Insulin Receptor Substrates-1 and -2 Are Both Depleted but via Different Mechanisms after Down-Regulation of Glucose Transport in Rat Adipoc
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内分泌学杂志 2005年第7期
Department of Medicine, Ume? University Hospital, SE-901 85 Ume?, Sweden
Address all correspondence and requests for reprints to: Jan W. Eriksson, M.D., Ph.D., Department of Medicine, Ume? University Hospital, SE-901 85 Ume?, Sweden. E-mail: jan.eriksson@medicin.umu.se.
Abstract
Alterations in muscle and adipose tissue insulin receptor substrate (IRS)-1 and IRS-2 are associated with, and commonly believed to contribute to, development of insulin resistance. In this study, we investigated the mechanisms behind previously observed reductions in IRS levels due to high concentrations of glucose and insulin and their significance in the impairment of glucose uptake capacity in primary rat adipocytes. Semiquantitative RT-PCR analysis showed that insulin (104 μU/ml) alone or in combination with glucose (15 mM) markedly suppressed IRS-2 gene expression, whereas IRS-1 mRNA was unaffected by the culture conditions. The negative effect of a high glucose/high insulin setting on IRS-1 protein level was still exerted when protein synthesis was inhibited with cycloheximide. Impairment of glucose uptake capacity after treatment with high glucose and insulin was most pronounced after 3 h, whereas IRS-1 and IRS-2 protein levels were unaffected up to 6 h but were reduced after 16 h. Moreover, impaired glucose uptake capacity could only partially be reversed by subsequent incubation at physiological conditions. These novel results suggest that: 1) in a high glucose/high insulin setting depletion of IRS-1 and IRS-2 protein, respectively, occurs via different mechanisms, and IRS-2 gene expression is suppressed, whereas IRS-1 depletion is due to posttranslational mechanisms; 2) IRS-1 and IRS-2 protein depletion is a secondary event in the development of insulin resistance in this model of hyperglycemia/hyperinsulinemia; and 3) depletion of cellular IRS in adipose tissue may be a consequence rather than a cause of insulin resistance and hyperinsulinemia in type 2 diabetes.
Introduction
THE METABOLIC SYNDROME and type 2 diabetes are diseases associated with insulin resistance and impaired glucose homeostasis, i.e. the balance between the addition of carbohydrates from the diet, glucose production by the liver, and glucose use by insulin-sensitive tissues, e.g. muscle and fat. Insulin plays a crucial role in glucose homeostasis by stimulating glucose uptake in muscle and fat, inhibiting glycogenolysis and gluconeogenesis in liver and inhibiting lipolysis in fat through the insulin receptor (IR) and the insulin receptor substrates (IRSs). The molecular mechanisms underlying insulin resistance are still largely unknown but seem to occur as a result of a combination of genetic predisposition and environmental factors. The IRSs are major substrates of the IR and important components in insulin signaling in the regulation of glucose homeostasis. Regarding the role of the IRSs in cellular insulin resistance, in vivo and in vitro studies in different human and animal models of insulin resistance and type 2 diabetes indicate tissue-specific alterations in gene expression and protein levels of IRS-1 and IRS-2 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Human skeletal muscle showed decreased expression of both IRS-1 and IRS-2 in response to insulin clamp but no difference between control and type 2 diabetic subjects (9), whereas adipocytes from type 2 diabetic individuals displayed a reduced IRS-1 protein content but unaffected levels of IRS-2 (8). Chronic insulin treatment in 3T3-L1 adipocytes resulted in both IRS-1 and IRS-2 depletion with unaffected IRS-1 gene expression (4, 10). The observed IRS depletions are commonly believed to contribute to the development of an insulin-resistant state, but it is not clear whether the observed IRS alterations are primary or secondary events. Our group has previously shown that long-term (24 h) treatment with high concentrations of glucose (15 mM) and insulin (104 μU/ml) in combination renders rat adipocytes insulin resistant with respect to the acute insulin effect to stimulate glucose uptake. This induction of cellular insulin resistance also coincided with a marked depletion in protein levels of IRS-1 and IRS-2 (11).
The aim of the present study was to further investigate the mechanisms behind the effect of chronically elevated glucose and insulin levels to produce cellular insulin resistance and alterations in IRS levels. We have more extensively elucidated the time course for induction as well as reversal of cellular insulin resistance. In particular, the role of gene expression and translation in the dysregulation of IRSs in induced cellular insulin resistance were studied. We also wished to address whether IRS alterations are preceding and contributing to insulin resistance. Primary epididymal rat adipocytes were used as a cell model for insulin’s target tissues, and the high glucose/high insulin setting was employed as an established insulin-resistant condition, mirroring the metabolic environment in early human type 2 diabetes (12, 13).
Materials and Methods
Chemicals
Collagenase A and adenosine deaminase were purchased from Boehringer Mannheim (Mannheim, Germany). BSA (fraction V) and N6-(R-phenylisopropyl)-adenosine were purchased from Sigma Chemical Co. (St. Louis, MO). Human insulin (Actrapid) was delivered by Novo Nordisk A/S (Copenhagen, Denmark). [14C]-U-D-glucose (specific activity 200–300 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Freiburg, Germany). DMEM, fetal calf serum, penicillin/streptomycin, medium 199, Ultrapure agarose electrophoresis grade, and TRIzol reagent were obtained from Life Technologies, Inc., (Paisley, UK). Primers and Superscript first-strand synthesis system for RT-PCR were purchased from Invitrogen, Life Technologies (Paisley, UK). Tag DNA polymerase (1 U/μl) and PCR nucleotide mix (10 mM) were delivered by Roche Diagnostics BmbH (Mannheim, Germany). Ethidium bromide tablets were obtained from KEBO Lab (Stockholm, Sweden). Anti-IRS-1 and anti-IRS-2 polyclonal antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY), and antiactin monoclonal antibody from Chemicon International Inc. (Temecula, CA). Secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Animals
Male Sprague Dawley rats were obtained from B & K (Sollentuna, Sweden). Rats were caged in groups of four and were given free access to pelleted food and water. Rats were allowed to acclimatize to their new surroundings for at least 5 d before they were killed. The Ume? Ethical Committee for Animal Research approved the study protocol.
Tissue collection and isolation of primary rat adipocytes
Rats weighing 150–200 g were decapitated, and the epididymal fat pads were immediately removed, minced, and treated with collagenase (0.6 mg/ml in medium 199 containing 5.6 mM glucose and 40 mg/ml BSA) for approximately 1 h at 37 C in a shaking water bath. The collagenase-treated tissue was filtered through a nylon mesh and isolated adipocytes were washed four times with fresh medium.
Cell culture
For long-term treatment with high glucose and/or insulin concentrations, dispersed cells from four rats were pooled and equally distributed to four Teflon flasks containing DMEM supplemented with 5 or 15 mM D-glucose, two flasks for each concentration, containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) with or without insulin (104 μU/ml). Cells were incubated at 37 C with gentle stirring (25 rpm) for 24 h under a gas phase of 95% O2-5% CO2. For cells treated with cycloheximide (CHX), a translation inhibitor isolated adipocytes from four rats were equally divided among four polystyrene flasks containing DMEM supplemented with 5 mM D-glucose or 15 mM D-glucose plus insulin with or without 18 μM CHX. Cells were incubated for 16 h before assessment of insulin-stimulated glucose uptake capacity and total cellular lysate preparation. Cell size was determined on isolated adipocytes as previously described (14) and did not differ between the incubation conditions used (data not shown). Cell viability after culture was confirmed by trypan blue exclusion test.
Glucose uptake assay
After incubation of isolated adipocytes, cells were washed four times, and glucose uptake was assessed as previously described (15, 16). In short, isolated adipocytes (lipocrit 3–5%) were incubated without glucose for 15 min in 37 C in medium 199 with 4% BSA, adenosine deaminase (1 U/ml), N6-(R-phenylisopropyl)-adenosine (1 μM), and various insulin concentrations ranging from 0 to 1000 μU/ml. Then D-[U-14C]glucose was added (0.21 mCi/liter, 0.7–1.0 μmol/liter), and cells were incubated for an additional 45 min after which cells were separated from the incubation medium by centrifugation through silicone oil. The adipocytes were collected and cell-associated radioactivity was measured by scintillation counting. The cellular clearance of glucose from medium was calculated according to the following formula and, under these conditions, it can be considered as an index of the rate of glucose transport (17): cellular clearance of medium glucose = [Cpm cells x volume]/[Cpm medium x cell number x time]. Measurements were performed in duplicate for all treatments.
