AJP - Renal Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 294: F881-F889, 2008. First published February 6, 2008; doi:10.1152/ajprenal.00373.2007
0363-6127/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/F881    most recent
00373.2007v1
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 ISI 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 Google Scholar
Google Scholar
Right arrow Articles by Cammisotto, P. G.
Right arrow Articles by Bendayan, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cammisotto, P. G.
Right arrow Articles by Bendayan, M.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats

Philippe G. Cammisotto, Irene Londono, Diane Gingras, and Moïse Bendayan

Department of Pathology and Cell Biology, University of Montreal, Montreal, Quebec, Canada

Submitted 8 August 2007 ; accepted in final form 31 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits {alpha}1 and {alpha}2, their phosphorylated active forms, and the glycogen-binding AMPK subunit β2. ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPK{alpha}1, AMPK{alpha}2, and AMPKβ2 were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits {alpha}1 and {alpha}2, their phosphorylated active forms, and the glycogen-binding subunit β2 were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 µg/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

adiponectin receptors; AMP-activated protein kinase; diabetes

GLYCOGEN NEPHROSIS (also known as Armanni-Ebstein lesions) is a feature of type 1 diabetes in human and rodents (1, 34, 49). In hyperglycemic conditions, cells of the distal tubule and thick ascending limb (TAL) absorb high amounts of glucose that accumulate into glycogen deposits (1, 3, 17, 34). Large glycogen accumulations disrupt cell function and alter expression and distribution of integrins with loss of basal infoldings and apical microvilli. Cells eventually undergo caspase-mediated apoptosis and detach (1, 26).

AMP-activated protein kinase (AMPK) is a master metabolic and antiapoptotic coordinator (33, 35, 41, 53). It is a heterotrimeric complex formed by a catalytic ({alpha}) and two regulatory (β and {gamma}) subunits (44). Catalytic AMPK subunits are activated by phosphorylation within a conserved sequence common to both isoforms {alpha}1 and {alpha}2 (9, 15). Once activated, they shutdown metabolic pathways consuming ATP (fatty acid and glycogen synthesis) and upregulate ATP-generating pathways (glycolysis and β-oxidation), therefore restoring energy levels (4, 5, 6). Inhibition of glycogen synthesis occurs through phosphorylation of glycogen synthase (GS) after activation of catalytic AMPK (11). The AMPK β-subunit, which possesses a glycogen binding site, is required for such regulation (32).

Adiponectin (also known as ACRP30 or adipoQ) is a major activator of AMPK. Its role in the control of glycogen synthesis in skeletal muscle and liver (4, 7, 8, 50) and in the regulation of inflammation and cellular apoptosis (33, 41) has been extensively studied. It is secreted essentially by white adipocytes (18, 39) and circulates in blood under various polymeric forms, ranging from globular (trimer) to high-molecular-weight structures (HMW) (13, 28). Globular adiponectin has a high affinity for receptor ADIPOR1, which activates AMPK, whereas HMW forms preferentially bind receptor ADIPOR2, leading to increased activity of peroxisome proliferator-activated receptor (PPAR)-{alpha} signaling pathways (51, 52).

Numerous correlation analyses carried out on patients with diabetic type 1 nephropathies revealed a link between renal tubular injuries and increases in plasma adiponectin levels (24, 37, 38). These increases have been considered as an attempt to limit renal microvascular damages and inflammatory changes in kidney (24, 38). Adiponectin receptors ADIPOR1 and ADIPOR2 mRNA as well as AMPK mRNA are expressed in rat and human renal tissues (44, 52); however, their localization and roles in renal physiology have been scarcely investigated. The aim of the present study was to assess whether adiponectin receptors and AMPK subunits are present in distal tubules and TAL and whether they participate in the control of glycogen metabolism. We found that adiponectin receptors ADIPOR1 and AMPK are located in these structures. In control animals, globular adiponectin potently activated AMPK, leading to the inhibition of GS. In hyperglycemic conditions, ADIPOR1 and AMPK signaling was altered, as was their regulation of GS, which may explain the large accumulations of glycogen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. The following antibodies were purchased: anti- ADIPOR1 (Affinity BioReagents, Golden, CO), anti-ADIPOR2 (Alpha Diagnostic, San Antonio, TX), anti-AMPKβ2 (H75) and anti-leptin receptor (K20) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-AMPK{alpha} (Thr172), anti-GS, and anti-phospho-GS (Ser641) (Cell Signaling, Boston, MA), anti-Tamm-Horsfall antigen (Cedarlane, Hornby, ON, Canada), and anti-AMPK{alpha}1 and anti-AMPK{alpha}2 (Bethyl Laboratory, Montgomery, TX). Secondary antibody to rabbit IgG (FITC labeled) was purchased from Chemicon International (Temecula, CA). The immunoperoxidase kit was obtained from Zymed Laboratories (San Francisco, CA). Collagenase type II, Ficoll type 400, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), streptozotocin, glucose-6-phosphate, β-NADP sodium salt hydrate, glucose-6-phosphate dehydrogenase (type XV), and anti-β-actin antibody (clone AC15) were obtained from Sigma-Aldrich (St. Louis, MO). Rat globular adiponectin was obtained from Biovision (Mountain View, CA).

