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Am J Physiol Endocrinol Metab 293: E783-E793, 2007. First published June 5, 2007; doi:10.1152/ajpendo.00685.2006
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Two phases of palmitate-induced insulin resistance in skeletal muscle: impaired GLUT4 translocation is followed by a reduced GLUT4 intrinsic activity

Hakam Alkhateeb,1 Adrian Chabowski,2 Jan F. C. Glatz,3 Joost F. P. Luiken,3 and Arend Bonen1

1Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada; 2Department of Physiology, Medical University of Bialystok, Bialystok, Poland; and 3Department of Molecular Genetics, Maastricht University, Maastricht, The Netherlands

Submitted 15 December 2006 ; accepted in final form 4 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined, in soleus muscle, the effects of prolonged palmitate exposure (0, 6, 12, 18 h) on insulin-stimulated glucose transport, intramuscular lipid accumulation and oxidation, activation of selected insulin-signaling proteins, and the insulin-stimulated translocation of GLUT4. Insulin-stimulated glucose transport was progressively reduced after 6 h (–33%), 12 h (–66%), and 18 h (–89%) of palmitate exposure. These decrements were closely associated with concurrent reductions in palmitate oxidation at 6 h (–40%), 12 h (–60%), and 18 h (–67%). In contrast, intramuscular ceramide (+24%) and diacylglycerol (+32%) concentrations, insulin-stimulated AS160 (–36%) and PRAS40 (–33%) phosphorylations, and Akt (–40%), PKC{theta} (–50%), and GLUT4 translocation (–40%) to the plasma membrane were all maximally altered within the first 6 h of palmitate treatment. No further changes were observed in any of these parameters after 12 and 18 h of palmitate exposure. Thus, the intrinsic activity of GLUT4 was markedly reduced after 12 and 18 h of palmitate treatment. During this reduced GLUT4 intrinsic activity phase at 12 and 18 h, the reduction in glucose transport was twofold greater compared with the early phase (≤6 h), when only GLUT4 translocation was impaired. Our study indicates that palmitate-induced insulin resistance is provoked by two distinct mechanisms: 1) an early phase (≤6 h), during which lipid-mediated impairments in insulin signaling and GLUT4 translocation reduce insulin-stimulated glucose transport, followed by 2) a later phase (12 and 18 h), during which the intrinsic activity of GLUT4 is markedly reduced independently of any further alterations in intramuscular lipid accumulation, insulin signaling and GLUT4 translocation.

glucose transport; glucose transporter 4; ceramide; diacylglycerol; Akt; AS160; protein kinase C{zeta}/{lambda}; protein kinase C{theta}; palmitate oxidation

IN INSULIN-SENSITIVE TISSUES, such as skeletal muscle, insulin activates a signaling cascade that induces the translocation of the glucose transporter GLUT4 from its intracellular depot(s) to the cell surface, where these transport proteins, dock, fuse, and become functionally active, thereby facilitating the transport of glucose into the cell (for review see Ref. 61). In recent years, dysregulated lipid metabolism has been associated with inducing insulin resistance. Infusion with fatty acids induces insulin resistance (5, 18, 42, 67), and there is a negative relationship between insulin-stimulated glucose uptake and intramuscular lipid accumulation, including tri- and diacylglycerols, long-chain fatty acyl-CoAs, and ceramides (12, 17, 31, 49). It has also been speculated that a reduction in skeletal muscle fatty acid oxidation contributes to the intracellular lipid accumulation, thereby inducing insulin resistance (36).

Impairments in the postreceptor insulin-signaling pathway are thought to be central in the development of fatty acid-induced insulin resistance. Fatty acid infusion (4–90 h) impaired the insulin-stimulated tyrosine phosphorylation of the insulin receptor, insulin receptor substrate (IRS)–1, and the activities of PI 3-kinase, IRS-1-associated PI 3-kinase (5, 18, 35, 42, 67), and atypical PKC{zeta}/{lambda} (38). Paradoxically, insulin resistance in skeletal muscle provoked by lipid (38, 42) or growth hormone infusion (32), as well as improved insulin sensitivity provoked by thiazolidinedione treatment in type 2 diabetics (33, 39), failed to alter insulin-stimulated Akt phosphorylation (32, 34, 38, 39, 42) and AS160 phosphorylation (34), downstream signals of PI 3-kinase. In high-fat-fed rats, complete inhibition of insulin-stimulated GLUT4 translocation in muscle only partially inhibited (–40%) insulin-stimulated glucose transport (62). Collectively, these findings illustrate that, although insulin sensitivity in muscle can be altered, the mechanisms involved are not completely understood. Mechanisms other than impaired insulin signaling may also contribute to lipid-induced insulin resistance.

To stimulate glucose transport, insulin appears to activate two mechanisms, 1) the well-known insulin-induced GLUT4 translocation from intracellular pools to the plasma membrane (61) and 2) an increased intrinsic activity of cell surface GLUT4 (3, 27, 30). In L6 muscle cells, insulin-stimulated glucose transport can be reduced by altering the intrinsic activity of cell surface GLUT4 in the absence of GLUT4 translocation and Akt phosphorylation (44, 59). Similarly, in rat skeletal muscle, insulin-stimulated glucose transport rates can be altered independently of the cell surface GLUT4 (11, 25, 62). Indeed, we have shown that epinephrine reduced insulin-stimulated glucose transport in a dose-dependent manner, whereas plasmalemmal GLUT4 remained unaltered (25). In addition, high-fat feeding appears to lower the intrinsic activity of GLUT4 (62). Thus, there is considerable evidence to suggest that the activity of cell surface GLUT4 can be regulated, a process that is not associated with the activation of p38 MAP kinase (3, 4, 63), as had been thought for some years. To date, there appear to be no studies, either in muscle cells or in mammalian tissues, that have examined whether fatty acids can impair glucose transport by interfering with GLUT4 activity at the cell surface.