RNA extraction and cDNA synthesis
After a 24-h incubation, the adipocytes were decanted into a sterile tube. Total RNA from each incubation was extracted using TRIzol and isolated in accordance with the manufacturer’s instructions. The RNA pellet was dissolved in diethylpyrocarbonate-treated water and stored at –20 C until further use. RNA concentration was determined spectrophotometrically (GeneQuant II; Pharmacia Biotech, Uppsala, Sweden) by the absorbance at 260 nm (1 A260 unit = 40 μg/ml single-stranded RNA). RNA integrity was assessed by electrophoresis in 1% (wt/vol) agarose minigels and ethidium bromide staining. Total RNA was reverse transcribed using the SuperScript first-strand synthesis system for RT-PCR. Four micrograms of total RNA was denatured for 5 min at 65 C together with 0.5 μl Oligo(dT)12–18 and 1 μl 10 mM deoxynucleotide triphosphate mix. Annealing was carried out by adding first-strand buffer, 0.1 M dithiothreitol, and 1 μl RNaseOut recombinant RNase inhibitor and incubating for 2 min at 42 C. First-strand synthesis was performed by adding 1 μl SuperScript II RT and incubating at 42 C for 50 min and thereafter at 70 C for 15 min. The reaction was terminated by incubating 20 min at 37 C with 1 μl RNase H. cDNA was stored at –20 C until further use.
Primers for PCR amplification
The nucleotide sequences and expected RT-PCR product sizes from oligonucleotide primer sets are given in Table 1. The primer set for IR and IRS-1 were designed to amplify bases 2474–2743 and 1382–1718 of the rat sequence M29014 and X58375, respectively (18). The primer set for IRS-2 was designed to amplify bases 220–370 of the rat IRS-2 coding sequence AF087674 using Primer3 Output available on the Internet (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primer set product was verified by sequencing. The primer sequences for phosphatidylinositol 3-kinase (PI3K) and glucose transporter 4 (GLUT)4 were designed to amplify bases 1–231 and 221–594 of the coding sequence D63325 and X14771, respectively (19). Primer set for protein kinase B (PKB) was designed to amplify bases 699-1679 of the rat PKB coding sequence D30041 (19). Primer set for ?-actin was designed to amplify bases 425-1016 of the rat ?-actin coding sequence NM_031144 (20).
TABLE 1. Sequences of each primer pair used in semiquantitative RT-PCR experiments
Semiquantitative PCR
Semiquantitative RT-PCR was performed using a method in which the cDNA from each incubation condition was divided in three equal parts and PCR was performed for different increasing numbers of cycles. The time course of appearance of different PCR products will give an estimate of their relative abundance, and their levels can thus be compared between different culture conditions (21). ?-Actin mRNA expression was used as internal control and was not affected by the different incubation conditions used. The PCR products were separated on a 1.2% agarose gel and analyzed by ethidium bromide staining. Gels were viewed and photographed under UV light illumination using Flour-SR MultiImager (Bio-Rad Laboratories, Hercules, CA).
Western analysis of total cellular lysates
After the incubation period, cells were washed four times and total cellular lysates were prepared essentially as previously described (11). In brief, cells were washed three times in PBS and centrifuged at 2300 x g for 5 min at 4 C, and the remaining supernatant was removed. Cells were lysed with lysis buffer containing 25 mM Tris-HCl (pH 7.4), 0.5 mM EGTA, 25 mM NaCl, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 0.2 mM leupeptin, 1 mM benzamidine, and 0.1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochlorine and were rocked for 2 h at 4 C. Detergent insoluble material was sedimented by centrifugation at 12000 x g for 15 min at 4 C, and the supernatant was collected. BCA reagent (Pierce Biotechnology, Inc., Rockford, IL) was used to determine protein concentration. The cell lysates were boiled for 5 min in SDS-PAGE loading buffer [0.1 M Tris-Cl (pH 6.8), 20% glycerol, 1% ?- mercaptoethanol, and bromophenol blue]. Samples were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA) and blocked overnight in 3% dry milk in 20 mM Tris, 137 mM NaCl, 0.5% Tween 20 (pH 7.6). The membranes were incubated with primary antibody, washed, and then incubated with appropriate secondary antibody. Immunoreactive proteins were visualized using the ECL plus Western blotting kit (Amersham Bioscience, Buckinghamshire, UK) according to the instructions from the manufacturer and quantified by densitometry (Molecular Analyst, Bio-Rad Laboratories). All protein quantifications were adjusted for the corresponding ?-actin level, which was not consistently changed by the different incubation conditions.
Statistical analysis
Statistical analyses were performed using the SPSS package (SPSS Inc., Chicago, IL). Results are given as means ± SEM, and statistical significance was determined using one-way or two-way ANOVA for glucose uptake and the Dunnett’s test as a post hoc test. Kruskal-Wallis nonparametric test with Mann-Whitney U test as post hoc was used when comparing protein concentrations. P < 0.05 was considered statistically significant.
Results
IRS-1 and -2 protein and mRNA levels after treatment with high glucose and insulin
In agreement with our previous studies, long-term treatment (24 h) with high concentrations of glucose (15 mM) and insulin (104 μU/ml) in combination markedly reduced both IRS-1 and IRS-2 protein content (Fig. 1). To elucidate the mechanism(s) underlying the observed alterations in IRS-1 and IRS-2, their relative mRNA levels were assessed on long-term treatment with high concentrations of glucose and/or insulin. We also measured mRNA levels for the IR, PI3K, PKB, and the GLUT4. Reverse-transcribed RNA was obtained from cells after each incubation condition, and PCR was performed for increasing numbers of cycles and the results are shown in Fig 2. High insulin concentration in the culture medium, regardless of the surrounding glucose concentration, consistently induced a decrease in IRS-2 mRNA. High glucose alone slightly increased the IRS-2 mRNA levels, whereas the combined effect of high glucose and high insulin was a decreased IRS-2 mRNA level, compared with the control situation, similar to that after high insulin alone. Exposure to high glucose and/or insulin for 24 h did not alter mRNA expression of IR, IRS-1, PI3K, or PKB. However, high insulin induced an increase in GLUT4 mRNA expression and when combined with high glucose, there was a further increase.
FIG. 1. IRS-1 (A) and -2 (B) content in total cellular lysates assessed by Western blotting. Isolated primary epididymal adipocytes were cultured for 24 h in 5 mM glucose or 15 mM glucose plus insulin (104 μU/ml). After washing, total cellular lysates were prepared as described in Materials and Methods. Protein from total cellular lysates (15 or 20 μg for IRS-1 and –2, respectively) was separated on SDS-PAGE, followed by immunoblotting using anti-IRS-1 (A) or anti-IRS-2 (B) antibody. One representative blot is shown together with results of densitometry analysis expressed as percent of amount of IRS protein after the control culture condition (i.e. 5 mM glucose = 100%). All proteins quantified were adjusted for the corresponding ?-actin level. Data are expressed as means ± SEM of five separate experiments.
FIG. 2. IR, IRS-1, IRS-2, PI3-K, PKB, GLUT4, and ?-actin mRNA assessed by semiquantitative RT-PCR. Isolated cells were cultured for 24 h in 5 or 15 mM glucose with or without insulin (104 μU/ml). After washing, total RNA was extracted and 4 μg were reverse transcribed as previously described in Materials and Methods. Ten percent (2 μl) of the cDNA was amplified using PCR and three PCRs were performed for each culture condition with increasing cycle numbers. The PCR products were separated on a 1.2% agarose gel and analyzed by ethidium bromide staining. Gels were viewed and photographed under UV light illumination. All experiments with subsequent RNA extraction and PCR analysis were repeated three times with similar results, and one representative gel is shown.
