AJP - Endo Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 287: E686-E695, 2004. First published May 27, 2004; doi:10.1152/ajpendo.00103.2004
0193-1849/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/4/E686    most recent
00103.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oliveira, R. L. G. S.
Right arrow Articles by Velloso, L. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oliveira, R. L. G. S.
Right arrow Articles by Velloso, L. A.

Cold-induced PGC-1{alpha} expression modulates muscle glucose uptake through an insulin receptor/Akt-independent, AMPK-dependent pathway

Rachel L. G. S. Oliveira, Mirian Ueno, Cláudio T. de Souza, Márcio Pereira-da-Silva, Alessandra L. Gasparetti, Rosangela M. N. Bezzera, Luciane C. Alberici, Aníbal E. Vercesi, Mario J. A. Saad, and Lício A. Velloso

Departments of Internal Medicine and Clinical Pathology, State University of Campinas, Campinas-SP 13083-970, Brazil

Submitted 3 March 2004 ; accepted in final form 21 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) participates in control of expression of genes involved in adaptive thermogenesis, muscle fiber type differentiation, and fuel homeostasis. The objective of the present study was to evaluate the participation of cold-induced PGC-1{alpha} expression in muscle fiber type-specific activity of proteins that belong to the insulin-signaling pathway. Rats were exposed to 4°C for 4 days and acutely treated with insulin in the presence or absence of an antisense oligonucleotide to PGC-1{alpha}. Cold exposure promoted a significant increase of PGC-1{alpha} and uncoupling protein-3 protein expression in type I and type II fibers of gastrocnemius muscle. In addition, cold exposure led to higher glucose uptake during a hyperinsulinemic clamp, which was accompanied by higher expression and membrane localization of GLUT4 in both muscle fiber types. Cold exposure promoted significantly lower insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and Ser473 phosphorylation of acute transforming retrovirus thymoma (Akt) and an insulin-independent increase of Thr172 phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Inhibition of PGC-1{alpha} expression in cold-exposed rats by antisense oligonucleotide treatment diminished glucose clearance rates during a hyperinsulinemic clamp and reduced expression and membrane localization of GLUT4. Reduction of PGC-1{alpha} expression resulted in no modification of insulin-induced tyrosine phosphorylation of the IR and Ser473 phosphorylation of Akt. Finally, reduction of PGC-1{alpha} resulted in lower Thr172 phosphorylation of AMPK. Thus cold-induced hyperexpression of PGC-1{alpha} participates in control of skeletal muscle glucose uptake through a mechanism that controls GLUT4 expression and subcellular localization independent of the IR and Akt activities but dependent on AMPK.

acute transforming retrovirus thymoma; adenosine 5'-monophosphate-activated protein kinase; cold exposure

PEROXISOME PROLIFERATOR-ACTIVATED receptor-{gamma} (PPAR{gamma}) coactivator-1{alpha} (PGC-1{alpha}) is a 795-amino acid protein with a molecular mass of 92 kDa. It has been identified in brown adipose tissue and skeletal muscle through a yeast two-hybrid system on the basis of its properties of induction by cold and binding to PPAR{gamma} (31). As a transcriptional coactivator, PGC-1{alpha} possesses several functional domains that participate in protein-protein interaction (30). Motifs that allow for interaction with PPAR{gamma} (31), nuclear respiratory factor (48), cAMP response element binding protein-binding protein (16), steroid receptor coactivator-1 (5), and myocyte enhancer factor-2 (22), have been identified and are thought to participate in the PGC-1{alpha}-dependent regulation of expression of genes involved in an array of specialized cell functions, such as the response to environmental thermal oscillations (31), mitochondrial biogenesis (31, 48), energy and nutrient homeostasis (27), muscle fiber type differentiation (19), adipocyte differentiation (31), cellular response to cytokines (29), and control of insulin secretion (7, 49).

Recent studies have implicated PGC-1{alpha} expression as a mechanism involved in the control of glucose uptake and insulin sensitivity in muscle cells (22). Accordingly, at least two clinical studies have found a correlation between mutations of the PGC-1{alpha} gene and insulin resistance or diabetes (8, 13). The mechanisms by which PGC-1{alpha} may regulate glucose uptake and insulin action are not completely understood. Hyperexpression of PGC-1{alpha} in muscle cells significantly induces the expression of glucose transporter-4 (GLUT4), but this seems to be an insulin-independent process (22). On the other hand, PGC-1{alpha} expression is upregulated by endurance training and by chronic treatment with the AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside, which lead to increased glucose uptake through insulin-independent mechanisms (1, 38).

The exposure of homeothermic animals to a cold environment has been widely used as a model for studying glucose homeostasis and insulin action (11, 45). Under this condition, animals mobilize glucose more efficiently, despite molecular resistance to insulin signal transduction in skeletal muscle and adipose tissue (11). Because cold exposure significantly induces PGC-1{alpha} expression in muscle, we decided to evaluate the effect of PGC-1{alpha} expression inhibition on glucose clearance and insulin signal transduction in type I and type II fiber-rich portions of gastrocnemius muscle from control and cold-exposed Wistar rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies, chemicals, and buffers. Reagents for SDS-PAGE and immunoblotting were obtained from Bio-Rad (Richmond, CA); HEPES, PMSF, aprotinin, DTT, Triton X-100, Tween 20, glycerol, BSA (fraction V), and adenine {beta}-D-arabinofuranoside (ARA-A) from Sigma (St. Louis, MO); protein A-Sepharose 6MB from Pharmacia (Uppsala, Sweden),125I-protein A, [U-14C]sucrose, and 2-deoxy-D-[3H]glucose (2-DG) from ICN Biomedicals (Costa Mesa, CA); nitrocellulose paper (BA85, 0.2 µm) from Amersham (Aylesbury, UK); and thiopental sodium and human recombinant insulin (Humulin R) from Lilly (Indianapolis, IN). Polyclonal antiphosphotyrosine antibodies were raised in rabbits and affinity purified on phosphotyramine columns. Anti-insulin receptor (IR; rabbit polyclonal, catalog no. sc-711), anti-GLUT4 (goat polyclonal, catalog no. sc-1608), anti-GLUT-1 (rabbit polyclonal, catalog no. sc-7903), antiphospho-ERK ({alpha}-pERK/Tyr204, detecting pERK42 and pERK44; goat polyclonal, catalog no. sc-7976), anti-phospho-Ser473 Akt (rabbit polyclonal, catalog no. sc-7985-R), anti-uncoupling protein (UCP)-3 (goat polyclonal, catalog no. sc-7756), and anti-PGC-1{alpha} (goat polyclonal, catalog no. sc-5816) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antiphospho-Thr172 AMPK-{alpha} (rabbit polyclonal, catalog no. 2531) and anti-AMPK-{alpha} (rabbit polyclonal, catalog no. 2532) from Cell Signaling Technology (Beverly, MA). Enzymatic colorimetric assay for the quantification of nonesterified fatty acids was obtained from Wako Chemicals (Richmond, VA), the leptin detection kit from Linco Research (St. Charles, MO), and corticosterone and thyroid-stimulating hormone RIA kits from Amersham Pharmacia Biotech-BIOTRAK (Aylesbury, UK). Insulin was determined by RIA (37).

Experimental animals and cold exposure protocols. Male Wistar rats (Rattus norvegicus; 8 wk old, 200–300 g body wt) were obtained from the University of Campinas Animal Breeding Center. The investigation followed the State University of Campinas guidelines for the use of animals in experimental studies and conforms to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). The animals were maintained on a 12:12-h artificial light-dark cycle and housed in individual cages. After the acclimatizing period (3 days), the animals were randomly divided into two groups: cold-exposed animals (4 ± 1°C for 4 days) and thermoneutrality-maintained (control) animals (23 ± 1°C). The animals were allowed free access to standard rodent chow and water. For tissue extraction (on day 4 of the experimental protocol), rats were anesthetized with thiopental sodium (15 mg/kg body wt ip), and the experiments were performed after the loss of corneal and pedal reflexes. After experimental procedures, the rats were euthanized under anesthesia following the recommendations of NIH Publication No. 85-23.