Since the mechanisms contributing to fatty acid-induced insulin resistance are not fully known, we have examined at selected time intervals (0, 6, 12 and 18 h), in isolated mammalian muscle (soleus), the effects of palmitate on 1) insulin-stimulated glucose transport, 2) intramuscular lipid accumulation and oxidation, 3) activation of selected insulin signaling proteins, and 4) the insulin-stimulated translocation of GLUT4. Our studies have revealed that palmitate induces insulin resistance in two ways: 1) during an early phase (≤6 h), when intracellular diacylglycerol (+32%) and ceramide (+24%) accumulate and likely impair insulin-stimulated glucose transport (–33%), in association with reductions in insulin-stimulated Akt activation (–55%) and GLUT4 translocation (–40%); 2) these events are followed by a subsequent later phase at 12 and 18 h, during which, independently of any further alterations in GLUT4 translocation, insulin signaling, and intramuscular lipid accumulation, the intrinsic activity of GLUT4 is markedly reduced.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

[1-14C]palmitate was purchased from Amersham Life Science (Oakville, ON, Canada). Collagenase type II was purchased from Worthington (Lakewood, NJ). Insulin (Humulin-R) was purchased from Eli-Lilly (Toronto, ON, Canada). Penicillin and streptomycin were purchased from Invitrogen (Grand island, NY). Silica plates (no. 60, 0.25 mm) were obtained from Merck KGaA (Darmstadt, Germany). Total and phosphorylated proteins were determined with commercially available antibodies from the following sources: anti-Akt1/2, anti-Akt2, anti-phospho-Akt Ser473, and anti-phospho-Akt Thr308 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-PKC{zeta}/{lambda} and anti-PKC{theta} from Santa Cruz Biotechnology (Santa Cruz, CA); anti-IRS-1, anti-PI 3-kinase, anti-AS160, and anti-phospho-AS160 (Thr642) from Upstate (Lake Placid, NY); anti-GLUT4 from Chemicon International (Temecula, CA); goat-anti-rabbit secondary antibodies from Chemicon International; and donkey-anti-rabbit secondary antibody from Amersham Biosciences (Oakville, ON, Canada). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Animals

All experiments were approved by the Committee on Animal Care at the University of Guelph. Male Sprague-Dawley rats (55–75 g), bred on site, were used in these studies. The animals consumed normal laboratory chow and water ad libitum. At the onset of each experiment, rats were anesthetized with Somnotol (6 mg/100 g body wt ip), and the soleus muscles were gently dissected free.

Muscle Incubation

We (2) have shown that soleus muscles (~20 mg) remain viable, ex vivo, for up to 18 h. In the present study, intact soleus muscles (~20 mg), after a 30 min preincubation, were incubated with (2 mM) or without palmitate (control) for 0, 6, 12, or 18 h. At t = 0 min, palmitate-treated muscles were exposed only briefly (5 min) to palmitate, and control muscles were also briefly (5 min) incubated without palmitate. All incubations (0–18 h) were performed in 10 ml of warmed (30°C), pregassed (95% O2-5% CO2) Medium 199 containing 5 mM glucose supplemented with 4% bovine serum albumin V (BSA), penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). Low concentrations of insulin (14.3 µU/ml) were also included, as failure to maintain low concentrations of this hormone during a prolonged incubation period reduces intramuscular phosphagens and glycogen (65). The low concentrations of insulin did not stimulate glucose transport (present study 14.3 µU/ml; data not shown). Incubation vials were shaken at 110 cycles/min, and the gas phase and temperature were maintained at 95% O2-5% CO2 and 30°C, respectively, throughout. The incubation medium was replenished every 6 h.

ATP and Phosphocreatine

To ascertain the viability of incubated muscles, ATP and phosphocreatine (PCr) concentrations were determined in muscles incubated with and without palmitate for up to 18 h, as we (2) have described in detail elsewhere.

Glucose Transport

At selected periods (0, 6, 12, and 18 h) insulin-stimulated glucose transport was determined. Standard procedures were employed as we have described previously (7, 9). Briefly, after the specified incubation periods, soleus muscles were incubated (30°C, 30 min, 95% O2-5% CO2) in 2 ml of palmitate-free Krebs-Henseleit buffer [8 mM glucose, 32 mM mannitol, and 0.1% BSA with or without insulin (20,000 µU/ml)]. Subsequently, muscles were washed [2 x 10 min, 30°C, glucose-free Krebs-Henseleit buffer, 40 mM mannitol, 0.1% BSA ± insulin (20,000 µU/ml)]. Glucose transport was then determined in palmitate-free Krebs-Henseleit buffer (2 ml) supplemented with 0.5 µCi 3-O-methyl-D-[3H]glucose (3-OMG), 1.0 µCi [14C]mannitol, 32 mM 3-OMG, 4 mM mannitol, 4 mM pyruvate, and 0.1% BSA in the presence (20,000 µU/ml) or absence of insulin for 20 min. Thereafter, muscles were blotted, weighed, and solubilized followed by scintillation counting of muscle extracts.