Role of protein synthesis in the impairment of glucose transport and depletion of IRS-1/2
Presence of 18 μM CHX [a concentration that effectively inhibits protein synthesis (22)] during 16 h treatment did not affect the ability of high glucose and insulin to suppress both basal and insulin-stimulated glucose uptake capacity (Fig. 3A). Addition of CHX in the control condition (5 mM glucose) exerted no effect on the insulin-stimulated glucose uptake capacity but caused a marked decrease (by 70%, P < 0.05) in the basal glucose uptake capacity. A higher concentration of CHX (36 μM) was also tested with the same result as for 18 μM CHX (data not shown). Addition of CHX in the control situation had no significant effect on the protein level of IRS-1 but induced a marked decrease in the level of IRS-2 (Fig. 3B). The decrease in protein level of IRS-1 after long-term treatment with high concentrations of glucose and insulin were still observed in the presence of CHX.
FIG. 3. Effect of protein synthesis inhibition by CHX on glucose uptake and IRS-1/2 protein levels. A, Adipocytes were incubated for 16 h in 5 mM glucose (G) or 15 mM glucose plus insulin (104 μU/ml) in the absence or presence of CHX (18 μM). After washing, cells were incubated for 15 min with different insulin concentrations (0–1000 μU/ml). 14C-glucose was then added and glucose uptake was measured during the next 45 min (see Materials and Methods). All data are expressed as means ± SEM of three separate experiments. , P < 0.05 vs. 5 mM G; ***, P < 0.001 vs. 5 mM G; ###, P < 0.001 vs. 5 mM G + CHX. G, Glucose; Ins, insulin. B, After 16 h incubation, cells were washed and total cellular lysates were prepared as described in Materials and Methods. Proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes followed by immunoblotting using appropriate antibody. One representative blot is shown. The results of densitometry analysis are shown and data are expressed as arbitrary units in relation to the amount of IRS-1/2 in the control culture condition (i.e. level of IRS-1/2 in 5 mM glucose = 100%). All proteins quantified were correlated against the corresponding ?-actin level. Data are means ± SEM of four separate experiments. *, P < 0.05 vs. control; #, P < 0.05 vs. 5 mM glucose plus CHX.
Time course for induction and reversal of impaired glucose transport and IRS levels
To investigate the time course of cellular insulin resistance due to culture in high glucose (15 mM) and insulin (104 μU/ml), adipocytes were cultured in total for 8 h, first in the presence of 5 mM glucose. During the last 1, 2, 3, 4, 6, or all 8 h, respectively, the culture medium conditions were changed to 15 mM glucose and 104 μU/ml insulin. Incubation for 2 h was sufficient to induce a marked cellular insulin resistance in the adipocytes and maximal effect was achieved after 3 h (Fig. 4A). To address whether the time course for IRS-1 and/or -2 depletion are primary or secondary to cellular insulin resistance, the protein levels of IRS-1/2 were analyzed after 3, 6, 16, and 24 h incubation with high concentrations of glucose and insulin (Fig. 4B). After 16 h a significant reduction in the protein levels of IRS-1 and IRS-2 is observed with no further reduction of IRS-1 after 24 h but a slight further suppression of IRS-2 levels. Incubation for 3 and 6 h showed no reduction in the protein levels of IRS-1/2 on the contrary protein levels of IRS-2 seemed to increase, however nonsignificantly.
FIG. 4. Time course for the effect of high concentrations of glucose and insulin on glucose uptake capacity. A, Adipocytes were cultured in medium containing 5 mM glucose for a total of 8 h. For the last 1, 2, 3, 4, 6, or 8 h, respectively, the adipocytes were exposed to high glucose (15 mM) and insulin (104 μU/ml). After washing, cells were incubated for 15 min with different insulin concentrations (0, 25, 1000 μU/ml as indicated). 14C-glucose was then added and glucose uptake was assessed during the following 45 min. Data are expressed as 14C-glucose clearance after different times of exposure to high glucose and insulin. All data are expressed as means ± SEM of three separate experiments. B, Effect on IRS-1 and -2 protein levels on incubation in high glucose- and insulin-containing medium for 3, 6, 16, and 24 h. After incubation cells were washed and total cellular lysates were prepared as described in Materials and Methods. Proteins were separated on SDS-PAGE and followed by immunoblotting using appropriate antibody. One representative blot is shown. The results of densitometry analysis are shown and data are expressed as arbitrary units in relation to the amount of IRS-1/2 in the control culture condition (i.e. level of IRS-1/2 in 5 mM glucose = 100%). All proteins quantified were adjusted for the corresponding ?-actin level. Data are means ± SEM of three to five separate experiments, respectively. *, P < 0.05.
After induction of cellular insulin resistance with high glucose and insulin pretreatment for at least 6 h, cells were transferred to medium containing 5 mM glucose for varying time periods. The high glucose/high insulin treatment itself suppressed glucose uptake capacity measured at the basal and insulin-stimulated (1000 μU/ml) state to 30 ± 10 and 41 ± 7% of control (pretreatment with 5 mM glucose, no insulin), respectively. After the high glucose/insulin treatment, a subsequent incubation at a physiological glucose concentration in the absence of insulin resulted in a gradual, but not complete, normalization but mainly of the insulin-stimulated glucose transport capacity (P < 0.05). Thus, after 3, 6, 12, and 16 h, basal glucose uptake rate was 17 ± 5, 40 ± 11, 62 ± 7, and 47 ± 3% of control, respectively, and maximal insulin-stimulated glucose uptake capacity was 58 ± 7, 69 ± 8, 79 ± 10, and 87 ± 14% of control, respectively (n = 3–7, Fig. 5). No further improvement was seen with 24 h incubation (data not shown).
FIG. 5. Time course for the effect on impaired glucose uptake capacity when culture conditions were switched from high concentrations of glucose and insulin to low glucose without insulin. Cells were pretreated with 5 mM glucose, no insulin for 0–16 h, so that the total incubation was finally 22 h. Then, the adipocytes were exposed to high glucose (15 mM) and insulin (104 μU/ml) for 6 h to induce cellular insulin resistance and subsequently transferred to medium containing low glucose (5 mM) without insulin for the indicated time periods. After washing and 15 min incubation with different insulin concentrations (0, 25, 1000 μU/ml as indicated), 14C-glucose was added and glucose uptake was assessed during the following 45 min. The result of one representative of seven similar experiments is shown (see Results for details). (C, Control situation, adipocytes cultured in 5 mM glucose without insulin).
Discussion
Using primary rat adipocytes, we have previously shown that reduction in insulin-stimulated glucose uptake capacity due to long-term treatment with high concentrations of glucose and insulin is accompanied by a reduction in the protein levels of IRS-1 and IRS-2 (11). A high concentration of insulin alone had no effect on insulin-stimulated glucose uptake but decreased the protein level of IRS-2. In contrast, a high concentration of glucose per se slightly reduced glucose uptake capacity, decreased protein level of IRS-1 but markedly increased the level of IRS-2. The present study focused on the underlying mechanisms and the physiological impact of IRS-1 and IRS-2 depletion in a high glucose/high insulin setting. It was confirmed that the IRS amounts were markedly decreased after 24 h treatment with high concentrations of glucose and insulin. For the first time, we show that high insulin concentrations reduce the level of IRS-2 mRNA in adipocytes independent of the surrounding glucose concentration, whereas high glucose alone slightly increased IRS-2 mRNA level. The net effect of high glucose and insulin in combination was a reduction in IRS-2 mRNA level, compared with the control condition, i.e. 5 mM glucose.