Animals. Sprague-Dawley male rats (100 g) were obtained from Charles River (St-Constant, QC, Canada) and handled following the guidelines of the Canadian Council on Animal Care. After a single intraperitoneal injection with streptozotocin (70 mg/Kg), they were kept for 3 mo in individual cages with 12:12 h light-dark cycles with free access to water and food. We previously determined that 3-mo diabetes constitutes the best time frame for the occurrence of glycogen-filled-Clear cells in the kidney (1). Animals became hyperglycemic 48 hrs after streptozotocin injection and remained hyperglycemic thereafter. No insulin treatment was required. Urine samples were collected and analyzed weekly for the presence of glucose, proteins, ketone bodies, blood and for pH using Multistix sticks (Multistix, Bayer/Ames, Berlex Canada, Pointe-Claire, Quebec, Canada). Fasting blood glucose levels were monitored monthly using an Accusoft monitoring system with Accu-Check Advantage sticks (Roche Diagnostics, Laval, Quebec, Canada). Animals showed strong glycosuria, proteinuria, and ketonuria throughout the experiment. The mean body mass of diabetic rats after 3 mo of diabetes was 386 ± 13 g with fasting blood glucose levels of 30.8 ± 1.4 mM. Nondiabetic control animals had a mean body mass of 423 ± 8 g with fasting blood glucose levels of 5.6 ± 0.3 mM. On the day of the experiment, animals were anesthetized by intraperitoneal injection of urethane (1 mg/kg). Kidneys were quickly removed and processed for immunohistochemistry and biochemistry. Blood was sampled from dorsal aorta. After centrifugation, serum was frozen and kept at –20°C until use. All protocols were approved by the Comite de Deontologie de l'Experimentation Sur les animaux de l'Universite de Montreal.

Light microscopy. Immunohistochemistry was carried out to reveal adiponectin receptors and AMPK subunits in renal tubules. Tissue samples were fixed by immersion in Bouin's fixative and embedded in paraffin. Sections 5 µm thick were mounted on Superfrost slides (Fisher Scientific, Montreal, QC, Canada). For fluorescence microscopy, deparaffinized tissue sections were washed twice in PBS (10 mM phosphate buffer, 150 mM NaCl, pH 7.4) and incubated for 5 min with 1 mg/ml pepsin in 0.01 M HCl. After being washed with PBS, they were covered with 1% ovalbumin for 20 min. Incubation with the primary antibodies (anti-AMPK{alpha}1, 1:10; anti-AMPK{alpha}2, 1;10; anti-ADIPOR1, 1:20; anti-ADIPOR2, 1:10) was carried out overnight at 4°C in a humidified chamber. The following morning, sections were washed twice with PBS and incubated with the secondary antibody for 1 h, washed three times in PBS, contrasted with Evan's blue, and mounted with 1% 1,4-diazabicyclo[2.2.2]octane (DABCO) in 50% glycerol. The tissue sections were examined with a Leitz DMRB fluorescence microscope (Leica, St-Laurent, QC, Canada). For peroxidase immunostaining, after deparaffinization, tissue sections were treated with methanol (90%) containing 3% H2O2 for 10 min to remove endogenous peroxidase activity. After being washed, tissue sections were digested with pepsin for 5 min (1 mg/ml in 0.01 M HCl), washed, and then incubated overnight at 4°C with the primary antibody (anti-AMPKβ2, 1:10; anti-phosphorylated AMPK, 1:10). The following day, they were washed with PBS and incubated with a biotinylated anti-rabbit IgG for 1 h at room temperature according to the manufacturer's instructions (Zymed). After three washings with PBS, the avidin-peroxidase complex was added. Tissue sections were washed with PBS, and the 3,3'-diaminobenzidine (DAB) substrate 0.05% in DAB buffer was added. Reaction was stopped with water after a significant staining was detected by light microscopy. Controls of specificity for both techniques were carried out by omitting the primary antibodies in the protocol.