Palmitate Oxidation

To determine the rates of fatty acid oxidation, our previously described method was used (19). Briefly, at the end of the incubation periods (0, 6, 12 and 18 h), isolated soleus muscles that had been incubated with and without palmitate for 0, 6, 12, and 18 h were transferred to other glass vials containing 2 ml of pregassed (95% O2-5% CO2) Medium 199 supplemented with 4% BSA and palmitate (2 mM, 0.5 µCi/ml [1-14C]palmitate; Amersham Life Science Oakville, ON, Canada). Palmitate oxidation occurred at 30°C for 40 min, and the 14CO2 released was captured in a benzothium hydroxide trap (400 µl, 1.0 M) In addition, at the end of the 40-min incubation period, dissolved CO2 was released by adding sulfuric acid (1.0 ml, 1 M) to a 1.0-ml aliquot of the incubating medium and trapping the 14CO2 in a benzothium hydroxide trap. Finally, water-soluble 14C-labeled intermediates were extracted from muscles homogenized after their incubation (19). After scintillation counting, the palmitate oxidation rate was determined as we have done previously (19), by summing the three sources of metabolized [14C]palmitate.

Intramuscular Triacylglycerol, Phospholipid, Diacylglycerol, and Ceramide Concentrations

The intramuscular lipids were determined in muscles incubated in Medium 199 with or without palmitate. At 0, 6, 12, and 18 h, muscles were freeze-clamped and stored at –80°C. To obtain sufficient muscle, five soleii were pooled for each independent determination.

Intramuscular lipids [triacylglycerol (TAG), diacylglycerol (DAG), phospholipids, ceramide] were extracted by the method of Folch et al. (20), as modified by van der Vusse et al. (64). Lipids were extracted from pulverized fat-free muscle samples in methanol (2 ml) and chloroform (4 ml) containing the antioxidant butylated hydroxytoluene (0.01%), and an internal standard (heptadecanoic acid) was added. Water (1.5 ml) was added to the extracting mixture. One portion of the chloroform layer was used to separate TAG, DAG, phospholipids, and lipid fractions, and the second portion was used for ceramide determinations. Thin-layer chromatography (silica plate no. 60, 0.25 mm; Merck, Darmstadt, Germany) was used to separate lipids. For TAG, DAG, and phospholipid separation a heptane-isopropyl ether-acetic acid (60:40:3, vol/vol/vol) resolving solution (54) was used, and for ceramide separation diethyl ether-hexane-acetic acid (90:10:1 vol/vol/vol) was used as described by Yano et al. (66). After being resolved and dried, lipid bands were visualized by spraying with a 0.2% solution of 2'7'-dichlorofluorescein in methanol and identified under UV light according to the standards on the plates. Each of the separated lipids was methylated (45, 66) in 14% boron trifluoride-methanol (14% BF3), and fatty acid methyl esters were extracted with pentane (64). Finally, samples were dissolved in hexane and analyzed by gas-liquid chromatography [Hewlett-Packard 5890 Series II gas chromatograph with Varian CP-SIL capillary column (50 m x 0.25 mm internal diameter)] and flame-ionization detector (Agilent Technologies, Santa Clara, CA). The oven temperature was programmed from 160°C to 225°C at 5°C/min and held at 225°C for 10 min. According to the retention times of standards, the individual long-chain fatty acids were quantified. The concentration of each lipid was obtained by summing the fatty acids in their chromatograph profile.

Plasma Membrane Preparation

The soleus muscle plasmalemmal content of selected proteins was determined in muscles incubated in Medium 199 for 0, 6, 12, and 18 h with or without palmitate. At these selected time points, the muscles were treated with (20,000 µU/ml) or without insulin for 70 min to mimic the time course in the foregoing glucose transport experiments. To obtain sufficient plasma membrane, 10 incubated soleii were pooled for each independent experiment. Giant vesicle plasma membranes were obtained as we have previously reported in detail (8, 10). In separate experiments, we established that insulin stimulated the appearance of GLUT4, Akt, and PKC{theta} and PKC{zeta}/{lambda} at the plasma membrane.

Protein Analysis

The soleus muscle total protein of selected proteins was determined in muscles incubated in Medium 199 for 0, 6, 12, and 18 h with or without palmitate. Thereafter, muscles were rapidly blotted, frozen in liquid nitrogen, and stored at –80°C until analyzed for selected proteins. To measure the phosphorylation status of Akt, PRAS40 (a proline-rich Akt substrate), and AS160, muscles were incubated for 0, 6, 12, and 18 h with and without palmitate followed by incubation in palmitate-free Krebs-Henseleit buffer in the presence of insulin (20,000 µU/ml) for 10 min, the time in which maximal phosphorylation was observed (H. Alkhateeb and A. Bonen, unpublished data, and Ref. 57). Thereafter, the muscles were rapidly blotted, frozen, and stored at –80°C for later analysis.

Muscle Protein Extraction and Western Blotting

For whole muscle protein determination, two frozen soleii were homogenized in 2 ml of buffer (8, 10). Muscle homogenate and plasma membrane protein concentrations were determined using the bicinchoninic acid assay. Proteins were separated using SDS-polyacrylamide gel electrophoresis and were detected using Western blotting. We (2, 8, 10) have reported these procedures previously.

Statistics

Data were analyzed using two-way analyses of variance, and when appropriate, a Fisher's least significant difference post hoc analysis was used. For some small experiments, the data were analyzed with a one-way analysis of variance when this was warranted by the experimental design. All data are reported as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The selection of the palmitate concentration in the present study was determined from dose response studies in which we examined the effects of palmitate on the inhibition of insulin-stimulated glucose transport after 12 h (Fig. 1A). Incubation with 1.5–2 mM palmitate induced the maximal effects. At a concentration of 2 mM, palmitate muscles remained metabolically viable for up to 18 h, as shown by the constancy of the ATP and PCr concentrations in all muscles (Fig. 1B).