Taken together our results suggest that the effects on IRS-2 mRNA levels exerted by the different incubation conditions occur in parallel with the alterations in IRS-2 protein levels. From the present data, it can, however, not be determined whether the effects of high glucose and insulin on IRS-2 mRNA levels are due to alterations in gene transcription or a change in mRNA stability. However, Zhang et al. (23) showed that insulin inhibited transcription of IRS-2 in rat liver. This was further supported by Hirashima et al. (24), who showed that insulin had no effect on the half-life of IRS-2 mRNA in Fao rat hepatoma cells. Insulin-induced suppression of IRS-2 mRNA and protein levels has also recently been reported in L6 muscle cells (25), and our results are in accordance with a similar effect of insulin in rat adipocytes. The specific mechanisms by which insulin regulates IRS-2 mRNA levels are not clarified, but a recent study indicated that insulin regulates IRS-2 gene transcription in a PI3K/Akt-dependent manner (24). The alterations in the protein levels of IRS-2 during hyperglycemia- and/or hyperinsulinemia-like conditions can probably thus be explained by alterations in gene expression, i.e. at the transcriptional level. The semiquantitative measurements of the relative mRNA levels of IR, IRS-1, PI3K, and PKB displayed no alterations on induction of cellular insulin resistance. This is compatible with previous reports indicating that insulin does not affect IRS-1 gene expression (4, 26). Hence, our finding of depleted IRS-1 protein levels should be explained by posttranscriptional mechanisms.
GLUT4 gene expression, on the other hand, was clearly affected by the different treatments and was enhanced by the presence of insulin, which is known to induce GLUT4 mRNA expression in adipose tissue (27, 28, 29, 30). Surprisingly, insulin in combination with high glucose induced a higher mRNA expression, compared with insulin at physiological glucose concentration. Animal studies indicate that GLUT4 content in fat tissue is unaffected by hyperglycemia, and a glucose-responsive element in the GLUT4 promoter region has not been found (31). Thus, the observed additive effect of high glucose on insulin’s ability to increase GLUT4 mRNA expression is an indirect effect of the high glucose concentration in this model.
To further address the mechanism behind the observed IRS-1 protein depletion, a protein synthesis inhibitor, CHX, was used. Protein synthesis inhibition had no effect on the cellular IRS-1 protein level during a 16-h incubation. Interestingly, high concentrations of glucose and insulin decreased the IRS-1 protein level to the same extent whether or not CHX was present. Accordingly, whatever mechanism that explains the observed depletion of IRS-1 in the high glucose/high insulin setting, it is not related to the rate of protein synthesis, and hence it should occur at a posttranslational level. In contrast, protein synthesis inhibition alone resulted in a marked decrease in the level of IRS-2, indicating that the turnover rate for IRS-2 is higher than for IRS-1. Because the insulin-stimulated glucose uptake in these cells was unaffected by CHX, these data also suggest that the IRS-2 level is not a critical rate-limiting factor in the insulin signaling cascade with respect to stimulation of glucose uptake and that the cellular mechanisms involved in down-regulation of insulin-stimulated glucose uptake is not dependent on the synthesis rate of other regulatory proteins. High glucose/high insulin in the presence of CHX failed to induce any further decrease in the IRS-2 level below that seen with CHX per se. This indicates that high glucose/high insulin leads to IRS-2 down-regulation at a transcriptional or translational level and that this is in accordance with the finding of suppressed mRNA expression.
The finding that insulin-stimulated glucose uptake capacity in control cells was not significantly affected by a 16-h inhibition of protein synthesis is in line with previous work in 3T3-L1 and rat adipocytes (32, 33, 34, 35, 36). Those authors also showed that the level of plasma membrane GLUT4 on insulin stimulation is not affected by CHX treatment. Our results indicate that the insulin-stimulated GLUT4 translocation is still functional after 16 h inhibition of protein synthesis, a result further supported by the fact that the half-life for GLUT4 is long, approximately 50 h (37). However, 16 h inhibition of protein synthesis reduced basal glucose uptake by approximately 70%, which was an unexpected finding. Previous data (35, 36, 38) showed that incubation of rat or 3T3-L1 adipocytes in the presence of CHX for 4 and up to 8 h increased basal glucose uptake owing to an increased intrinsic activity of GLUT1 due to regulatory protein(s) with rapid turnover. The reason for this discrepancy is most likely the differences in incubation periods because it has been reported that the half-life for GLUT1 is 19 h (37), and a 16-h incubation period with inhibited protein synthesis should have reduced GLUT1 levels significantly. Protein synthesis inhibition most likely also affects recycling of GLUT1, which might contribute to the observed impairment of basal glucose uptake capacity (39).
Taken together our present results indicate that the protein levels of IRS-1 and IRS-2, respectively, are regulated via different mechanisms in a model of insulin resistance, i.e. adipocytes cultured in a high glucose/high insulin setting. The observed decrease in the protein level of IRS-2 after 24 h treatment with high concentrations of glucose and insulin seems to be the result of a suppressing effect by insulin on gene transcription. On the contrary, the decrease in IRS-1 appears to be caused by posttranslational mechanism(s), most likely alterations in the protein degradation pathway (5). This is supported by previous results indicating that chronic insulin exposure increases serine/threonine phosphorylation of IRS-1 enhancing its degradation by the proteasome pathway (5, 40). We are currently addressing possible alterations in the IRS-1 degradation pathways in ongoing studies.
It is an interesting and novel finding that the observed depletion of IRS-1 and IRS-2 most likely can be ruled out as a primary cause for the impaired glucose uptake capacity in this model because the effect of a high glucose/high insulin setting on glucose uptake capacity reached a maximum already after 3 h when there was no reduction in either IRS-1 or IRS-2 protein levels. A significant reduction in IRS-1 and IRS-2 was instead demonstrated after 16 h incubation with high concentrations of glucose and insulin. This indicates that the observed depletion in IRS-1 and IRS-2 protein in adipocytes cultured in a high glucose/high insulin environment develops after and possibly as a result of the impaired glucose uptake capacity or, alternatively, as a result of insulin resistance per se. It is not unlikely, however, that the observed IRS depletion occurring as a secondary event in turn might aggravate the impaired glucose uptake capacity (11). Moreover, it can obviously not be excluded that IRS depletion might be of primary importance in other cell systems or other models of insulin resistance. Furthermore, we did not address the activity of IRSs, and alterations in the phosphorylation status can occur as a result of exposure to high glucose and insulin (40, 41). We have previously shown that the dose-response curve for insulin-stimulated PKB phosphorylation was shifted to the right after 24 h treatment in a high glucose/high insulin setting (11), and this effect could possibly be attributed to the observed IRS-1 and IRS-2 depletion.
Although little is known about the mechanism(s) behind insulin resistance in our model, recent studies present possible pathways. Hyperglycemia has been shown to activate protein kinase C isoforms via increased flux through the hexosamine biosynthesis pathway (42). Protein kinase C can induce serine phosphorylation of IRS-1 that impairs P13 kinase activation and insulin-mediated glucose uptake (40, 41, 43). Interestingly, hyperglycemia has also been shown to induce reactive oxygen species in adipocytes (43). Reactive oxygen species may trigger an inflammatory response including a rise in the levels of TNF, which is known to induce insulin resistance through impairment of GLUT4 synthesis and phosphorylation of IRS-1 Ser307 (43). Chronic exposure to high insulin levels down-regulates cell surface insulin receptors and can lead to increased IRS-1 serine phosphorylation (40, 44).
In contrast to the rapid time course for development of insulin resistance in the presence of high concentrations of glucose and insulin, the restoration of responsiveness to insulin on normalization of glucose and insulin levels appears to be sluggish and incomplete. The ability to restore basal compared with insulin-stimulated glucose transport capacity appears to be smaller.
In summary, long-term exposure to a high glucose/high insulin environment leads to reduction of IRS-1 and IRS-2 content in primary rat adipocytes. For the first time, we demonstrate that IRS-2 depletion can be explained by suppression of gene expression, whereas IRS-1 depletion appears to be caused by posttranslational mechanisms, presumably alterations in the protein degradation pathway. A high glucose/high insulin setting leads to impaired glucose uptake capacity, but the underlying mechanisms do not seem to involve alterations in IRS-1 and IRS-2 content. On the contrary, IRS-1 and IRS-2 depletion develops after the impairment of glucose uptake capacity and can potentially be secondary to insulin resistance. Although it cannot be excluded that alterations in IRS levels in adipocytes contribute to insulin resistance in human type 2 diabetes, they probably occur as a consequence of a diabetic tissue environment.