In vitro incubation of isolated tubules. To examine the in vitro activation of renal tubule AMPK by adiponectin and AICAR, we prepared isolated distal tubules and TAL following techniques slightly modified from those previously described (10, 26). Briefly, kidneys from anesthetized rats were removed and washed in a Krebs-Henseleit bicarbonate buffer (KHB) of the following composition: 118 mM NaCl, 4.7 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 1.9 mM CaCl2, 25 mM NaHCO3, and 5.5 mM glucose, pH 7.4, supplemented with 10 mM glutamine, 1 mM glutamic acid, 1 mM pyruvate, and 10 mM lactate. KHB was bubbled with 95% O2-5% CO2 before use. Kidneys were then sliced using a Thomas tissue slicer (Thomas Scientific, Swedesboro, NJ) and incubated in fresh KHB medium containing 0.5 mg/ml collagenase at 37°C for 50 min in a shaking bath. At the end of the incubation, the suspension was passed through a 250-µm nylon filter to remove nondigested material. Tubules were washed four times by centrifugation in KHB (800 g for 4 min at room temperature) to remove collagenase. They were then carefully dropped on a 2.6–30% Ficoll gradient and centrifuged at 800 g for 40 min at 20°C. Four phases were obtained. The bottom phase containing distal tubules and TAL was carefully recovered and washed twice in KHB to remove Ficoll.

Samples of isolated tubules were processed for morphological examination by light and electron microscopy and for dot-blot analysis using an antibody specific for the Tamm-Horsfall antigen, a marker for distal tubules and TAL (43). Isolated tubules were incubated at 37°C for 40 min in the presence or absence of globular adiponectin (10 µg/ml final) or AICAR (2 mM) in fresh KHB (4). At the end of the incubation, homogenization buffer RIPA [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholate, 1 mM sodium orthovanadate, and 1% Nonidet P-40 containing protease inhibitor cocktail Complete Mini (Roche Diagnostics)] was added volume/volume to the tubule suspension. After 1 h at 4°C, samples were sonicated and centrifuged at 10,000 g for 3 min to remove insoluble material, and the supernatants were frozen and kept at –80°C until use.

Measurement of glucose-6-phosphate. An enzymatic method was used (27). Incubation medium consisted of 10 mM MgCl2, 250 µM NADP, and 1 U/ml glucose-6-phosphate dehydrogenase in 100 mM Tris, pH 7.5. Samples of tubule homogenates were incubated for 30 min at 37°C in medium, and then absorbance was read at 340 nm. Results were reported to a standard curve of glucose-6-phosphate performed under the same conditions.