Figure 1
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  		Fig. 1. Dose-response relationship of palmitate on insulin-stimulated 3-O-methylglucose (3-OMG) transport in soleus muscle (A) and ATP and phosphocreatine (PCr) concentrations in isolated soleus muscle incubated for 0, 6, 12, and 18 h in the absence and presence of 2 mM palmitate (B). Data are total 3-OMG transport (basal + insulin-stimulated) and are presented as means ± SE; n = 4–5 muscles per data point. In some cases error bars are less than the plot symbol. For both A and B, at t = 0, control muscles were briefly incubated, while palmitate-treated muscles were briefly exposed to palmitate (see METHODS). A: incubations without (0 mM) or with palmitate (0.5–2.0 mM) occurred for 12 h, after which muscles were exposed to insulin. *P < 0.05, 0.5 vs. 0 mM; **P < 0.05, 1.0 vs. 0.5 mM; ***P < 0.05, 1.5 vs. 1.0 mM, and 2.0 vs 1.0 mM (asterisks denote comparisons based on post hoc tests). B: n = 8–10 muscles per data point; there were no significant differences at any point.

 
Effect of Palmitate on Basal and Insulin-Stimulated Glucose Transport in Soleus Muscles

In the absence of palmitate, basal 3-OMG transport rates were not altered during the 18-h incubation period (P > 0.05; Fig. 2). In the palmitate-treated muscles, the basal rates of 3-OMG transport were reduced only in the first 6 h (P < 0.05). Thereafter (6–18 h), basal 3-OMG transport remained unaltered in palmitate-treated muscles (P > 0.05; Fig. 2).


Figure 2
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  		Fig. 2. Effect of prolonged palmitate exposure (0–18 h) on basal and insulin-stimulated 3-OMG transport in soleus muscle. At t = 0, control muscles were briefly incubated, while palmitate-treated muscles were briefly exposed to palmitate (see methods). Data are presented as means ± SE; n = 8 muscles per data point. In some cases error bars are less than the plot symbol. Note that insulin was present for only 70 min (see METHODS) after 0, 6, 12, or 18 h of muscle incubation. Control: no differences among time points (P > 0.05). Palmitate treatment: *P < 0.05, 6 vs. 0 h; **P < 0.05, 12 vs. 6 h; ***P < 0.05, 18 vs. 12 h (asterisks denote comparisons based on post hoc tests).

 
Insulin-stimulated glucose transport was not altered in the control muscles (P > 0.05; Fig. 2). In contrast, palmitate induced a very marked, progressive reduction in insulin-stimulated 3-OMG transport over 18 h. After 6, 12, and 18 h, glucose transport was reduced by 33% (P < 0.05), 66% (P < 0.05), and 89% (P < 0.05), respectively, relative to t = 0 h. Notably, after 18 h of palmitate exposure, insulin-stimulated glucose transport did not differ from basal glucose transport at t = 0 h (P > 0.05; Fig. 2).

Effects of Insulin on Plasmalemmal GLUT4, Akt, and PKC{theta} and PKC{zeta}/{lambda}

Although it had been reported some years ago that insulin-stimulated GLUT4 appearance at the plasma membrane of giant vesicles could not be detected (50), we found that insulin increased plasmalemmal GLUT4 (2.3-fold, P < 0.05; Fig. 3). In addition, insulin also increased plasmalemmal Akt (1.5-fold), PKC{theta} (3.3-fold), and PKC{zeta}/{lambda} (4.8-fold, P < 0.05; Fig. 3).


Figure 3
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  		Fig. 3. Effects of insulin on plasma membrane GLUT4, Akt, PKC{theta}, and PKC{zeta}/{lambda}. Data are presented as means ± SE; GLUT4, n = 9 independent determinations; Akt, n = 3 independent determinations; PKC{theta}, n = 4 independent determinations; PKC{zeta}/{lambda}, n = 4 independent determinations. To obtain sufficient plasma membrane for each independent determination, 10 solei (~20 mg each) were incubated with or without insulin for 30 min. These 10 muscles were then pooled for each treatment (basal and insulin stimulation) to obtain sufficient giant vesicles plasma membrane for Western blotting. *P < 0.05, insulin vs. basal.

 
Effect of Palmitate on Protein Expression of GLUT4 and Insulin-Stimulated Plasmalemmal GLUT4

The expression of GLUT4 protein was not altered during the 18-h incubation period in the control and palmitate-treated muscles, (P > 0.05; Fig. 4A). Basal levels of plasma membrane GLUT4 were also not altered during the 18-h incubation period in either the control or palmitate-treated muscles, (P > 0.05; Fig. 4B).


Figure 4
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  		Fig. 4. Effect of prolonged palmitate exposure (0–18 h) on soleus muscle protein expressions of GLUT4 (A), on basal plasma membrane GLUT4 and insulin-stimulated plasma membrane GLUT4 (B), and on the ratio of 3-OMG transport to plasma membrane GLUT4 (C). At t = 0, control muscles were briefly incubated, while palmitate-treated muscles were briefly exposed to palmitate (see METHODS). Data are presented as means ± SE. In some cases error bars are less than the plot symbol. Note that insulin was present for only 70 min (see METHODS) after 0, 6, 12, or 18 h of muscle incubation. A: n = 5–6 muscles per data point. B: n = 7 independent experiments for each data point. To obtain sufficient plasma membrane, 10 solei were pooled for each independent experiment at each time point. C: ratio was calculated from means in insulin-stimulated muscles in Fig. 2 and here in B. Basal plasma membrane GLUT4 levels in control (P > 0.05) and palmitate treated muscle (P > 0.05) were unaltered. Insulin stimulation in control muscle resulted in a 2.5-fold increase in plasma membrane GLUT4 at 0 to 18 h, P < 0.05. Palmitate treatment + insulin-stimulation: *P < 0.05 at 6, 12, and 18 h vs. control muscles at corresponding time points (asterisks denote comparisons between control and palmitate treated conditions based on post hoc tests). In palmitate-treated muscle at 12 and 18 h, insulin-stimulated plasma membrane GLUT4 does not differ from basal plasma membrane GLUT4.