Metabolic, hormone, and biochemical measurements. Food intake, rectal temperature, and body weight (during the light cycle) were measured at time 0, 2 h from the beginning of the experimental period, and daily during the 4 experimental days in control and cold-exposed rats. Rectal temperature was measured with a high-precision digital thermometer (Hanna Instruments, Woonsocket, RI) inserted 1.5 cm from the anus. In rats treated with sense or antisense oligonucleotides, 24-h food intake and body temperature were determined and compared with controls. Blood samples were obtained from rats fasted for 2 h. Plasma glucose was measured by the glucose oxidase method in samples collected from the tail at time 0, 2 h, and daily (42). Nonesterified fatty acid was determined by ELISA according to the manufacturer's directions in samples collected at time 0 and 2 h and on days 2 and 4. Insulin was detected by RIA with a guinea pig anti-rat insulin antibody and rat insulin as standard (37) on samples collected at time 0 and 2 h and on days 2 and 4. Serum leptin concentrations were measured by ELISA on samples collected at time 0 and 2 h and on days 2 and 4. Corticosterone and thyroid-stimulating hormone were measured by RIA according to the manufacturer's specifications on samples collected at time 0 and 2 h and on days 2 and 4. Plasma norepinephrine levels here determined by reverse-phase HPLC according to a method previously described (4). Samples were collected at the end of the experimental period.

2-DG studies. Tissue uptake of 2-DG was measured in vivo according to a method described previously (44) with minor modifications (11). The rats were anesthetized and then injected through the portal vein with 6.0 µCi of 2-DG and [14C]sucrose in the presence or absence of 0.1 U of insulin in 0.4 ml of isotonic phosphate buffer (pH 7.4) with 0.1% defatted BSA. After 16 min, slices of gastrocnemius muscle tissue were quickly removed and solubilized in NCS-II tissue solubilizer (Amersham, Buckinghamshire, UK). The radioactivity of 3H in the resulting supernatant was measured in liquid scintillation fluid (ACS-II, Amersham, Tokyo, Japan) using a scintillation counter (model LSC-1000, Aloka, Kyoto, Japan). Cellular uptake of 2-DG was calculated as the difference between the total tissue radioactivity and the amount of radioactivity in the tissue extracellular space. The extracellular space volume (in µl/mg) was calculated by dividing the amount of 14C (dpm/mg) in tissue by the amount of 14C (dpm/µl) in plasma. The concentration of extracellular 3H (dpm/mg) in tissue could then be obtained by multiplying the extracellular volume (in µl/mg) of tissue by the concentration of 3H (dpm/µl) in plasma. The cellular radioactivity was converted to picomoles of 2-DG using the specific activity, and the results were expressed per milligram of dry tissue.

Mitochondrial respiration rates and phosphorylation efficiency. Muscle mitochondria were isolated from rat hindlimb skeletal muscle by homogenization in ice-cold medium containing 100 mM sucrose, 100 mM KCl, 50 mM Tris·HCl, 1 mM K2HPO4, 0.1 mM EGTA, and 0.2% BSA (pH 7.4) followed by differential centrifugation (40). The final mitochondrial pellet was resuspended in ice-cold storage buffer containing 0.2 M mannitol, 0.1 M sucrose, 10 mM Tris·HCl, and 0.1 mM EDTA (pH 7.4). The presence of fatty acid-free 0.2% BSA in the buffers throughout the isolation procedure depleted the mitochondria of endogenous free-fatty acids. Oxygen consumption was measured using a Clark-type electrode (Hansatech Instruments; OXIGRAPH software, version 1.10) in a 0.5-ml glass chamber equipped with magnetic stirring. Mitochondria (0.5 mg/ml) from type I and type II fiber-rich gastrocnemius muscle were added to the standard medium containing 225 mM mannitol, 75 mM sucrose, 10 mM Tris·HCl (pH 7.4), 10 mM KH2PO4, and 0.1 mM EDTA at 28°C. Respiration rates are given in nanomoles of oxygen per milligram per minute. Phosphorylating (state 3) respiration was initiated by addition of 100 nmol ADP/mg protein. Phosphorylation efficiency (ADP-to-O ratio) was calculated from the added amount of ADP and the total amount of oxygen consumed during state 3. Preparations were treated with NAD-linked substrates, {alpha}-ketoglutarate, pyruvate, malate, and glutamate at 5 mM and with 1 µM carboxyatractiloside, 6 µM linoleic acid, 500 µM GDP, and 0.1% BSA.

Clamp studies. All procedures for clamp studies followed a previously published description of the method (2, 6). After 6 h of fasting, a 2-h hyperinsulinemic-euglycemic clamp study was performed in the lower limb. Under thiopental sodium anesthesia and aseptic conditions, a monoocclusive polyethylene catheter was inserted into the femoral artery for infusion of insulin and glucose. A second polyvinyl catheter was inserted into the femoral vein for blood sampling, and the animal was kept in a heated box (37°C) throughout the study. During the first phase of the study (30 min), a priming dose of insulin was infused followed glucose infusion at a rate necessary to reach a plateau. After glucose equilibration, insulin infusion (3.0 mU·kg–2·min–1) was maintained for 2 h at a constant rate (0.20 ml/h), and a variable infusion of glucose (5% solution) was adjusted to maintain the plasma glucose concentration at ~120 mg/dl. Blood samples were collected from the femoral vein every 5 min for plasma glucose and every 30 min for plasma insulin determinations. Insulin was measured in duplicate by RIA and oscillated between 4.0 and 9.0 ng/ml in samples collected from animals of both experimental groups.

Sense and antisense oligonucleotide treatment protocols. Sense and antisense oligonucleotides (Invitrogen, Carlsbad, CA) were diluted to a final concentration of 20 µmol/l in dilution buffer containing 10 mmol/l Tris·HCl and 1 mmol/l EDTA. The rats were injected intraperitoneally with two doses (24 h and 4 h before the experimental procedure) of 200 µl of dilution buffer with or without sense or antisense oligonucleotides. Phosphothioate-modified oligonucleotides were designed according to the R. norvegicus PGC-1{alpha} sequence deposited at the National Center for Biotechnology Information under the designation NM 031347 and were composed of sense (5'-TCA GGA GCT GGA TGG C-3') and antisense (5'-GCC ATC CAG CTC CTG A-3'). These oligonucleotides have been tested previously, and the antisense was capable of reducing the expression of PGC-1{alpha} by ~60% in pancreatic islets (7). In the present studies, we have employed Western blot analyses of total protein extracts from muscle to evaluate PGC-1{alpha} protein expression in control and sense and antisense oligonucleotide-treated rats. We also evaluated UCP-3 expression by Western blot and mitochondrial respiration in muscle from rats of the same experimental groups.

ARA-A in vivo treatment protocol. In some experiments, rats were treated with the AMPK inhibitor ARA-A. The drug (25 mmol/dose) was injected intraperitoneally 12 and 2 h before the experimental procedure (25).