Acknowledgments
We express our appreciation for the skillful experimental work and assistance by Ewa Str?mqvist-Engbo.
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Address all correspondence and requests for reprints to: Jan W. Eriksson, M.D., Ph.D., Department of Medicine, Ume? University Hospital, SE-901 85 Ume?, Sweden. E-mail: jan.eriksson@medicin.umu.se.
Abstract
Alterations in muscle and adipose tissue insulin receptor substrate (IRS)-1 and IRS-2 are associated with, and commonly believed to contribute to, development of insulin resistance. In this study, we investigated the mechanisms behind previously observed reductions in IRS levels due to high concentrations of glucose and insulin and their significance in the impairment of glucose uptake capacity in primary rat adipocytes. Semiquantitative RT-PCR analysis showed that insulin (104 μU/ml) alone or in combination with glucose (15 mM) markedly suppressed IRS-2 gene expression, whereas IRS-1 mRNA was unaffected by the culture conditions. The negative effect of a high glucose/high insulin setting on IRS-1 protein level was still exerted when protein synthesis was inhibited with cycloheximide. Impairment of glucose uptake capacity after treatment with high glucose and insulin was most pronounced after 3 h, whereas IRS-1 and IRS-2 protein levels were unaffected up to 6 h but were reduced after 16 h. Moreover, impaired glucose uptake capacity could only partially be reversed by subsequent incubation at physiological conditions. These novel results suggest that: 1) in a high glucose/high insulin setting depletion of IRS-1 and IRS-2 protein, respectively, occurs via different mechanisms, and IRS-2 gene expression is suppressed, whereas IRS-1 depletion is due to posttranslational mechanisms; 2) IRS-1 and IRS-2 protein depletion is a secondary event in the development of insulin resistance in this model of hyperglycemia/hyperinsulinemia; and 3) depletion of cellular IRS in adipose tissue may be a consequence rather than a cause of insulin resistance and hyperinsulinemia in type 2 diabetes.
Introduction
THE METABOLIC SYNDROME and type 2 diabetes are diseases associated with insulin resistance and impaired glucose homeostasis, i.e. the balance between the addition of carbohydrates from the diet, glucose production by the liver, and glucose use by insulin-sensitive tissues, e.g. muscle and fat. Insulin plays a crucial role in glucose homeostasis by stimulating glucose uptake in muscle and fat, inhibiting glycogenolysis and gluconeogenesis in liver and inhibiting lipolysis in fat through the insulin receptor (IR) and the insulin receptor substrates (IRSs). The molecular mechanisms underlying insulin resistance are still largely unknown but seem to occur as a result of a combination of genetic predisposition and environmental factors. The IRSs are major substrates of the IR and important components in insulin signaling in the regulation of glucose homeostasis. Regarding the role of the IRSs in cellular insulin resistance, in vivo and in vitro studies in different human and animal models of insulin resistance and type 2 diabetes indicate tissue-specific alterations in gene expression and protein levels of IRS-1 and IRS-2 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Human skeletal muscle showed decreased expression of both IRS-1 and IRS-2 in response to insulin clamp but no difference between control and type 2 diabetic subjects (9), whereas adipocytes from type 2 diabetic individuals displayed a reduced IRS-1 protein content but unaffected levels of IRS-2 (8). Chronic insulin treatment in 3T3-L1 adipocytes resulted in both IRS-1 and IRS-2 depletion with unaffected IRS-1 gene expression (4, 10). The observed IRS depletions are commonly believed to contribute to the development of an insulin-resistant state, but it is not clear whether the observed IRS alterations are primary or secondary events. Our group has previously shown that long-term (24 h) treatment with high concentrations of glucose (15 mM) and insulin (104 μU/ml) in combination renders rat adipocytes insulin resistant with respect to the acute insulin effect to stimulate glucose uptake. This induction of cellular insulin resistance also coincided with a marked depletion in protein levels of IRS-1 and IRS-2 (11).
The aim of the present study was to further investigate the mechanisms behind the effect of chronically elevated glucose and insulin levels to produce cellular insulin resistance and alterations in IRS levels. We have more extensively elucidated the time course for induction as well as reversal of cellular insulin resistance. In particular, the role of gene expression and translation in the dysregulation of IRSs in induced cellular insulin resistance were studied. We also wished to address whether IRS alterations are preceding and contributing to insulin resistance. Primary epididymal rat adipocytes were used as a cell model for insulin’s target tissues, and the high glucose/high insulin setting was employed as an established insulin-resistant condition, mirroring the metabolic environment in early human type 2 diabetes (12, 13).
Materials and Methods
Chemicals
Collagenase A and adenosine deaminase were purchased from Boehringer Mannheim (Mannheim, Germany). BSA (fraction V) and N6-(R-phenylisopropyl)-adenosine were purchased from Sigma Chemical Co. (St. Louis, MO). Human insulin (Actrapid) was delivered by Novo Nordisk A/S (Copenhagen, Denmark). [14C]-U-D-glucose (specific activity 200–300 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Freiburg, Germany). DMEM, fetal calf serum, penicillin/streptomycin, medium 199, Ultrapure agarose electrophoresis grade, and TRIzol reagent were obtained from Life Technologies, Inc., (Paisley, UK). Primers and Superscript first-strand synthesis system for RT-PCR were purchased from Invitrogen, Life Technologies (Paisley, UK). Tag DNA polymerase (1 U/μl) and PCR nucleotide mix (10 mM) were delivered by Roche Diagnostics BmbH (Mannheim, Germany). Ethidium bromide tablets were obtained from KEBO Lab (Stockholm, Sweden). Anti-IRS-1 and anti-IRS-2 polyclonal antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY), and antiactin monoclonal antibody from Chemicon International Inc. (Temecula, CA). Secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Animals
Male Sprague Dawley rats were obtained from B & K (Sollentuna, Sweden). Rats were caged in groups of four and were given free access to pelleted food and water. Rats were allowed to acclimatize to their new surroundings for at least 5 d before they were killed. The Ume? Ethical Committee for Animal Research approved the study protocol.
Tissue collection and isolation of primary rat adipocytes
Rats weighing 150–200 g were decapitated, and the epididymal fat pads were immediately removed, minced, and treated with collagenase (0.6 mg/ml in medium 199 containing 5.6 mM glucose and 40 mg/ml BSA) for approximately 1 h at 37 C in a shaking water bath. The collagenase-treated tissue was filtered through a nylon mesh and isolated adipocytes were washed four times with fresh medium.
Cell culture
For long-term treatment with high glucose and/or insulin concentrations, dispersed cells from four rats were pooled and equally distributed to four Teflon flasks containing DMEM supplemented with 5 or 15 mM D-glucose, two flasks for each concentration, containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) with or without insulin (104 μU/ml). Cells were incubated at 37 C with gentle stirring (25 rpm) for 24 h under a gas phase of 95% O2-5% CO2. For cells treated with cycloheximide (CHX), a translation inhibitor isolated adipocytes from four rats were equally divided among four polystyrene flasks containing DMEM supplemented with 5 mM D-glucose or 15 mM D-glucose plus insulin with or without 18 μM CHX. Cells were incubated for 16 h before assessment of insulin-stimulated glucose uptake capacity and total cellular lysate preparation. Cell size was determined on isolated adipocytes as previously described (14) and did not differ between the incubation conditions used (data not shown). Cell viability after culture was confirmed by trypan blue exclusion test.
Glucose uptake assay
After incubation of isolated adipocytes, cells were washed four times, and glucose uptake was assessed as previously described (15, 16). In short, isolated adipocytes (lipocrit 3–5%) were incubated without glucose for 15 min in 37 C in medium 199 with 4% BSA, adenosine deaminase (1 U/ml), N6-(R-phenylisopropyl)-adenosine (1 μM), and various insulin concentrations ranging from 0 to 1000 μU/ml. Then D-[U-14C]glucose was added (0.21 mCi/liter, 0.7–1.0 μmol/liter), and cells were incubated for an additional 45 min after which cells were separated from the incubation medium by centrifugation through silicone oil. The adipocytes were collected and cell-associated radioactivity was measured by scintillation counting. The cellular clearance of glucose from medium was calculated according to the following formula and, under these conditions, it can be considered as an index of the rate of glucose transport (17): cellular clearance of medium glucose = [Cpm cells x volume]/[Cpm medium x cell number x time]. Measurements were performed in duplicate for all treatments.