 
In control muscles, insulin induced a 2.5-fold increase in plasmalemmal GLUT4 at the end of each of the incubation periods (0, 6, 12, and 18 h, P < 0.05; Fig. 4B). The insulin-stimulated plasmalemmal GLUT4 content was not altered throughout the 18-h period in the control muscles (P > 0.05; Fig. 4B). In contrast, in palmitate-treated muscles, the insulin-stimulated plasmalemmal GLUT4 was decreased by 40% within the first 6 h (P < 0.05; Fig. 4B). Thereafter, palmitate treatment did not further reduce the insulin-stimulated GLUT4 content at the plasma membrane at 12 and 18 h (P > 0.05; Fig. 4B), despite the continued reduction in insulin-stimulated glucose transport (Fig. 2).

At 6 h, the concurrent decrease in 3-OMG transport and plasma membrane GLUT4 in the palmitate-treated muscles yielded a ratio (Fig. 4C) that did not differ from that at t = 0, before the onset of insulin resistance. However, after 6 h, there was a sharp progressive decrease in the ratio of 3-OMG transport to plasma membrane GLUT4 in the palmitate treated muscles (Fig. 4C).

Effect of Palmitate on Expression and Insulin-Stimulated Phosphorylation of Signaling Proteins

The protein expressions of IRS-1, PI 3-kinase, Akt1/2, Akt2, AS160, PRAS40, PKC{zeta}/{lambda}, and PKC{theta} remained unchanged during the 18-h incubation period in either presence or absence of palmitate (n = 5–7 muscles at all time points in control and palmitate-treated muscles, P > 0.05; data not shown). Under basal conditions, the phosphorylation states of Akt (Thr308 and Ser473) were not altered (P > 0.05; Fig. 5) in either control or palmitate-treated muscles. Relative to t = 0, the basal phosphorylation states of AS160 and PRAS40 were reduced somewhat, and to a similar extent, in both control and palmitate-treated muscles (Fig. 5, C and D).


Figure 5
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  		Fig. 5. Phosphorylation of Akt Thr308 (A), Akt Ser473 (B), AS160 (C), and PRAS40 (a proline-rich Akt substrate; D) in control and palmitate-treated muscles under basal and insulin stimulated (10 min) conditions. At t = 0, control muscles were briefly incubated, while palmitate-treated muscles were briefly exposed to palmitate (see METHODS). Data are presented as means ± SE; n = 3–4 muscles for basal conditions and n = 5–7 muscles for insulin treatment for each data point. In some cases error bars are less than the plot symbol. As figure is already crowded, blots for basal phosphorylation states are not shown. Note that insulin was present for only 10 min (see METHODS) after 0, 6, 12, or 18 h of muscle incubation. Note in D, the reversal of +palmitate and –palmitate loading compared with A–C. In all instances, basal phosphorylations are less than in muscles treated with insulin. In addition, basal control and basal palmitate-treated muscles did not differ at any point except at 6 h in D. Insulin treatment: *P < 0.05, 6, 12, or 18 h vs. 0 h; **P < 0.05, 12 vs. 0 h; ***P < 0.05 18 vs. 12 h. Basal: {dagger}P < 0.05, 12 vs. 0 h for control and palmitate-treated; {dagger}{dagger}P < 0.05, 18 vs. 12 h, for control and palmitate-treated; +P < 0.05, 6 vs. 0 h for control. Symbols denote comparisons based on post hoc tests.

 
In the control muscles, the insulin-induced phosphorylation states of Akt Thr308 (Fig. 5A) and Ser473 (Fig. 5B) and AS160 (Fig. 5C) were not changed (P > 0.05) during the 18-h incubation period. In contrast, PRAS40 phosphorylation, relative to t = 0 h, was increased after 12 h (+14%) and 18 h (+27%) (P < 0.05; Fig. 5D). Palmitate did not alter the insulin-induced phosphorylation of Akt Thr308 (Fig. 5A) and Ser473 (Fig. 5B) at any time point (P > 0.05). In contrast, palmitate treatment reduced the insulin-stimulated phosphorylation of AS160 in the first 6 h (–36%, P < 0.05; Fig. 5C), but it was not changed further thereafter, as AS160 phosphorylation remained stably depressed at 12 h (–32%) and 18 h (–40%) (Fig. 5C). A similar reduction pattern was observed for insulin-stimulated phosphorylation of the proline-rich Akt substrate PRAS40 (40), in palmitate-treated muscles (reduction relative to t = 0 h: –23% at 6 h, –18% at 12 h, –22% at 18 h; Fig. 5D).

Effect of Palmitate on Insulin-Stimulated Plasmalemmal Akt, PKC{zeta}/{lambda}, and PKC{theta}

Akt. In control muscles, during the 18-h incubation period the insulin-stimulated plasmalemmal Akt was not altered in the first 6 h (P > 0.05; Fig. 6A) but was increased thereafter at 12 h (+55%, P < 0.05; Fig. 5A) and 18 h (+77%, P < 0.05; Fig. 6A). These increments in plasmalemmal Akt at 12 and 18 h did not differ (P < 0.05; Fig. 6A). In contrast, palmitate treatment was associated with an impaired insulin-stimulated Akt translocation to the plasma membrane within the first 6 h (–55%, P < 0.05; Fig. 6A). Relative to the 6-h time point, this insulin-stimulated translocation of Akt to the plasma membrane had recovered slightly at 12 and 18 h (P < 0.05; Fig. 6A). However, relative to t = 0, insulin-stimulated translocation of Akt to the plasma membrane was still inhibited in palmitate-treated muscles at 12 h (–32%, P < 0.05; Fig. 6A) and at 18 h (–36%, P < 0.05; Fig. 6A).