Semiquantitative RT-PCR. Seven micrograms of total RNA from type I or type II fiber-rich portions of the gastrocnemius muscle of rats exposed to cold for 4 days or unexposed rats were reverse transcribed with SuperScript RT (200 U/µl) using 50 mM oligo(dT) in a 30-µl reaction volume (5x RT buffer, 10 mM dNTP, and 40 U/µl RNase-free inhibitor). The reverse transcriptions consisted of incubation for 50 min at 42°C and incubation for 15 min at 70°C. After reverse transcription, 0.75 µl of the RT products were used in each PCR at a final volume of 50 µl (10x PCR buffer, 1.0 mM dNTP, 50 mM MgCl2, Taq polymerase, and sense and antisense primers for PGC-1{alpha} and {beta}-actin). The expression of mRNA was determined by PCR using the primers 5'-TTT GGG AGG GTG AGG GAC TCC-3' (antisense) and 5'-TGA GCG CAA GTA CTC TGT GTG G-3' (sense), amplifying a 489-bp DNA fragment of {beta}-actin, and the primers 5'-GGA TCT TGA AGA GGA TCT AC-3' (antisense) and 5'-TGA GTG TTC TGG TAC CCA AG-3' (sense), amplifying a 804-bp DNA fragment of PGC-1{alpha}. Triplicate reactions were performed using an initial incubation at 94°C for 5 min and denaturation at 94°C for 1 min followed by annealing at 50°C and 57°C (for PGC-1{alpha} and {beta}-actin, respectively) for 50 s, extension at 72°C for 1 min, and final extension at 72°C for 7 min. Titration between 16 and 32 cycles revealed that 25 cycles for {beta}-actin and 35 cycles for PGC-1{alpha} were within the logarithmic phase of amplification. These PCR conditions were therefore used in subsequent experiments. All PCR experiments included a control tube with no RT step. PCR-amplified products were run on 2% Tris-acetic acid-EDTA-agarose gels, and the DNA was visualized by ethidium bromide staining. The size of the products was determined using a 1-kb plus DNA ladder (Life Technologies) as standard size markers. Images of the bands were captured using a UV transluminator (TFX 35M, Life Technologies), and band intensity was quantified by digital densitometry (Scion Image software).

Tissue extraction, immunoblotting, and immunoprecipitation. The abdominal cavities of anesthetized rats were opened, and the experimental animals received an infusion of insulin (0.2 ml, 10–6 M) or saline (0.2 ml) through the cava vein. After different intervals (see RESULTS), fragments (4.0 x 4.0 x 4.0 mm) of type I or type II fiber-rich portions of gastrocnemius muscle were excised and immediately homogenized in solubilization buffer at 4°C [1% Triton X-100, 100 mM Tris·HCl (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium orthovanadate, 2.0 mM PMSF, and 0.1 mg aprotinin/ml] with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY) operated at maximum speed for 30 s. Insoluble material was removed by centrifugation for 20 min at 9,000 g in a 70.Ti rotor (Beckman) at 4°C. The protein concentration of the supernatants was determined by the Bradford dye-binding method. Aliquots of the resulting supernatants containing 5.0 mg of total protein were used for immunoprecipitation with antibodies against IR at 4°C overnight followed by SDS-PAGE, transfer to nitrocellulose membranes, and blotting with antiphosphotyrosine or anti-IR antibodies. In direct immunoblot experiments, 0.2 mg of protein extracts from each tissue was separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-IR, antiphospho-Akt, antiphospho-ERK, anti-GLUT4, anti-GLUT-1, anti-PGC-1{alpha}, anti-UCP-3, antiphospho-AMPK, and antiphosphoacetyl-CoA carboxylase antibodies, as described previously (3).

Subcellular fractionation. To characterize the expression and subcellular localization of GLUT4, a subcellular fractionation protocol was employed as described previously (24) with minor modifications. Fragments of type I or type II fiber-rich portions of gastrocnemius muscle from untreated rats or rats treated with insulin (0.2 ml, 10–6 M, tissue obtained 15 min after insulin infusion) according to the protocols described above were minced and homogenized in 2 vol of STE buffer [0.32 M sucrose, 20 mM Tris·HCl (pH 7.4), 2 mM EDTA, 1 mM DTT, 100 mM sodium fluoride, 100 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 1 mM PMSF, and 0.1 mg aprotinin/ml] at 4°C with a Polytron homogenizer. The homogenates were centrifuged (1,000 g, 25 min, 4°C) to obtain pellets. The pellets were washed once with STE buffer (1,000 g, 10 min, 4°C) and suspended in Triton buffer [1% Triton X-100, 20 mM Tris·HCl (pH 7.4), 150 mM NaCl, 200 mM EDTA, 10 mM sodium orthovanadate, 1 mM PMSF, 100 mM NaF, 100 mM sodium pyrophosphate, and 0.1 mg aprotinin/ml], kept on ice for 30 min, and centrifuged (15,000 g, 30 min, 4°C) to obtain the nuclear fraction. The supernatant was centrifuged (100,000 g, 60 min, 4°C) to obtain the cytosol fraction and the pellet, which was suspended in STE buffer + 1% NP-40, kept on ice for 20 min, and centrifuged (100,000 g, 20 min) to obtain the membrane fraction. The fractions were treated with Laemmli buffer with 100 mM DTT and heated in a boiling water bath for 5 min, and aliquots (0.2 mg of protein) were subjected to SDS-PAGE and Western blotting with anti-GLUT4 antibodies, as described elsewhere (11).

Data presentation and statistical analysis. Values are means ± SE of the indicated number of experiments. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry using Scion Image software (ScionCorp). Student's t-test for unpaired samples and ANOVA for multiple comparisons were used for statistical analysis as appropriate. The post hoc test was employed when required. The level of significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Metabolic characterization of rats exposed to cold. Table 1 shows the results of hormonal and biochemical measurements along with determinations of daily food intake, body weight variation, and body temperature of rats exposed or not exposed (control) to a cold (4°C) environment for 4 days. Cold-exposed rats presented a significant increase in food intake, accompanied by weight loss, hypoinsulinemia, hypoleptinemia, and increase of norepinephrine. All the remaining parameters were similar in both experimental groups.


View this table:
[in this window]
[in a new window]
  		Table 1. Metabolic characterization of experimental animals

 
Cold exposure induces the expression of PGC-1{alpha} and UCP-3 in muscle. The concentrations of PGC-1{alpha} and UCP-3 were determined in total protein extracts of type II and type I fiber-rich portions of gastrocnemius muscle from cold-exposed and control rats. The extracts were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and blotted with specific antibodies. Cold exposure induced increases of 1.5-fold (P < 0.05) and 2.8-fold (P < 0.05) in PGC-1{alpha} expression in type II (Fig. 1A)and type I (Fig. 1B) fibers, respectively. Similarly, cold exposure induced 4.3-fold (P < 0.05) and 4.6-fold (P < 0.05) increases in UCP-3 expression in type II (Fig. 1A) and type I (Fig. 1B) fibers, respectively. Cold-induced hyperexpression of PGC-1{alpha} in both fiber types was also evident at the mRNA level, as detected by RT-PCR (Fig. 1C): 2.1-fold (P < 0.05) and 5.3-fold (P < 0.05) increase of PGC-1{alpha} in type II and type I fibers, respectively.



View larger version (42K):
[in this window]
[in a new window]
  		Fig. 1. Evaluation of peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) and uncoupling protein-3 (UCP-3) expression in gastrocnemius muscle of control and cold-exposed (4°C) rats by immunoblot (A and B) and RT-PCR (C). Samples containing 0.2 mg of total protein extracts of type II (T-II) or type I (T-I) fiber-rich portions of gastrocnemius were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-PGC-1{alpha} or anti-UCP-3 antibodies. C: total RNA samples from gastrocnemius muscle portions containing predominantly type II or type I fibers were reverse transcribed and used in PCR to amplify {beta}-actin (489 bp) and PGC-1{alpha} (804 bp, C). Values are means ± SE; n = 4. *P < 0.05.

 
Acute insulin treatment does not modulate PGC-1{alpha} protein expression in muscle. Because the objective of the present study was to evaluate the possible involvement of PGC-1{alpha} in insulin signal transduction, we decided to look first for any possible effect of an acute dose of insulin on the expression of PGC-1{alpha}. Control and cold-exposed rats were injected with a single intravenous (through the vena cava) dose of saline with or without insulin (200 µl, 10–6 M), and after 10 min, fragments of type I and type II fiber-rich portions of gastrocnemius muscle were excised, homogenized, and subjected to SDS-PAGE, transferred to nitrocellulose membranes, and blotted with PGC-1{alpha}-specific antibodies. An acute dose of insulin promoted no significant changes in PGC-1{alpha} expression in either type of muscle fiber (Fig. 2).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 2. Effect of acute insulin treatment on PGC-1{alpha} expression in gastrocnemius muscle of control and cold-exposed rats. Control and cold-exposed rats were acutely treated with a single dose of saline with (+) or without (–) insulin (200 µl, 10–6 M), and, after 10 min, type II (A) and type I (B) fiber-rich portions of gastrocnemius were excised and homogenized. Samples containing 0.2 mg of total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-PGC-1{alpha} antibody. Blots are representative of results from 4 experiments.

