insulin action in vivo. Studies using cell culture systems have shown distinct effects of different FAs on impairing insulin signaling in skeletal muscle cells in vitro (1). The current results appear contrary to these observations; however, the presentation of excess lipids to cells or tissue is very different in the two experimental situations. In culture systems, cells are exposed to only one FA, whereas in vivo all FA species are still present in the circulation even if there is enrichment of one subtype of FA because of the composition of the diet. It would seem likely that the in vivo effects of increased SFA or PUFA will always result in a combination of any individual effects of specific FAs because it is not possible to produce an experimental situation in vivo that generates exposure of muscle tissue to only one FA, and there can be interconversion of FA species in vivo (26, 27).
The results of the current study show a major defect in insulin signaling with chronic high-fat diet-induced insulin resistance at the level of IRS-1 and a lower Ser473-PKB phosphorylation downstream. It is difficult to be definitive, however, about whether one defect is primary to the other. It is possible that the decrease in PKB phosphorylation is secondary to the reduced phosphorylation of IRS-1, but it is also possible that the high-fat diets have direct effects on both signaling molecules or that a decrease in the phosphorylation and activity of PKB reduces the phosphorylation of specific serine residues on IRS-1 that normally protects it from protein-tyrosine phosphatases (28).
In conclusion, we have used a method of physiological insulin stimulation designed to study the onset of insulin signaling events in vivo to show that rats fed a high-fat diet enriched in either PUFA or SFA developed similar impairments in the phosphorylation of insulin signaling intermediates in skeletal muscle. There was no defect at the level of IR, reduced insulin-stimulated tyrosine phosphorylation at IRS-1 Tyr612 (but not at IRS-1 Tyr895 or ERK), and reduced insulin-stimulated PKB Ser473 phosphorylation in both PUFA and SFA rats. This suggests that impairments in skeletal muscle insulin signal transduction in vivo after physiological insulin stimulation are a consequence of chronic high-fat treatment, independent of fat subtype, and that decreased IRS-1 Tyr612 and PKB Ser473 phosphorylations are key components of the reduced insulin action observed in situations of chronic lipid oversupply.
Acknowledgments
We thank Elaine Preston, Donna Wilks, Joanna Edema, and the staff of Garvan Institute’s Biological Testing Facility for technical assistance with this study.
Footnotes
This work was supported by the National Health and Medical Research Council of Australia and the Diabetes Australia Research Trust. G.F. was the recipient of an Australian Postgraduate Research Award.
First Published Online September 8, 2005
Abbreviations: FA, Fatty acid; Grb-2, growth receptor-bound-2; IR, insulin receptor; IRS-1, IR substrate-1; PI3-K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PUFA, n-6 polyunsaturated fatty acid; SFA, saturated fatty acid.
Accepted for publication September 1, 2005.
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