Figure 6
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  		Fig. 6. Soleus muscle plasma membrane Akt (A), PKC{zeta}/{lambda} (B), and PKC{theta} (C) in control and palmitate-treated muscles exposed to insulin. At t = 0, control muscles were briefly incubated, while palmitate-treated muscles were briefly exposed to palmitate (see METHODS). Data are presented as means ± SE; n = 5–7 independent experiments for each data point. In some cases error bars are less than the plot symbol. Note that insulin was present for only 70 min (see METHODS) after 0, 6, 12, or 18 h of muscle incubation. To obtain sufficient plasma membrane 10 solei were pooled for each independent experiment. *P < 0.05 vs. 0 h; **P < 0.05, control vs. palmitate-treated muscles; ***P < 0.05 12 or 18 h vs. 6 h (asterisks denote comparisons based on post hoc tests).

 
PKC{zeta}/{lambda}. In the control muscles, the insulin-stimulated translocation of PKC{zeta}/{lambda} to the plasma membrane was not altered (P > 0.05; Fig. 6B). In the palmitate-treated muscles, insulin-stimulated translocation of PKC{zeta}/{lambda} to the plasma membrane was also not altered, except for an increase at 12 h (+23%, P < 0.05; Fig. 6B).

PKC{theta}. In the control muscles, there was a reduction in the insulin-stimulated PKC{theta} translocation to the plasma membrane within the first 6 h (–20%, P < 0.05; Fig. 6C). This was renormalized by 12 h, whereas after 18 h there was an increase in the insulin-stimulated plasmalemmal PKC{theta} (+50%, P < 0.05; Fig. 6C). In contrast, in muscles that were treated with palmitate, insulin-stimulated plasmalemmal PKC{theta} was reduced at 6 h (–50%, P < 0.05; Fig. 6C), at 12 h (–45%, P < 0.05; Fig. 6C) and at 18 h (–35%, P < 0.05;, Fig. 6C) compared with insulin-stimulated PKC{theta} translocation at t = 0.

Intramuscular Lipid Accumulation and Palmitate Oxidation

In control muscles, the concentrations of the intramuscular lipids were relatively stable during the 18-h incubation. However, in control muscles, TAG concentrations were increased somewhat at 18 h (P < 0.05; Fig. 7A). Phospholipid concentrations decreased somewhat (–22%) in both control and palmitate-treated muscles over the 18-h period, with most of the reduction (–15%) occurring during the last 6 h (P < 0.05, data not shown). DAG (Fig. 7B) and ceramide (Fig. 7C) concentrations were not altered in control muscles, except at 12 h when a reduction was observed in DAG (–19%, P < 0.05; Fig. 7B).


Figure 7
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  		Fig. 7. Effects of prolonged palmitate exposure (0–18 h) on intramuscular concentrations of triacylglycerol (A), diacylglycerol (B), and ceramide (C), and palmitate oxidation (D) in soleus muscle. Data are presented as means ± SE; for A–C, n = 5 independent experiments, each independent observation consisting of 5 pooled solei; for D, n = 8 muscles per data point. In some cases error bars are less than the plot symbol. At t = 0, control muscles were briefly incubated, while palmitate-treated muscles were briefly exposed to palmitate (see METHODS). *P < 0.05, 6 h vs. 0 h; **P < 0.05, 12 or 18 h vs. 6 h; ***P < 0.05, control vs. palmitate-treated (asterisks denote comparisons based on post hoc tests).

 
Incubation with palmitate increased the intramuscular TAG concentration, but only when incubated for more than 6 h with palmitate (Fig. 7A). By 12 h, the intramuscular TAG concentration had increased 53% (P < 0.05) and remained at that level until 18 h (Fig. 7A). Palmitate incubation increased DAG concentrations by 31% after 6 h (P < 0.05; Fig. 7B). After 12 and 18 h of incubation with palmitate, DAG concentrations were 11% (P = 0.075) and 19% (P < 0.05) greater, respectively, than at t = 0 (Fig. 7B). Palmitate treatment also increased the ceramide concentrations after 6 h (+24%, P < 0.05; Fig. 7C) and relative to t = 0 these concentrations remained elevated at that level at 12 h (+17%, P < 0.05; Fig. 7C) and at 18 h (+33%, P < 0.05; Fig. 7C).

The rates of palmitate (2 mM) oxidation were not altered in control muscles (P > 0.05; Fig. 7D). In contrast, in the palmitate-treated muscles, palmitate oxidation decreased progressively (P < 0.05; Fig. 7D), by 40% and 60% at 6 h and 12 h, respectively (P < 0.05; Fig. 6D). Relative to the 12-h reduction, palmitate oxidation was not reduced any further at 18 h in the palmitate-treated muscles (P > 0.05; Fig. 7D).

Interrelationships Among 3-OMG Transport, Plasma Membrane GLUT4, and Selected Variables

Many factors have been implicated in fatty acid-induced impairments in insulin-stimulated glucose transport. Palmitate treatment altered a number of parameters only within the first 6 h, with no further changes thereafter (i.e., DAG and ceramide concentrations, insulin-stimulated phosphorylation of AS160 and PRAS40, and insulin-stimulated plasmalemmal Akt, PKC{theta}, and GLUT4; Fig. 8A). However, these changes were not closely associated with the progressively greater reduction in insulin-stimulated glucose transport, particularly during the 6- to 18-h period of palmitate exposure. Among all parameters examined over the 18-h period, there was a strong relationship between the reductions in the rate of palmitate oxidation and the rates of insulin-stimulated glucose transport in palmitate-treated muscles (Fig. 8, B and C).