 
Cold exposure leads to a reduction of insulin-induced activation of the IR and Akt. Insulin activates IR kinase activity by promoting conformational changes that ultimately lead to tyrosine phosphorylation of the IR. Akt and ERK are intermediate, indirect substrates of the IR that, once activated, participate in transduction of the insulin signal toward metabolic, antiapoptotic, growth-promoting, and promitogenic pathways. In type I and type II muscle fiber, cold exposure led to a reduction of the insulin-induced (single intravenous 200-µl dose of 10–6 M insulin through the vena cava, 90 s for evaluation of IR and 5 min for evaluation of Akt and ERK) tyrosine phosphorylation of the IR [0.5-fold (P < 0.05) and 0.4-fold (P < 0.05) for type II and type I fiber-rich portions of gastrocnemius muscle, respectively; Fig. 3, A and B]and of the Ser473 phosphorylation of Akt [0.2-fold (P < 0.05) and 0.6-fold (P < 0.05) for type II and type I fiber-rich portions of gastrocnemius muscle, respectively; Fig. 3, C and D]. However, under the same conditions, no modulation of basal or insulin-stimulated tyrosine phosphorylation of 42- and 44-kDa ERK was detected (Fig. 3, C and D).



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 3. Effect of cold exposure on initial and intermediate steps of the insulin signal transduction cascade. A and B: 0.2 mg of total protein extracts from type II and type I fiber-rich portions of gastrocnemius from control and cold-exposed rats acutely treated (90 s) with a single dose of saline with (+) or without (–) insulin (200 µl, 10–6 M) was separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-insulin receptor (IR) antibodies, or 5.0 mg of total protein extracts were used in immunoprecipitation experiments with anti-insulin receptor (IR) antibodies, producing immunoprecipitates that were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antiphosphotyrosine (pY) antibodies. C and D: total protein extracts (0.2 mg) from type II and type I fiber-rich portions of gastrocnemius from control and cold-exposed rats acutely treated (5 min) with a single dose of saline with (+) or without (–) insulin (200 µl, 10–6 M) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antiphospho-Ser473 Akt (p473Akt; C and D, top blots) or antiphospho-ERK (pERK) antibodies. Blots are representative of results from 4 experiments.

 
Cold exposure regulates glucose uptake and expression and subcellular localization of GLUT4. Exposure to cold for 4 days promoted a significant increase of basal and insulin-stimulated glucose uptake by gastrocnemius muscle (Fig. 4A).This was accompanied by a significant increase of GLUT4 expression in whole gastrocnemius (containing type I and type II fiber types) protein extracts (1.4-fold, P < 0.05; Fig. 4B). In subcellular fractionation studies, cold exposure promoted an insulin-independent increase of GLUT4 in the membrane fraction of both fiber types (Fig. 4, E and F), while in control rats, higher basal levels of GLUT4 were observed in cytosol fractions, which migrated, on an insulin stimulus-dependent basis, to the membrane: insulin induced a 0.8-fold (P < 0.05) and a 0.7-fold (P < 0.05) reduction of GLUT4 in cytosol fractions of type II and type I fiber-rich portions of the gastrocnemius muscle of control rats, respectively (Fig. 4, C and D). No significant modulation of GLUT1 expression in gastrocnemius muscle was induced by cold exposure (not shown).



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4. Effect of cold exposure on 2-deoxy-D-glucose uptake and GLUT4 expression and translocation. A: 2-[3H]deoxyglucose (2-DG) uptake by gastrocnemius muscle was determined in control and cold-exposed rats acutely injected with saline with (+) or without (–) insulin. Protein extract samples (0.2 mg) from total gastrocnemius (B) or from type II (C and E) and type I (D and F) fiber-rich portions of gastrocnemius from control and cold-exposed rats acutely treated (in B, rats were not treated with insulin) with a single dose of saline with (+) or without (–) insulin (200 µl, 10–6 M) were directly separated by SDS-PAGE (B), or, after a subcellular fractionation protocol, cytosol and membrane fractions were separated by SDS-PAGE (C–F). SDS-PAGE-separated proteins were transferred to nitrocellulose membranes and blotted with anti-GLUT4 antibodies. Bands were scanned and densitometrically evaluated. Relative amount of GLUT4 is shown as a direct comparison with samples from control (B) or from cytosol of control not treated with insulin (C–F). Values are means ± SE; n = 4. *P < 0.05.

 
PGC-1{alpha} protein expression reduction inhibits glucose uptake in cold-exposed rats. To evaluate the participation of cold-induced PGC-1{alpha} expression on insulin signal transduction in skeletal muscle, control and cold-exposed rats were treated (4.0 nmol) with antisense or sense (not shown) phosphorthioate-modified oligonucleotides specific for PGC-1{alpha}, in parallel with rats not treated with oligonucleotide. The treatment with sense or antisense oligonucleotide did not modulate 24-h food intake and body temperature (not shown). Initially, the blocking capacity of the oligonucleotide was evaluated by immunoblot of SDS-PAGE-separated, nitrocellulose-transferred proteins extracted from type I and type II fiber-rich portions of gastrocnemius muscle. PGC-1{alpha} antisense treatment led to significant reduction of basal [control; 0.3-fold (P < 0.05) and 0.15-fold (P < 0.05)] and cold-induced [0.2-fold (P < 0.05) and 0.1-fold (P < 0.05)] PGC-1{alpha} protein expression in type II (Fig. 5A)and type I (Fig. 5B) fiber-rich portions of gastrocnemius muscle, respectively. This was accompanied by significant reduction of cold-induced [0.5-fold (P < 0.05) and 0.65-fold (P < 0.05) for type II and type I fiber-rich portions of gastrocnemius, respectively], but not basal, UCP-3 expression in both fiber types (Fig. 5, A and B). To further evaluate the effect of PGC-1{alpha} blockade on UCP-3, a mitochondrial respiration assay was performed (Fig. 5, C and D). The quality of mitochondria preparation was optimal, presenting ADP-to-O ratios of 2.9 ± 0.2, 2.9 ± 0.1, 2.8 ± 0.2, and 2.9 ± 0.1 for mitochondria from type II muscle without oligonucleotide, type II muscle with PGC-1{alpha} antisense oligonucleotide, type I muscle without oligonucleotide, and type I muscle with PGC-1{alpha} antisense oligonucleotide, respectively, and respiratory control (state 3-to-state 4 ratio) of 4.87 ± 1.26, 4.37 ± 0.32, 4.46 ± 0.46, and 4.38 ± 0.39 for mitochondria from type II muscle without oligonucleotide, type II muscle with PGC-1{alpha} antisense oligonucleotide, type I muscle without oligonucleotide, and type I muscle with PGC-1{alpha} antisense oligonucleotide, respectively. The blockade of PGC-1{alpha} expression resulted in improved mitochondrial respiratory coupling in type II, but not type I, fiber-rich portions of gastrocnemius muscle, because the post/prelinoleic acid oxygen consumption rate ratio was 2.2 for mitochondria from the type II fiber-rich portion of the gastrocnemius not treated with oligonucleotide and 1.4 for mitochondria from the type II fiber-rich portion of the gastrocnemius treated with PGC-1{alpha} antisense oligonucleotide (Fig. 5, C and D).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 5. Effect of treatment with antisense PGC-1{alpha} oligonucleotide on PGC-1{alpha} and uncoupling protein-3 (UCP-3) protein expression (A and B) and mitochondrial respiration (C) in gastrocnemius of control and cold-exposed rats. Rats were injected intraperitoneally with 2 doses (24 h and 4 h before experimental procedure) of 200 µl (20 µmol/l) of antisense oligonucleotide specific for PGC-1{alpha} (as) or were left untreated (wo). Samples containing 0.2 mg of total protein extracts of type II (A) or type I (B) fiber-rich portions of gastrocnemius were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-PGC-1{alpha} or anti-UCP-3 antibodies. C: mitochondria from type II or type I fiber-rich portions of gastrocnemius from cold-exposed rats were prepared as described in MATERIALS AND METHODS and employed in experiments for determination of mitochondrial respiration. O2 consumption (nmol) was measured using a Clark-type electrode in a 0.5-ml glass chamber equipped with magnetic stirring. Preparations were treated with NAD-linked substrates, {alpha}-ketoglutarate, pyruvate, malate, and glutamate, at 5 mM and with 1 µM carboxyatractiloside (CAT), 6 µM linoleic acid (LA), 500 µM GDP, and 0.1% BSA. D: pre/post-linoleic acid O2 consumption rates for type II and type I fiber-rich portions of gastrocnemius. Values are means ± SE; n = 4. P < 0.05.