RNA extraction and cDNA synthesis
After a 24-h incubation, the adipocytes were decanted into a sterile tube. Total RNA from each incubation was extracted using TRIzol and isolated in accordance with the manufacturer’s instructions. The RNA pellet was dissolved in diethylpyrocarbonate-treated water and stored at –20 C until further use. RNA concentration was determined spectrophotometrically (GeneQuant II; Pharmacia Biotech, Uppsala, Sweden) by the absorbance at 260 nm (1 A260 unit = 40 μg/ml single-stranded RNA). RNA integrity was assessed by electrophoresis in 1% (wt/vol) agarose minigels and ethidium bromide staining. Total RNA was reverse transcribed using the SuperScript first-strand synthesis system for RT-PCR. Four micrograms of total RNA was denatured for 5 min at 65 C together with 0.5 μl Oligo(dT)12–18 and 1 μl 10 mM deoxynucleotide triphosphate mix. Annealing was carried out by adding first-strand buffer, 0.1 M dithiothreitol, and 1 μl RNaseOut recombinant RNase inhibitor and incubating for 2 min at 42 C. First-strand synthesis was performed by adding 1 μl SuperScript II RT and incubating at 42 C for 50 min and thereafter at 70 C for 15 min. The reaction was terminated by incubating 20 min at 37 C with 1 μl RNase H. cDNA was stored at –20 C until further use.
Primers for PCR amplification
The nucleotide sequences and expected RT-PCR product sizes from oligonucleotide primer sets are given in Table 1. The primer set for IR and IRS-1 were designed to amplify bases 2474–2743 and 1382–1718 of the rat sequence M29014 and X58375, respectively (18). The primer set for IRS-2 was designed to amplify bases 220–370 of the rat IRS-2 coding sequence AF087674 using Primer3 Output available on the Internet (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primer set product was verified by sequencing. The primer sequences for phosphatidylinositol 3-kinase (PI3K) and glucose transporter 4 (GLUT)4 were designed to amplify bases 1–231 and 221–594 of the coding sequence D63325 and X14771, respectively (19). Primer set for protein kinase B (PKB) was designed to amplify bases 699-1679 of the rat PKB coding sequence D30041 (19). Primer set for ?-actin was designed to amplify bases 425-1016 of the rat ?-actin coding sequence NM_031144 (20).
TABLE 1. Sequences of each primer pair used in semiquantitative RT-PCR experiments
Semiquantitative PCR
Semiquantitative RT-PCR was performed using a method in which the cDNA from each incubation condition was divided in three equal parts and PCR was performed for different increasing numbers of cycles. The time course of appearance of different PCR products will give an estimate of their relative abundance, and their levels can thus be compared between different culture conditions (21). ?-Actin mRNA expression was used as internal control and was not affected by the different incubation conditions used. The PCR products were separated on a 1.2% agarose gel and analyzed by ethidium bromide staining. Gels were viewed and photographed under UV light illumination using Flour-SR MultiImager (Bio-Rad Laboratories, Hercules, CA).
Western analysis of total cellular lysates
After the incubation period, cells were washed four times and total cellular lysates were prepared essentially as previously described (11). In brief, cells were washed three times in PBS and centrifuged at 2300 x g for 5 min at 4 C, and the remaining supernatant was removed. Cells were lysed with lysis buffer containing 25 mM Tris-HCl (pH 7.4), 0.5 mM EGTA, 25 mM NaCl, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 0.2 mM leupeptin, 1 mM benzamidine, and 0.1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochlorine and were rocked for 2 h at 4 C. Detergent insoluble material was sedimented by centrifugation at 12000 x g for 15 min at 4 C, and the supernatant was collected. BCA reagent (Pierce Biotechnology, Inc., Rockford, IL) was used to determine protein concentration. The cell lysates were boiled for 5 min in SDS-PAGE loading buffer [0.1 M Tris-Cl (pH 6.8), 20% glycerol, 1% ?- mercaptoethanol, and bromophenol blue]. Samples were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA) and blocked overnight in 3% dry milk in 20 mM Tris, 137 mM NaCl, 0.5% Tween 20 (pH 7.6). The membranes were incubated with primary antibody, washed, and then incubated with appropriate secondary antibody. Immunoreactive proteins were visualized using the ECL plus Western blotting kit (Amersham Bioscience, Buckinghamshire, UK) according to the instructions from the manufacturer and quantified by densitometry (Molecular Analyst, Bio-Rad Laboratories). All protein quantifications were adjusted for the corresponding ?-actin level, which was not consistently changed by the different incubation conditions.
Statistical analysis
Statistical analyses were performed using the SPSS package (SPSS Inc., Chicago, IL). Results are given as means ± SEM, and statistical significance was determined using one-way or two-way ANOVA for glucose uptake and the Dunnett’s test as a post hoc test. Kruskal-Wallis nonparametric test with Mann-Whitney U test as post hoc was used when comparing protein concentrations. P < 0.05 was considered statistically significant.
Results
IRS-1 and -2 protein and mRNA levels after treatment with high glucose and insulin
In agreement with our previous studies, long-term treatment (24 h) with high concentrations of glucose (15 mM) and insulin (104 μU/ml) in combination markedly reduced both IRS-1 and IRS-2 protein content (Fig. 1). To elucidate the mechanism(s) underlying the observed alterations in IRS-1 and IRS-2, their relative mRNA levels were assessed on long-term treatment with high concentrations of glucose and/or insulin. We also measured mRNA levels for the IR, PI3K, PKB, and the GLUT4. Reverse-transcribed RNA was obtained from cells after each incubation condition, and PCR was performed for increasing numbers of cycles and the results are shown in Fig 2. High insulin concentration in the culture medium, regardless of the surrounding glucose concentration, consistently induced a decrease in IRS-2 mRNA. High glucose alone slightly increased the IRS-2 mRNA levels, whereas the combined effect of high glucose and high insulin was a decreased IRS-2 mRNA level, compared with the control situation, similar to that after high insulin alone. Exposure to high glucose and/or insulin for 24 h did not alter mRNA expression of IR, IRS-1, PI3K, or PKB. However, high insulin induced an increase in GLUT4 mRNA expression and when combined with high glucose, there was a further increase.
FIG. 1. IRS-1 (A) and -2 (B) content in total cellular lysates assessed by Western blotting. Isolated primary epididymal adipocytes were cultured for 24 h in 5 mM glucose or 15 mM glucose plus insulin (104 μU/ml). After washing, total cellular lysates were prepared as described in Materials and Methods. Protein from total cellular lysates (15 or 20 μg for IRS-1 and –2, respectively) was separated on SDS-PAGE, followed by immunoblotting using anti-IRS-1 (A) or anti-IRS-2 (B) antibody. One representative blot is shown together with results of densitometry analysis expressed as percent of amount of IRS protein after the control culture condition (i.e. 5 mM glucose = 100%). All proteins quantified were adjusted for the corresponding ?-actin level. Data are expressed as means ± SEM of five separate experiments.
FIG. 2. IR, IRS-1, IRS-2, PI3-K, PKB, GLUT4, and ?-actin mRNA assessed by semiquantitative RT-PCR. Isolated cells were cultured for 24 h in 5 or 15 mM glucose with or without insulin (104 μU/ml). After washing, total RNA was extracted and 4 μg were reverse transcribed as previously described in Materials and Methods. Ten percent (2 μl) of the cDNA was amplified using PCR and three PCRs were performed for each culture condition with increasing cycle numbers. The PCR products were separated on a 1.2% agarose gel and analyzed by ethidium bromide staining. Gels were viewed and photographed under UV light illumination. All experiments with subsequent RNA extraction and PCR analysis were repeated three times with similar results, and one representative gel is shown.