Figure 8
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  		Fig. 8. Relationship between the relative (%) changes in intramuscular lipids, insulin-stimulated AS160 phosphorylation (A), and insulin-stimulated plasma membrane PKC{theta}, Akt, and GLUT4 in 0- to 18-h palmitate-treated soleus muscles (B) between rates of insulin-stimulated glucose transport and rates of palmitate oxidation, and between relative (%) changes in insulin-stimulated glucose transport and rates of palmitate oxidation (C). In B, a nonlinear regression line was obtained by fitting mean values of palmitate acid oxidation vs. insulin-stimulated glucose transport, as these data were obtained from different muscles. Means ± SE were from data obtained in palmitate-treated muscles at 0, 6, 12, and 18 h and are shown in Figs. 2 and 7D.

 

   DISCUSSION
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 ABSTRACT
 METHODS
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 DISCUSSION
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 REFERENCES
 
We have examined the mechanisms involved in palmitate-induced insulin resistance in skeletal muscle over a prolonged period of time (0, 6, 12, and 18 h). The key novel finding is that palmitate provokes insulin resistance in skeletal muscle by two separate mechanisms: one linked to an intracellular, lipid-associated impairment in insulin signaling and GLUT4 translocation and another that is associated with a reduced intrinsic activity of plasmalemmal GLUT4, independent of further inhibition of insulin signaling. Moreover, the disorders in insulin signaling and GLUT4 translocation precede the reduction in GLUT4 intrinsic activity. Compared with the impaired GLUT4 translocation, the reduced intrinsic activity of GLUT4 accounted for a far greater (2-fold) impairment in insulin-stimulated glucose transport.

The muscles in the present study, as in other long-term incubation studies (2, 65), were metabolically viable and remained insulin responsive. The use of a high palmitate concentration to induce insulin resistance is similar to previous approaches [2 mM (60); 1.6 mM (48)].

Palmitate Impairs Insulin-Stimulated Glucose Transport and GLUT4 Translocation and Activity

The palmitate-induced impairment in insulin-stimulated glucose transport consisted of an early and a late phase. Each of these appeared to be attributable to different mechanisms.

Early-phase (<6 h) impaired glucose transport and GLUT4 translocation. The palmitate (2 mM)-induced reduction (–33%) in insulin-stimulated glucose transport after 6 h parallels the observations in two other, similar studies, in which palmitate also reduced insulin-stimulated glucose transport in isolated soleus [–25% after 6 h with 2 mM palmitate (60)] and extensor digitorum longus muscles [–22%, after 5 h with 1.6 mM palmitate (48)]. These changes were not attributable to the minimal, palmitate-induced reduction in basal glucose transport (present study and Ref. 48) or to an altered GLUT4 protein expression (present study). Instead, the impaired insulin-stimulated GLUT4 translocation (–44%) appeared to account for the similar reductions in glucose transport (–33%) within the first 6 h. This is also suggested by the similar ratio of insulin-stimulated glucose transport to plasma membrane GLUT4 in the 6-h palmitate-treated muscles compared with the control muscle ratios at every time point (see Fig. 4C).

Late-phase impaired insulin-stimulated glucose transport and GLUT4 activity. With prolonged palmitate incubation (≤18 h), the insulin-stimulated glucose transport was reduced progressively further at 12 h (–66%) and 18 h (–89%). These reductions were not attributable either 1) to changes in GLUT4 protein expression or 2) to further reductions in the insulin-stimulated GLUT4 translocation to the plasma membrane. Thus, the late-phase, twofold greater insulin resistance was associated with a reduced GLUT4 intrinsic activity. We recognize that preparative procedures designed to isolate plasma membrane fractions may not be fully representative of the plasma membrane in intact muscle in which the glucose transport rates were determined. Nevertheless, our study strongly suggests that a second, somewhat delayed mechanism is involved in palmitate-induced insulin resistance, namely a reduced intrinsic activity of plasmalemmal GLUT4 (see Fig. 4C).

Changes In Intramuscular Lipids During Long-Term Palmitate Incubation (0–18h)

Fatty acids have been widely implicated in the induction of insulin resistance (present study and refs. 6, 31, 49). Our study supports the idea that the intramuscular accumulation of triacylglycerols per se does not induce insulin resistance, as their accumulation occurred well after insulin resistance had been observed. Ceramides and DAG are more likely to inhibit insulin signaling (1, 16, 28, 43). In palmitate-treated muscles, intramuscular ceramide and DAG accumulations coincided with the onset of insulin resistance within the first 6 h. However, it appears that these metabolites are not directly involved with the late-phase (12 and 18 h), more severe insulin resistance that developed, since the intramuscular ceramide and DAG accumulations were not increased further at 12 and 18 h.

The reduced rate of palmitate oxidation in the palmitate-treated muscles corresponded most closely with the reduced rate of insulin-stimulated glucose transport during both the early (≤6 h) and late phases (12 and 18 h). The reason for this is unclear; however, others (36) have argued that the impaired fatty acid oxidation is closely associated with skeletal muscle insulin resistance.

Palmitate-Mediated Inhibition Of Insulin Signaling

Impairments in the post-receptor insulin-signaling pathway are thought to be central in the development of fatty acid-induced insulin resistance. (5, 18, 26, 35, 38, 41, 42, 47, 62, 67). In the present study, palmitate treatment impaired the insulin-induced activation of Akt, PRAS40, AS160, and PKC{theta} t PKC{zeta}/{lambda}. However, these impairments were all observed within the first 6 h, with no further impairment thereafter at 12 and18 h. Therefore, we focus our discussion on the changes in the early-phase (≤6 h) impairments in insulin signaling.