 
To assess the effect of PGC-1{alpha} blockade on insulin-induced glucose uptake, cold-exposed and control rats were treated with PGC-1{alpha} antisense oligonucleotide and subjected to a hyperinsulinemic-euglycemic clamp. Cold exposure induced a significant increase (4.8-fold, P < 0.05) of insulin-induced glucose consumption (Fig. 6),which was reversed by the blockade of PGC-1{alpha} (0.6-fold, P < 0.05). In control rats, the inhibition of PGC-1{alpha} expression did not modulate glucose clearance rates during the clamp.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 6. Effect of inhibition of PGC-1{alpha} protein expression on glucose consumption during hyperinsulinemic-euglycemic clamp. Control and cold-exposed rats were injected intraperitoneally with 2 doses (24 h and 4 h before experimental procedure) of 200 µl (20 µmol/l) of antisense oligonucleotide specific for PGC-1{alpha} or left untreated, and insulin action was evaluated. Values are means ± SE; n = 6. *P < 0.05.

 
PGC-1{alpha} blockade reduces GLUT4 expression in membrane fraction independently of IR and Akt functional status. Antisense oligonucleotide blockade of PGC-1{alpha} produced no significant modulation of basal (not shown) and insulin-induced tyrosine phosphorylation of the IR and of the Ser473 phosphorylation of Akt in type I (Fig. 7A)and type II (Fig. 7B) fiber-rich portions of gastrocnemius muscle. Nevertheless, blockade of PGC-1{alpha} expression significantly reduced the membrane expression of GLUT4 in type II (0.8-fold, P < 0.05; Fig. 7E) and type I (0.75-fold, P < 0.05; Fig. 7G) fiber-rich portions of gastrocnemius muscle from cold-exposed (Fig. 7, E and G), but not control (Fig. 7, D and F), rats. This effect was independent of whole muscle content of GLUT4 (Fig. 7C).



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 7. Effect of inhibition of PGC-1{alpha} protein expression on insulin-induced insulin receptor (IR) tyrosine phosphorylation and acute transforming retrovirus thymoma (Akt) serine phosphorylation and on GLUT4 expression and translocation in rats injected intraperitoneally with 2 doses (24 h and 4 h before experimental procedure) of 200 µl (20 µmol/l) of antisense oligonucleotide specific for PGC-1{alpha} and in untreated rats. A and B: 0.2 mg of total protein extracts from type II and type I fiber-rich portions of gastrocnemius from control or cold-exposed rats, treated (+) with a single acute [90 s (top blots) and 5 min (bottom blots)] intravenous dose of insulin (200 µl, 10–6 M) was separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antiphospho-Ser473 Akt (p473Akt) antibodies (A and B, bottom blots), or 5.0 mg of total protein extracts were used in immunoprecipitation experiments with anti-IR antibodies, producing immunoprecipitates that were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antiphosphotyrosine antibodies. C–G: protein extract samples (0.2 mg) from total gastrocnemius (C) or from type II (D and E) and type I (F and G) fiber-rich portions of gastrocnemius from control and cold-exposed rats injected (+) with a single acute (15 min) intravenous dose of insulin (200 µl, 10–6 M) were directly separated by SDS-PAGE (C) or subjected to a subcellular fractionation protocol followed by SDS-PAGE separation of cytosol and membrane fractions (D–G). SDS-PAGE-separated proteins were transferred to nitrocellulose membranes and blotted with anti-GLUT4 antibodies. Bands were scanned and densitometrically evaluated. Relative amount of GLUT4 is shown as a direct comparison with samples from control (C) or cytosol of control treated with insulin and not treated with antisense oligonucleotide (D–G). Values are means ± SE; n = 4. *P < 0.05.

 
Glucose uptake and GLUT4 subcellular distribution in skeletal muscle of cold-exposed rats depend on PGC-1{alpha} expression and AMPK activity. AMPK is an intracellular fuel sensor that has recently been shown to play a role in the physiological control of insulin-independent glucose uptake (36). To evaluate the possible participation of AMPK in the mechanisms that regulate glucose uptake in cold-exposed animals, we initially performed immunoblot determinations of AMPK protein expression and AMPK Thr172 phosphorylation in type II and type I portions of gastrocnemius muscle. Cold exposure promoted a significant increase in Thr172 phosphorylation of AMPK in type II (2.8-fold, P < 0.05; Fig. 8A,bottom blot) and type I (3.4-fold, P < 0.05; Fig. 8B, bottom blot) fiber-rich portions of gastrocnemius muscle without affecting AMPK protein expression (Fig. 8, A and B, top blots). The inhibition of PGC-1{alpha} expression with PGC-1{alpha} antisense oligonucleotide, as well as the inhibition of AMPK activity by in vivo treatment with ARA-A, significantly reduced cold-induced Thr172 phosphorylation of AMPK in type II (0.4- and 0.3-fold for antisense and ARA-A treatments, respectively, P < 0.05) and type I (0.3- and 0.2-fold for antisense and ARA-A treatments, respectively, P < 0.05; Fig. 8, bottom blots) fiber-rich portions of gastrocnemius muscle but did not affect AMPK protein expression (Fig. 8, top blots). To evaluate the participation of phospho-Thr172 AMPK in glucose uptake during a hyperinsulinemic-euglycemic clamp, we treated control and cold-exposed rats with ARA-A and performed clamp studies. ARA-A was capable of significantly reducing control (0.4-fold, P < 0.05) and cold-induced (0.3-fold, P < 0.05) glucose consumption (Fig. 8C).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 8. Effect of cold exposure and inhibition of PGC-1{alpha} protein expression on 5'-adenine monophosphate-activated protein kinase (AMPK) expression and phosphorylation and effect of AMPK pharmacological inhibition on glucose consumption during a hyperinsulinemic-euglycemic clamp. A and B: total protein extracts (0.2 mg) from type II and type I fiber-rich portions of gastrocnemius were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-AMPK or antiphospho-Thr172 AMPK-{alpha} antibodies. Experimental conditions were as follows: control or cold-exposed rats were injected intraperitoneally with 2 doses (24 h and 4 h before experimental procedure) of 200 µl (20 µmol/l) of antisense oligonucleotide specific for PGC-1{alpha} or with adenine {beta}-D-arabinofuranoside (ARA-A, 25 mmol/dose) 12 and 2 h before the experimental procedure or were not pretreated (npt). Blots are representative of results from 4 experiments. C: control and cold-exposed rats were injected intraperitoneally with ARA-A (25 mmol/dose) 12 and 2 h before the experimental procedure or left untreated and subjected to a clamp evaluation of insulin action measured as the rate of glucose consumption. Values are means ± SE; n = 6. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The characterization of novel mechanisms that participate in the control of glucose uptake is of special interest as a starting point for selecting candidate targets for therapeutic approaches in diabetes mellitus and related diseases. Studies of insulin production and action and of glucose uptake in homeothermic animals exposed to cold revealed that, in this particular physiological condition, glucose is efficiently mobilized, despite low blood insulin levels and an apparent tissue-specific resistance to insulin's action (7, 11, 41, 45). Therefore, it became clear that mechanisms that act independently of insulin signaling through the IR-IR substrates (IRSs)-Akt pathway play an important role in the control of glucose disposal in cold-exposed animals.