Role of protein synthesis in the impairment of glucose transport and depletion of IRS-1/2
Presence of 18 μM CHX [a concentration that effectively inhibits protein synthesis (22)] during 16 h treatment did not affect the ability of high glucose and insulin to suppress both basal and insulin-stimulated glucose uptake capacity (Fig. 3A). Addition of CHX in the control condition (5 mM glucose) exerted no effect on the insulin-stimulated glucose uptake capacity but caused a marked decrease (by 70%, P < 0.05) in the basal glucose uptake capacity. A higher concentration of CHX (36 μM) was also tested with the same result as for 18 μM CHX (data not shown). Addition of CHX in the control situation had no significant effect on the protein level of IRS-1 but induced a marked decrease in the level of IRS-2 (Fig. 3B). The decrease in protein level of IRS-1 after long-term treatment with high concentrations of glucose and insulin were still observed in the presence of CHX.
FIG. 3. Effect of protein synthesis inhibition by CHX on glucose uptake and IRS-1/2 protein levels. A, Adipocytes were incubated for 16 h in 5 mM glucose (G) or 15 mM glucose plus insulin (104 μU/ml) in the absence or presence of CHX (18 μM). After washing, cells were incubated for 15 min with different insulin concentrations (0–1000 μU/ml). 14C-glucose was then added and glucose uptake was measured during the next 45 min (see Materials and Methods). All data are expressed as means ± SEM of three separate experiments. , P < 0.05 vs. 5 mM G; ***, P < 0.001 vs. 5 mM G; ###, P < 0.001 vs. 5 mM G + CHX. G, Glucose; Ins, insulin. B, After 16 h incubation, cells were washed and total cellular lysates were prepared as described in Materials and Methods. Proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes followed by immunoblotting using appropriate antibody. One representative blot is shown. The results of densitometry analysis are shown and data are expressed as arbitrary units in relation to the amount of IRS-1/2 in the control culture condition (i.e. level of IRS-1/2 in 5 mM glucose = 100%). All proteins quantified were correlated against the corresponding ?-actin level. Data are means ± SEM of four separate experiments. *, P < 0.05 vs. control; #, P < 0.05 vs. 5 mM glucose plus CHX.
Time course for induction and reversal of impaired glucose transport and IRS levels
To investigate the time course of cellular insulin resistance due to culture in high glucose (15 mM) and insulin (104 μU/ml), adipocytes were cultured in total for 8 h, first in the presence of 5 mM glucose. During the last 1, 2, 3, 4, 6, or all 8 h, respectively, the culture medium conditions were changed to 15 mM glucose and 104 μU/ml insulin. Incubation for 2 h was sufficient to induce a marked cellular insulin resistance in the adipocytes and maximal effect was achieved after 3 h (Fig. 4A). To address whether the time course for IRS-1 and/or -2 depletion are primary or secondary to cellular insulin resistance, the protein levels of IRS-1/2 were analyzed after 3, 6, 16, and 24 h incubation with high concentrations of glucose and insulin (Fig. 4B). After 16 h a significant reduction in the protein levels of IRS-1 and IRS-2 is observed with no further reduction of IRS-1 after 24 h but a slight further suppression of IRS-2 levels. Incubation for 3 and 6 h showed no reduction in the protein levels of IRS-1/2 on the contrary protein levels of IRS-2 seemed to increase, however nonsignificantly.
FIG. 4. Time course for the effect of high concentrations of glucose and insulin on glucose uptake capacity. A, Adipocytes were cultured in medium containing 5 mM glucose for a total of 8 h. For the last 1, 2, 3, 4, 6, or 8 h, respectively, the adipocytes were exposed to high glucose (15 mM) and insulin (104 μU/ml). After washing, cells were incubated for 15 min with different insulin concentrations (0, 25, 1000 μU/ml as indicated). 14C-glucose was then added and glucose uptake was assessed during the following 45 min. Data are expressed as 14C-glucose clearance after different times of exposure to high glucose and insulin. All data are expressed as means ± SEM of three separate experiments. B, Effect on IRS-1 and -2 protein levels on incubation in high glucose- and insulin-containing medium for 3, 6, 16, and 24 h. After incubation cells were washed and total cellular lysates were prepared as described in Materials and Methods. Proteins were separated on SDS-PAGE and followed by immunoblotting using appropriate antibody. One representative blot is shown. The results of densitometry analysis are shown and data are expressed as arbitrary units in relation to the amount of IRS-1/2 in the control culture condition (i.e. level of IRS-1/2 in 5 mM glucose = 100%). All proteins quantified were adjusted for the corresponding ?-actin level. Data are means ± SEM of three to five separate experiments, respectively. *, P < 0.05.
After induction of cellular insulin resistance with high glucose and insulin pretreatment for at least 6 h, cells were transferred to medium containing 5 mM glucose for varying time periods. The high glucose/high insulin treatment itself suppressed glucose uptake capacity measured at the basal and insulin-stimulated (1000 μU/ml) state to 30 ± 10 and 41 ± 7% of control (pretreatment with 5 mM glucose, no insulin), respectively. After the high glucose/insulin treatment, a subsequent incubation at a physiological glucose concentration in the absence of insulin resulted in a gradual, but not complete, normalization but mainly of the insulin-stimulated glucose transport capacity (P < 0.05). Thus, after 3, 6, 12, and 16 h, basal glucose uptake rate was 17 ± 5, 40 ± 11, 62 ± 7, and 47 ± 3% of control, respectively, and maximal insulin-stimulated glucose uptake capacity was 58 ± 7, 69 ± 8, 79 ± 10, and 87 ± 14% of control, respectively (n = 3–7, Fig. 5). No further improvement was seen with 24 h incubation (data not shown).
FIG. 5. Time course for the effect on impaired glucose uptake capacity when culture conditions were switched from high concentrations of glucose and insulin to low glucose without insulin. Cells were pretreated with 5 mM glucose, no insulin for 0–16 h, so that the total incubation was finally 22 h. Then, the adipocytes were exposed to high glucose (15 mM) and insulin (104 μU/ml) for 6 h to induce cellular insulin resistance and subsequently transferred to medium containing low glucose (5 mM) without insulin for the indicated time periods. After washing and 15 min incubation with different insulin concentrations (0, 25, 1000 μU/ml as indicated), 14C-glucose was added and glucose uptake was assessed during the following 45 min. The result of one representative of seven similar experiments is shown (see Results for details). (C, Control situation, adipocytes cultured in 5 mM glucose without insulin).
Discussion
Using primary rat adipocytes, we have previously shown that reduction in insulin-stimulated glucose uptake capacity due to long-term treatment with high concentrations of glucose and insulin is accompanied by a reduction in the protein levels of IRS-1 and IRS-2 (11). A high concentration of insulin alone had no effect on insulin-stimulated glucose uptake but decreased the protein level of IRS-2. In contrast, a high concentration of glucose per se slightly reduced glucose uptake capacity, decreased protein level of IRS-1 but markedly increased the level of IRS-2. The present study focused on the underlying mechanisms and the physiological impact of IRS-1 and IRS-2 depletion in a high glucose/high insulin setting. It was confirmed that the IRS amounts were markedly decreased after 24 h treatment with high concentrations of glucose and insulin. For the first time, we show that high insulin concentrations reduce the level of IRS-2 mRNA in adipocytes independent of the surrounding glucose concentration, whereas high glucose alone slightly increased IRS-2 mRNA level. The net effect of high glucose and insulin in combination was a reduction in IRS-2 mRNA level, compared with the control condition, i.e. 5 mM glucose.