Early-Phase (≤6 H) Lipid-Mediated Impairment in Akt

There is considerable disagreement as to whether fatty acids impair insulin-stimulated glucose transport via a reduction in Akt phosphorylation (13–15, 22, 38, 42, 52, 56, 58, 60). Although insulin effects on Akt Ser473 and Thr308 phosphorylations were not altered by palmitate treatment, it did appear that palmitate contributed to decreasing Akt activity, since 1) reductions occurred in the insulin-induced phosphorylations of AS160 and PRAS40 (downstream Akt targets), and 2) reductions occurred in the insulin-stimulated appearance of Akt at the plasma membrane, as has been observed in palmitate-treated L6 muscle cells (52). The different effects of palmitate on insulin-stimulated Akt phosphorylation and Akt translocation may be attributable to the discrepancies that can arise between Akt Ser473 and Thr308 phosphorylation and Akt activity (41, 62).

It is thought that ceramides, derived from saturated fatty acids, interfere with insulin action by reducing Akt phosphorylation/activation and translocation (14, 15, 24, 28, 51, 52, 56). Therefore, in the palmitate-treated muscles, the ceramide accumulation (+24%) within the first 6 h likely accounted for the impaired Akt activation. Presumably, this resulted in the impaired AS160 phosphorylation, which then inhibited GLUT4 translocation. However, this sequence of actions cannot account for the further reductions in insulin-stimulated glucose transport observed at 12 and 18 h, as neither Akt activation nor GLUT4 translocation were changed beyond that observed within the first 6 h.

Increased ceramide concentrations that occur with lipid infusion (38) or high-fat feeding (26, 41) can also contribute to the inhibition of insulin-stimulated Akt activity via the activation of PKC{zeta}/{lambda} (26, 38, 41), although this is not always observed (62). In the present study, it appears that PKC{zeta}/{lambda} was not activated by ceramide accumulation, but the absence of a direct measurement of PKC enzyme activity precludes a definitive conclusion on this matter. Nevertheless, it is known that ceramide-induced PKC{zeta}/{lambda} activation is not the only means for inhibiting Akt activity, as this can be inhibited by an alternative, ceramide-mediated mechanism, namely the activation of a type 2A-like phosphatase, which plays a role in the dephosphorylation of Akt (13).

It has been proposed that DAG accumulation induces insulin resistance via the activation of PKC{theta} (23, 31, 67), which antagonizes insulin signaling by increasing serine phosphorylation of IRS-1 (23, 67). Moreover, PKC{theta}-null mice do not develop insulin resistance when placed on a high-fat diet (37). Although an increase in plasmalemmal PKC{theta} is indicative of its activation (55), in the present study the insulin-stimulated appearance of PKC{theta} was not altered. This suggests that insulin resistance in the palmitate-treated muscles was not associated with DAG-mediated activation of PKC{theta}.

Late-Phase (12 And 18 h) Impaired Plasmalemmal GLUT4 Activity

The data in the present study suggest strongly that prolonged palmitate treatment (>6 h) interfered with insulin action by mechanisms that are independent of 1) altered insulin signaling and 2) impaired GLUT4 translocation to the plasma membrane. One of these mechanisms may be the reduction of the intrinsic activity of the plasmalemmal GLUT4.

In early studies in skeletal muscle (11, 21), a discrepancy was found between the changes in cell surface GLUT4 and rates of glucose transport. Therefore, it was proposed that glucose transport was regulated via both GLUT4 translocation and intrinsic activity. However, this concept fell into disfavor for a number of years, only to be revived in recent years (3, 25, 27, 29, 30, 46). We (25) have shown that epinephrine induces insulin resistance in skeletal muscle in a dose-dependent manner by inhibiting GLUT4 activity. The present study also supports the idea that GLUT4 intrinsic activity can be altered. The mechanism by which the intrinsic activity of cell surface GLUT4 is altered remains obscure. Contrary to some earlier studies, recent work has shown that p38 MAPK activation is not involved in altering the intrinsic activity of GLUT4 (3, 4, 53, 63). Nevertheless, there is substantial evidence to indicate that cell surface GLUT4 activity can be altered to regulate glucose transport (3, 11, 21, 25, 27, 30, 53, 63). The present study indicates that the palmitate-induced reduction in the intrinsic activity of GLUT4 occurs after insulin-stimulated GLUT4 translocation has been impaired.

Summary

Our study has shown that palmitate-induced insulin resistance is provoked by two distinct mechanisms. First, in the early phase (<6 h), GLUT4 translocation is impaired, resulting from the intramuscular accumulation of diacylglycerol and ceramide and the concurrent impairments in insulin-stimulated Akt, PRAS40, and AS160 activation. Second, another phase is evident after 12 and 18 h of palmitate exposure, during which the intrinsic activity of GLUT4 is markedly reduced. This later phase is independent of any further alterations in GLUT4 translocation, insulin signaling, and intramuscular lipid accumulation. Compared with the impaired GLUT4 translocation, the reduced intrinsic activity of GLUT4 accounted for a far greater (2-fold) impairment in insulin-stimulated glucose transport.


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 ABSTRACT
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Studies in our laboratories are supported by grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, The Heart and Stroke Foundation of Ontario, the Netherlands Heart Foundation (2002T049), the European Community (Integrated Project LSHM-CT-2004-005272, Exgenesis), KBN 3P05B 19022 (Poland), and the Canada Research Chair program. H. Alkhateeb was supported by a scholarship from The Hashemite University, Zarqa, Jordan. J. J. F. P. Luiken is a recipient of a VIDI-Innovation Research Grant from the Netherlands Organization for Scientific Research (NWO-ZonMw Grant 016.036.305). A. Bonen is the Canada Research Chair in Metabolism and Health.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Bonen,Dept. of Human Health and Nutritional Sciences, Univ. of Guelph, Guelph, ON N1G 2W1, Canada (e-mail: abonen{at}uoguelph.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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