The high adrenergic tonus generated by exposure of homeothermic animals to cold is thought to play a central role in the control of glucose uptake driven by insulin-independent mechanisms (20, 21). This fact is reinforced by the demonstration of the antidiabetic effect of {beta}3-adrenergic compounds such as CL-316243 (17, 21) and BRL-26830A (34). However, the molecular mechanisms by which the adrenergic stimulus improves glucose uptake independently of insulin action through the IR-IRSs-Akt pathway are not known. Studies concerned with the possible direct cross talk between insulin and {beta}-adrenergic intracellular signaling systems have shown that insulin is able to induce tyrosine phosphorylation of residues at the COOH terminus of the {beta}2-adrenergic receptor (12), whereas a {beta}-adrenergic stimulus inhibits insulin signal transduction (15, 26, 47). As a rule, most of the sympathetic signals are thought to be deleterious for insulin signaling and participate negatively in the pathogenesis of diabetes mellitus (33). However, after the characterization of the {beta}3-adrenergic receptor, it became clear that sympathetic inputs could play dual roles in this complex scenario (9).

Along with the recent characterization of PGC-1{alpha} (31), the demonstration of its induction by cold-induced adrenergic stimulus (30), and its properties of controlling GLUT4 expression and glucose uptake by insulin-independent mechanisms (22), we decided to evaluate its possible participation in cold-induced modulation of insulin signaling in skeletal muscle of rats.

As expected, cold exposure promoted a significant increase of PGC-1{alpha} expression in type I and type II fibers of gastrocnemius muscle that was accompanied by an increase of UCP-3 expression. Because the main objective of the present study was to evaluate the effect of PGC-1{alpha} on insulin signal transduction, we decided to begin by determining whether an acute dose of insulin would exert any modulation on the expression of PGC-1{alpha}. As expected, the protein levels of PGC-1{alpha} were not affected by a single intravenous dose of insulin.

Next, we evaluated the effect of cold exposure on key steps of the insulin signal transduction machinery. As previously reported, exposure to cold promotes a tissue-specific modulation of insulin signaling (11). In skeletal muscle, there is an apparent impairment of the insulin signal transduction (11), which was confirmed here. Furthermore, we demonstrate that the phenomenon occurs similarly in type I and type II fibers of gastrocnemius muscle. Only the metabolic-related Akt pathway is affected by cold exposure, whereas the growth-related ERK pathway is preserved. Although during cold exposure the insulin-signaling machinery in muscle is impaired, an increase of basal and insulin-induced glucose uptake and of whole body glucose clearance is detected. This phenomenon seems to be mediated, at least in part, by an insulin-independent accumulation of GLUT4 in the membrane fraction of type II and type I fiber-rich portions of gastrocnemius muscle (11, 18). No modulation of GLUT1 expression occurs.

To evaluate the participation of cold-induced PGC-1{alpha} in glucose uptake, insulin signal transduction, and GLUT4 localization in muscle, we utilized an antisense oligonucleotide to PGC-1{alpha} that promoted a significant reduction of PGC-1{alpha} expression in both fiber types of skeletal muscle. This oligonucleotide has been utilized in previous experiments and provided reproducible and effective inhibition of PGC-1{alpha} expression (7). In Western blot experiments, we observed that not only was PGC-1{alpha} protein expression reduced, but the levels of UCP-3 were reduced as well. This was accompanied by a significant change in the coupling of mitochondrial respiration, reinforcing the property of the antisense oligonucleotide to inhibit PGC-1{alpha} expression and, consequently, modulate UCP-3 expression and activity. In a recent study, Fan and co-workers (10) demonstrated that the inhibition of PGC-1{alpha} transcriptional activity by hyperexpression and recruitment of p160 Myb binding protein reduces mitochondrial respiration. This effect was accompanied by reduced expression of cytochrome c and {beta}-ATP synthase. UCP expression was not evaluated. In the present study, UCP-3 expression was evaluated to provide further validation for the property of the antisense oligonucleotide to inhibit PGC-1{alpha} expression. However, the results also further support a role for skeletal muscle UCP-3 in thermogenesis, as recently described by Mills and co-workers (23), and evidence that, at least under cold exposure conditions, UCP-3 expression is controlled by the levels of PGC-1{alpha}. When PGC-1{alpha} expression was inhibited by the antisense oligonucleotide treatment, glucose uptake during the clamp was reduced and GLUT4 migration to the membrane fraction of muscle was significantly impaired. This close connection between PGC-1{alpha} expression levels, GLUT4 distribution, and glucose clearance strongly suggests that PGC-1{alpha} plays an important role in the whole body control of glucose disposal during cold exposure. Michael et al. (22) demonstrated that, in cultured myocytes that do not express GLUT4, the adenoviral-mediated hyperexpression of PGC-1{alpha} induces GLUT4 expression with a preferential localization in the cell membrane. Previous studies found a direct relation between uncoupling and GLUT4 expression in cultured cells and muscle of living mammals (18, 43). Because under conditions that promote mitochondrial uncoupling there are increased cellular needs for energy, a physiological rationale for a parallel regulation of GLUT4 and UCPs may be proposed. According to our results, PGC-1{alpha} may play a pivotal role in the simultaneous control of GLUT4 and UCP-3 expression. Finally, because inhibition of PGC-1{alpha} did not modulate insulin-induced IR and Akt phosphorylation, we believe that PGC-1{alpha} affects glucose uptake through mechanisms other than the those dependent on classic insulin signaling through the IR-Akt signal transduction pathway.

AMPK is a trimeric enzyme present in membrane and cytosolic fractions of cells from several tissues (36, 46). It acts as a metabolic switch in insulin-sensitive and non-insulin-sensitive cells (14), favoring the shutdown of fatty acid synthetic pathways to control the waste of energy under stressful conditions. Recent studies have demonstrated that AMPK activation may participate in the control of glucose uptake by insulin-independent mechanisms (28, 35). In the present study, cold exposure did not modulate AMPK expression but significantly increased its phosphorylation in Thr172, a phenomenon closely related to its catalytic activity. When PGC-1{alpha} expression was inhibited in cold-exposed rats, no modulation of AMPK expression was observed, but a significant decrease of its Thr172 phosphorylation was determined. Furthermore, blockade of AMPK activity with the chemical inhibitor ARA-A mimicked the action of PGC-1{alpha} inhibition on glucose consumption during the hyperinsulinemic-euglycemic clamp.

Pharmacological and exercise-induced activation of AMPK has been reported to lead to stimulation of PGC-1{alpha} and UCP-3 protein expression (32, 38, 39, 50). Thus, according to current concepts, PGC-1{alpha} is a downstream target of AMPK. In the present study, we show that inhibition of PGC-1{alpha} expression modulates AMPK activity. Because the intracellular levels of AMP are among the most important physiological regulators of AMPK activity and because, in the present report, we show that PGC-1{alpha} increases mitochondrial uncoupling, it would be expected that intracellular levels of AMP would increase on PGC-1{alpha} activation and, therefore, promote a positive modulation of AMPK activity.