Taken together our results suggest that the effects on IRS-2 mRNA levels exerted by the different incubation conditions occur in parallel with the alterations in IRS-2 protein levels. From the present data, it can, however, not be determined whether the effects of high glucose and insulin on IRS-2 mRNA levels are due to alterations in gene transcription or a change in mRNA stability. However, Zhang et al. (23) showed that insulin inhibited transcription of IRS-2 in rat liver. This was further supported by Hirashima et al. (24), who showed that insulin had no effect on the half-life of IRS-2 mRNA in Fao rat hepatoma cells. Insulin-induced suppression of IRS-2 mRNA and protein levels has also recently been reported in L6 muscle cells (25), and our results are in accordance with a similar effect of insulin in rat adipocytes. The specific mechanisms by which insulin regulates IRS-2 mRNA levels are not clarified, but a recent study indicated that insulin regulates IRS-2 gene transcription in a PI3K/Akt-dependent manner (24). The alterations in the protein levels of IRS-2 during hyperglycemia- and/or hyperinsulinemia-like conditions can probably thus be explained by alterations in gene expression, i.e. at the transcriptional level. The semiquantitative measurements of the relative mRNA levels of IR, IRS-1, PI3K, and PKB displayed no alterations on induction of cellular insulin resistance. This is compatible with previous reports indicating that insulin does not affect IRS-1 gene expression (4, 26). Hence, our finding of depleted IRS-1 protein levels should be explained by posttranscriptional mechanisms.
GLUT4 gene expression, on the other hand, was clearly affected by the different treatments and was enhanced by the presence of insulin, which is known to induce GLUT4 mRNA expression in adipose tissue (27, 28, 29, 30). Surprisingly, insulin in combination with high glucose induced a higher mRNA expression, compared with insulin at physiological glucose concentration. Animal studies indicate that GLUT4 content in fat tissue is unaffected by hyperglycemia, and a glucose-responsive element in the GLUT4 promoter region has not been found (31). Thus, the observed additive effect of high glucose on insulin’s ability to increase GLUT4 mRNA expression is an indirect effect of the high glucose concentration in this model.
To further address the mechanism behind the observed IRS-1 protein depletion, a protein synthesis inhibitor, CHX, was used. Protein synthesis inhibition had no effect on the cellular IRS-1 protein level during a 16-h incubation. Interestingly, high concentrations of glucose and insulin decreased the IRS-1 protein level to the same extent whether or not CHX was present. Accordingly, whatever mechanism that explains the observed depletion of IRS-1 in the high glucose/high insulin setting, it is not related to the rate of protein synthesis, and hence it should occur at a posttranslational level. In contrast, protein synthesis inhibition alone resulted in a marked decrease in the level of IRS-2, indicating that the turnover rate for IRS-2 is higher than for IRS-1. Because the insulin-stimulated glucose uptake in these cells was unaffected by CHX, these data also suggest that the IRS-2 level is not a critical rate-limiting factor in the insulin signaling cascade with respect to stimulation of glucose uptake and that the cellular mechanisms involved in down-regulation of insulin-stimulated glucose uptake is not dependent on the synthesis rate of other regulatory proteins. High glucose/high insulin in the presence of CHX failed to induce any further decrease in the IRS-2 level below that seen with CHX per se. This indicates that high glucose/high insulin leads to IRS-2 down-regulation at a transcriptional or translational level and that this is in accordance with the finding of suppressed mRNA expression.
The finding that insulin-stimulated glucose uptake capacity in control cells was not significantly affected by a 16-h inhibition of protein synthesis is in line with previous work in 3T3-L1 and rat adipocytes (32, 33, 34, 35, 36). Those authors also showed that the level of plasma membrane GLUT4 on insulin stimulation is not affected by CHX treatment. Our results indicate that the insulin-stimulated GLUT4 translocation is still functional after 16 h inhibition of protein synthesis, a result further supported by the fact that the half-life for GLUT4 is long, approximately 50 h (37). However, 16 h inhibition of protein synthesis reduced basal glucose uptake by approximately 70%, which was an unexpected finding. Previous data (35, 36, 38) showed that incubation of rat or 3T3-L1 adipocytes in the presence of CHX for 4 and up to 8 h increased basal glucose uptake owing to an increased intrinsic activity of GLUT1 due to regulatory protein(s) with rapid turnover. The reason for this discrepancy is most likely the differences in incubation periods because it has been reported that the half-life for GLUT1 is 19 h (37), and a 16-h incubation period with inhibited protein synthesis should have reduced GLUT1 levels significantly. Protein synthesis inhibition most likely also affects recycling of GLUT1, which might contribute to the observed impairment of basal glucose uptake capacity (39).
Taken together our present results indicate that the protein levels of IRS-1 and IRS-2, respectively, are regulated via different mechanisms in a model of insulin resistance, i.e. adipocytes cultured in a high glucose/high insulin setting. The observed decrease in the protein level of IRS-2 after 24 h treatment with high concentrations of glucose and insulin seems to be the result of a suppressing effect by insulin on gene transcription. On the contrary, the decrease in IRS-1 appears to be caused by posttranslational mechanism(s), most likely alterations in the protein degradation pathway (5). This is supported by previous results indicating that chronic insulin exposure increases serine/threonine phosphorylation of IRS-1 enhancing its degradation by the proteasome pathway (5, 40). We are currently addressing possible alterations in the IRS-1 degradation pathways in ongoing studies.
It is an interesting and novel finding that the observed depletion of IRS-1 and IRS-2 most likely can be ruled out as a primary cause for the impaired glucose uptake capacity in this model because the effect of a high glucose/high insulin setting on glucose uptake capacity reached a maximum already after 3 h when there was no reduction in either IRS-1 or IRS-2 protein levels. A significant reduction in IRS-1 and IRS-2 was instead demonstrated after 16 h incubation with high concentrations of glucose and insulin. This indicates that the observed depletion in IRS-1 and IRS-2 protein in adipocytes cultured in a high glucose/high insulin environment develops after and possibly as a result of the impaired glucose uptake capacity or, alternatively, as a result of insulin resistance per se. It is not unlikely, however, that the observed IRS depletion occurring as a secondary event in turn might aggravate the impaired glucose uptake capacity (11). Moreover, it can obviously not be excluded that IRS depletion might be of primary importance in other cell systems or other models of insulin resistance. Furthermore, we did not address the activity of IRSs, and alterations in the phosphorylation status can occur as a result of exposure to high glucose and insulin (40, 41). We have previously shown that the dose-response curve for insulin-stimulated PKB phosphorylation was shifted to the right after 24 h treatment in a high glucose/high insulin setting (11), and this effect could possibly be attributed to the observed IRS-1 and IRS-2 depletion.
Although little is known about the mechanism(s) behind insulin resistance in our model, recent studies present possible pathways. Hyperglycemia has been shown to activate protein kinase C isoforms via increased flux through the hexosamine biosynthesis pathway (42). Protein kinase C can induce serine phosphorylation of IRS-1 that impairs P13 kinase activation and insulin-mediated glucose uptake (40, 41, 43). Interestingly, hyperglycemia has also been shown to induce reactive oxygen species in adipocytes (43). Reactive oxygen species may trigger an inflammatory response including a rise in the levels of TNF, which is known to induce insulin resistance through impairment of GLUT4 synthesis and phosphorylation of IRS-1 Ser307 (43). Chronic exposure to high insulin levels down-regulates cell surface insulin receptors and can lead to increased IRS-1 serine phosphorylation (40, 44).
In contrast to the rapid time course for development of insulin resistance in the presence of high concentrations of glucose and insulin, the restoration of responsiveness to insulin on normalization of glucose and insulin levels appears to be sluggish and incomplete. The ability to restore basal compared with insulin-stimulated glucose transport capacity appears to be smaller.
In summary, long-term exposure to a high glucose/high insulin environment leads to reduction of IRS-1 and IRS-2 content in primary rat adipocytes. For the first time, we demonstrate that IRS-2 depletion can be explained by suppression of gene expression, whereas IRS-1 depletion appears to be caused by posttranslational mechanisms, presumably alterations in the protein degradation pathway. A high glucose/high insulin setting leads to impaired glucose uptake capacity, but the underlying mechanisms do not seem to involve alterations in IRS-1 and IRS-2 content. On the contrary, IRS-1 and IRS-2 depletion develops after the impairment of glucose uptake capacity and can potentially be secondary to insulin resistance. Although it cannot be excluded that alterations in IRS levels in adipocytes contribute to insulin resistance in human type 2 diabetes, they probably occur as a consequence of a diabetic tissue environment.
Acknowledgments
We express our appreciation for the skillful experimental work and assistance by Ewa Str?mqvist-Engbo.
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