Thus, as a whole, this study shows that ≥50% of the cold exposure-induced increment of glucose consumption determined by the clamp method is reversed by the inhibition of PGC-1{alpha} expression and is associated with the modulation of expression and membrane localization of GLUT4. These effects are not related to the functional status of the IR and Akt but accompany AMPK activity. These data show, for the first time, the participation of PGC-1{alpha} in the control of glucose uptake by muscle during cold exposure and reinforce the role of AMPK in the modulation of glucose homeostasis through mechanisms that act independently of insulin-induced activation of the IR-IRSs-Akt pathway.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Fundação de Amparo à Pesquisa de Estado de São Paulo and the Conselho Nacional de Pesquisa.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Velloso, Dept. of Internal Medicine, Faculty of Medical Sciences (FCM), State Univ. of Campinas (UNICAMP), Campinas-SP 13083-970, Brazil (E-mail: lavelloso{at}fcm.unicamp.br)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Baar K, Wende AR, Jones TE, Marison M, Nolte LA, Chen M, Kelly DP, and Holloszy JO. Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1. FASEB J 16: 1879–1886, 2002.[Abstract/Free Full Text]
  2. Bertelli DF, Ueno M, Amaral ME, Toyama MH, Carneiro EM, Marangoni S, Carvalho CR, Saad MJ, Velloso LA, and Boschero AC. Reversal of denervation-induced insulin resistance by SHIP2 protein synthesis blockade. Am J Physiol Endocrinol Metab 284: E679–E687, 2003.[Abstract/Free Full Text]
  3. Calegari VC, Bezerra RM, Torsoni MA, Torsoni AS, Franchini KG, Saad MJ, and Velloso LA. Suppressor of cytokine signaling 3 is induced by angiotensin II in heart and isolated cardiomyocytes and participates in desensitization. Endocrinology 144: 4586–4596, 2003.[Abstract/Free Full Text]
  4. Cassis L, Laughter A, Fettinger M, Akers S, Speth R, Burke G, King V, and Dwoskin L. Cold exposure regulates the renin-angiotensin system. J Pharmacol Exp Ther 286: 718–726, 1998.[Abstract/Free Full Text]
  5. Chang E, Bartholomeusz G, Pimental R, Chen J, Lai H, Wang L, Yang P, and Marcus S. Direct binding and in vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein scd2. Mol Cell Biol 19: 8066–8074, 1999.[Abstract/Free Full Text]
  6. DeFronzo RA, Tobin JD, and Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol Endocrinol Metab Gastrointest Physiol 237: E214–E223, 1979.[Abstract/Free Full Text]
  7. De Souza CT, Gasparetti AL, Pereira-da-Silva M, Araujo EP, Carvalheira JB, Saad MJ, Boschero AC, Carneiro EM, and Velloso LA. Peroxisome proliferator-activated receptor-{gamma} coactivator-1-dependent uncoupling protein-2 expression in pancreatic islets of rats: a novel pathway for neural control of insulin secretion. Diabetologia 46: 1522–1531, 2003.[CrossRef][Web of Science][Medline]
  8. Ek J, Andersen G, Urhammer SA, Gaede PH, Drivsholm T, Borch-Johnsen K, Hansen T, and Pedersen O. Mutation analysis of peroxisome proliferator-activated receptor-{gamma} coactivator-1 (PGC-1) and relationships of identified amino acid polymorphisms to type II diabetes mellitus. Diabetologia 44: 2220–2226, 2001.[CrossRef][Web of Science][Medline]
  9. Emorine LJ, Marullo S, Briend-Sutren MM, Patey G, Tate K, Delavier-Klutchko C, and Strosberg AD. Molecular characterization of the human {beta}3-adrenergic receptor. Science 245: 1118–1121, 1989.[Abstract/Free Full Text]
  10. Fan M, Rhee J, St-Pierre J, Handschin C, Puigserver P, Lin J, Jaeger S, Erdjument-Bromage H, Tempst P, and Spiegelman BM. Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1{alpha}: modulation by p38 MAPK. Genes Dev 18: 278–289, 2004.[Abstract/Free Full Text]
  11. Gasparetti AL, de Souza CT, Pereira-da-Silva M, Oliveira RL, Saad MJ, Carneiro EM, and Velloso LA. Cold exposure induces tissue-specific modulation of the insulin-signalling pathway in Rattus norvegicus. J Physiol 552: 149–162, 2003.
  12. Hadcock JR, Port JD, Gelman MS, and Malbon CC. Cross-talk between tyrosine kinase and G-protein-linked receptors. Phosphorylation of {beta}2-adrenergic receptors in response to insulin. J Biol Chem 267: 26017–26022, 1992.[Abstract/Free Full Text]
  13. Hara K, Tobe K, Okada T, Kadowaki H, Akanuma Y, Ito C, Kimura S, and Kadowaki T. A genetic variation in the PGC-1 gene could confer insulin resistance and susceptibility to type II diabetes. Diabetologia 45: 740–743, 2002.[CrossRef][Web of Science][Medline]
  14. Hardie DG, Carling D, and Carlson M. The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67: 821–855, 1998.[CrossRef][Web of Science][Medline]
  15. Haring H, Kirsch D, Obermaier B, Ermel B, and Machicao F. Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vitro. Biochem J 234: 59–66, 1986.[Web of Science][Medline]
  16. Herzig S, Long F, Jhala US, Hedrick S, Quinn R, Bauer A, Rudolph D, Schutz G, Yoon C, Puigserver P, Spiegelman B, and Montminy M. CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature 413: 179–183, 2001.[CrossRef][Medline]
  17. Jost P, Fasshauer M, Kahn CR, Benito M, Meyer M, Ott V, Lowell BB, Klein HH, and Klein J. Atypical {beta}-adrenergic effects on insulin signaling and action in {beta}3-adrenoceptor-deficient brown adipocytes. Am J Physiol Endocrinol Metab 283: E146–E153, 2002.[Abstract/Free Full Text]
  18. Lin B, Coughlin S, and Pilch PF. Bidirectional regulation of uncoupling protein-3 and GLUT-4 mRNA in skeletal muscle by cold. Am J Physiol Endocrinol Metab 275: E386–E391, 1998.[Abstract/Free Full Text]
  19. Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, Michael LF, Puigserver P, Isotani E, Olson EN, Lowell BB, Bassel-Duby R, and Spiegelman BM. Transcriptional co-activator PGC-1{alpha} drives the formation of slow-twitch muscle fibres. Nature 418: 797–801, 2002.[CrossRef][Medline]
  20. Liu X, Perusse F, and Bukowiecki LJ. Chronic norepinephrine infusion stimulates glucose uptake in white and brown adipose tissues. Am J Physiol Regul Integr Comp Physiol 266: R914–R920, 1994.[Abstract/Free Full Text]
  21. Liu X, Perusse F, and Bukowiecki LJ. Mechanisms of the antidiabetic effects of the {beta}3-adrenergic agonist CL-316243 in obese Zucker-ZDF rats. Am J Physiol Regul Integr Comp Physiol 274: R1212–R1219, 1998.[Abstract/Free Full Text]
  22. Michael LF, Wu Z, Cheatham RB, Puigserver P, Adelmant G, Lehman JJ, Kelly DP, and Spiegelman BM. Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1. Proc Natl Acad Sci USA 98: 3820–3825, 2001.[Abstract/Free Full Text]
  23. Mills EM, Banks ML, Sprague JE, and Finkel T. Pharmacology: uncoupling the agony from ecstasy. Nature 426: 403–404, 2003.[CrossRef][Medline]
  24. Mizukami Y and Yoshida K. Mitogen-activated protein kinase translocates to the nucleus during ischaemia and is activated during reperfusion. Biochem J 323: 785–790, 1997.
  25. Musi N, Hayashi T, Fujii N, Hirshman MF, Witters LA, and Goodyear LJ. AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle. Am J Physiol Endocrinol Metab 280: E677–E684, 2001.[Abstract/Free Full Text]
  26. Obermaier B, Ermel B, Kirsch D, Mushack J, Rattenhuber E, Biemer E, Machicao F, and Haring HU. Catecholamines and tumour promoting phorbol esters inhibit insulin receptor kinase and induce insulin resistance in isolated human adipocytes. Diabetologia 30: 93–99, 1987.[Web of Science][Medline]
  27. Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, Miyazaki Y, Kohane I, Costello M, Saccone R, Landaker EJ, Goldfine AB, Mun E, DeFronzo R, Finlayson J, Kahn CR, and Mandarino LJ. Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: potential role of PGC1 and NRF1. Proc Natl Acad Sci USA 100: 8466–8471, 2003.[Abstract/Free Full Text]
  28. Paulsen SR, Rubink DS, and Winder WW. AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4. J Appl Physiol 91: 2102–2108, 2001.[Abstract/Free Full Text]
  29. Puigserver P, Rhee J, Lin J, Wu Z, Yoon JC, Zhang CY, Krauss S, Mootha VK, Lowell BB, and Spiegelman BM. Cytokine stimulation of energy expenditure through p38 MAP kinase activation of PPAR-{gamma} coactivator-1. Mol Cell 8: 971–982, 2001.[CrossRef][Web of Science][Medline]
  30. Puigserver P and Spiegelman BM. Peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}): transcriptional coactivator and metabolic regulator. Endocr Rev 24: 78–90, 2003.[Abstract/Free Full Text]
  31. Puigserver P, Wu Z, Park CW, Graves R, Wright M, and Spiegelman BM. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92: 829–839, 1998.[CrossRef][Web of Science][Medline]
  32. Putman CT, Kiricsi M, Pearcey J, MacLean IM, Bamford JA, Murdoch GK, Dixon WT, and Pette D. AMPK activation increases uncoupling protein-3 expression and mitochondrial enzyme activities in rat muscle without fibre type transitions. J Physiol 551: 169–178, 2003.[Abstract/Free Full Text]
  33. Reaven GM, Lithell H, and Landsberg L. Hypertension and associated metabolic abnormalities—the role of insulin resistance and the sympathoadrenal system. N Engl J Med 334: 374–381, 1996.[Free Full Text]
  34. Rochet N, Tanti JF, Gremeaux T, Van Obberghen E, and Le Marchand-Brustel Y. Effect of a thermogenic agent, BRL 26830A, on insulin receptors in obese mice. Am J Physiol Endocrinol Metab 255: E101–E109, 1988.[Abstract/Free Full Text]
  35. Russell RR III, Bergeron R, Shulman GI, and Young LH. Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol Heart Circ Physiol 277: H643–H649, 1999.[Abstract/Free Full Text]
  36. Rutter GA, Da Silva Xavier G, and Leclerc I. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J 375: 1–16, 2003.[CrossRef][Web of Science][Medline]
  37. Scott AM, Atwater I, and Rojas E. A method for the simultaneous measurement of insulin release and B cell membrane potential in single mouse islets of Langerhans. Diabetologia 21: 470–475, 1981.[Web of Science][Medline]
  38. Suwa M, Nakano H, and Kumagai S. Effects of chronic AICAR treatment on fiber composition, enzyme activity, UCP3, and PGC-1 in rat muscles. J Appl Physiol 95: 960–968, 2003.[Abstract/Free Full Text]
  39. Terada S, Goto M, Kato M, Kawanaka K, Shimokawa T, and Tabata I. Effects of low-intensity prolonged exercise on PGC-1 mRNA expression in rat epitrochlearis muscle. Biochem Biophys Res Commun 296: 350–354, 2002.[CrossRef][Web of Science][Medline]
  40. Tonkonogi M and Sahlin K. Rate of oxidative phosphorylation in isolated mitochondria from human skeletal muscle: effect of training status. Acta Physiol Scand 161: 345–353, 1997.[CrossRef][Web of Science][Medline]
  41. Torsoni MA, Carvalheira JB, Pereira-Da-Silva M, de Carvalho-Filho MA, Saad MJ, and Velloso LA. Molecular and functional resistance to insulin in hypothalamus of rats exposed to cold. Am J Physiol Endocrinol Metab 285: E216–E223, 2003.[Abstract/Free Full Text]
  42. Trinder P. Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J Clin Pathol 22: 158–161, 1969.[Abstract/Free Full Text]
  43. Tsakiridis T, Vranic M, and Klip A. Phosphatidylinositol 3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action. Biochem J 309: 1–5, 1995.
  44. Turinsky J. Glucose metabolism in the region recovering from burn injury: effect of insulin on 2-deoxyglucose uptake in vivo. Endocrinology 113: 1370–1376, 1983.[Abstract/Free Full Text]
  45. Vallerand AL, Perusse F, and Bukowiecki LJ. Cold exposure potentiates the effect of insulin on in vivo glucose uptake. Am J Physiol Endocrinol Metab 253: E179–E186, 1987.[Abstract/Free Full Text]
  46. Warden SM, Richardson C, O'Donnell J Jr, Stapleton D, Kemp BE, and Witters LA. Post-translational modifications of the {beta}1-subunit of AMP-activated protein kinase affect enzyme activity and cellular localization. Biochem J 354: 275–283, 2001.[CrossRef][Web of Science][Medline]
  47. Weber TM, Joost HG, Kuroda M, Cushman SW, and Simpson IA. Subcellular distribution and phosphorylation state of insulin receptors from insulin- and isoproterenol-treated rat adipose cells. Cell Signal 3: 51–58, 1991.[CrossRef][Web of Science][Medline]
  48. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla RC, and Spiegelman BM. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98: 115–124, 1999.[CrossRef][Web of Science][Medline]
  49. Yoon JC, Xu G, Deeney JT, Yang SN, Rhee J, Puigserver P, Levens AR, Yang R, Zhang CY, Lowell BB, Berggren PO, Newgard CB, Bonner-Weir S, Weir G, and Spiegelman BM. Suppression of beta cell energy metabolism and insulin release by PGC-1{alpha}. Dev Cell 5: 73–83, 2003.[CrossRef][Web of Science][Medline]
  50. Zhou M, Lin BZ, Coughlin S, Vallega G, and Pilch PF. UCP-3 expression in skeletal muscle: effects of exercise, hypoxia, and AMP-activated protein kinase. Am J Physiol Endocrinol Metab 279: E622–E629, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Physiol.Home page
E. R. Ropelle, J. R. Pauli, D. E. Cintra, M. J. S. Frederico, R. A. de Pinho, Líc. A. Velloso, and C.áu. T. De Souza
Acute exercise modulates the Foxo1/PGC-1\#945; pathway in the liver of diet-induced obesity rats
J. Physiol., May 1, 2009; 587(9): 2069 - 2076.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
P. W. Franks, R. J. F. Loos, S. Brage, S. O'Rahilly, N. J. Wareham, and U. Ekelund
Physical activity energy expenditure may mediate the relationship between plasma leptin levels and worsening insulin resistance independently of adiposity
J Appl Physiol, May 1, 2007; 102(5): 1921 - 1926.
[Abstract] [Full Text] [PDF]

Home page
 J Gerontol A Biol Sci Med SciHome page
K. A. Al-Regaiey, M. M. Masternak, M. S. Bonkowski, J. A. Panici, J. J. Kopchick, and A. Bartke
Effects of Caloric Restriction and Growth Hormone Resistance on Insulin-Related Intermediates in the Skeletal Muscle
J Gerontol A Biol Sci Med Sci, January 1, 2007; 62(1): 18 - 26.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
Y.-C. Hsieh, M. A. Choudhry, H.-P. Yu, T. Shimizu, S. Yang, T. Suzuki, J. Chen, K. I. Bland, and I. H. Chaudry
Inhibition of cardiac PGC-1{alpha} expression abolishes ER{beta} agonist-mediated cardioprotection following trauma-hemorrhage
FASEB J, June 1, 2006; 20(8): 1109 - 1117.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/4/E686    most recent
00103.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oliveira, R. L. G. S.
Right arrow Articles by Velloso, L. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oliveira, R. L. G. S.
Right arrow Articles by Velloso, L. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.