Western blot. Protein content was measured using a Non-Interfering Protein Assay (BioLynx, Brockville, ON, Canada). For SDS-PAGE, samples (20 µg of proteins) were heated at 95°C for 5 min in Laemmli buffer before being loaded on a 7 or 10% SDS-polyacrylamide gel, depending on the protein. Transfer to nitrocellulose membrane was performed at 4°C in the presence of methanol. Incubation with antibodies (anti-AMPK{alpha}1, 1:1,000; anti-AMPK{alpha}2, 1:1,000; anti-AMPKβ2, 1:200; ADIPOR1, 1:200; ADIPOR2, 1:50; GS, 1:200; phospho-GS, 1:100) was carried out in TBST (10 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 1% nonfat dry milk. For phosphorylated protein [phospho-AMPK{alpha} (Thr172), 1:100; and phospho-GS (Ser641), 1:100], milk was replaced by 5% bovine serum albumin and 5 mM fluoride sodium. In all cases, actin was revealed using the anti-actin antibody (1:20,000) and used as a loading control. The Lumi-light Plus kit (Roche Diagnostics) was used to reveal proteins. Intensity of bands was evaluated using Scion Image software (Scion, Frederick, MD). Densities were measured and normalized to β-actin. Data were analyzed, and statistics (Student's t-test) were analyzed using Microsoft Excel software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adiponectin receptors. Distal tubules and TAL are particularly targeted in diabetic tubulopathy due to the accumulation of intracellular glycogen (1,3, 17, 34). Adiponectin, through its receptors, is a major regulator of glycogen synthesis (7, 8, 50). To establish the presence of adiponectin receptors in distal tubules and TAL, we carried out Western blotting and immunohistochemistry on isolated distal tubules and TAL homogenates and on sections of renal tissue, respectively. Enrichment and purity of the distal tubule suspension was confirmed by morphological examination and by dot-blot analysis, using the antibody against the Tamm-Horsfall antigen (results not shown) (43). Electron microscopy further confirmed the typical morphology of the isolated distal tubules and TAL in the preparation (results not shown). Homogenates of isolated tubules from control and diabetic animals were resolved on a 10% acrylamide gel as described in MATERIALS AND METHODS. ADIPOR1 was revealed as a single band of 42 kDa in both groups of animals (Fig. 1A). ADIPOR2 was not found in renal tubules; however, it was present as a band of 34 kDa in a sample of rat liver tissue taken as a positive control (results not shown) (52). Quantitation of band densities showed that levels of ADIPOR1 were significantly higher in isolated tubules of diabetic rats (15.5 ± 3.10 as normalized to β-actin) compared with controls (0.86 ± 0.26 as normalized to β-actin) (Fig. 1A). Immunohistochemistry revealed that ADIPOR1 was localized to the apical portion of distal tubular and TAL cells in tissues of normal animals (Fig. 1, B and C). Plasma membranes at the base of the cells were devoid of staining. In tissues of diabetics animals, most of the tubules were altered with the appearance of Clear cells (1). These Clear cells display characteristic clear, empty cytoplasmic spaces left by the extraction of the large glycogen deposits. The Clear cells displayed strong stainings for ADIPOR1 at the level of the apical and basolateral regions as well as in the cytoplasm (Fig. 1C). A few tubules were not affected to the same degree with glycogen deposition, and their staining was not as intense. Although Yamauchi et al. (52) have reported, using molecular biology, the presence of ADIPOR2 in total renal homogenate, we were unable to detect ADIPOR2 in our isolated tubule preparations from either group. With immunocytochemistry, ADIPOR2 was only found in glomeruli and capillary endothelial cells, not in TAL and distal tubules (results not shown). Controls performed by omitting primary antibodies in the histochemical protocol displayed no staining (Fig. 1D).


Figure 1
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 1. Presence of adiponectin receptor ADIPOR1 in distal tubules and thick ascending limb (TAL) of control (CTL) and streptozotocin (STZ)-induced diabetic rats. ADIPOR1 is detected as a single band of 42 kDa in the isolated tubule preparation (A). Actin is shown as a loading control. Quantification of bands is expressed in reference to β-actin (means ± SE; n = 4) and indicates significant differences between tissues of control and diabetic animals (**P < 0.01). Immunofluorescence positive staining for ADIPOR1 is present at the luminal portion (arrow) of distal tubule and TAL cells of normal (B) and diabetic animals (C). In tissues of diabetic animals, Clear cells (arrowheads) are intensely stained (C). Omission of the primary antibody resulted in absence of staining (D). Bars, 50 µm (B and D) and 100 µm (C).

 
AMPK subunits {alpha}1 and {alpha}2. AMPK is the main second messenger of ADIPOR1. To assess its presence in distal tubules and TAL, we carried out detection of the catalytic AMPK subunits {alpha}1 and {alpha}2. Both isoforms were found in isolated tubule homogenates as bands of 64 kDa, as previously reported for total kidney homogenates (Fig. 2, A and D) (12). Tubules from diabetic animals displayed a much enhanced expression for both isoforms {alpha}1 (32.2 ± 6.75 as normalized to β-actin) and {alpha}2 (67.4 ± 12.4 as normalized to β-actin) compared with tubules of control animals (3.64 ± 1.3 and 19.1 ± 6.74 as normalized to β-actin, respectively) (Fig. 2, A and D). Light microscopy showed that AMPK{alpha}1 and AMPK{alpha}2 are present in distal tubules and TAL of control animals at the level of the apical membrane and cytoplasm (Fig. 2, B and E). In diabetic animals, Clear cells also displayed a positive staining for AMPK{alpha}1 and AMPK{alpha}2 in the peripheral rim of cytoplasm (Fig. 2, C and F). Intensities of the staining for AMPK{alpha}1 and AMPK{alpha}2 appeared rather similar in tissues from normal and diabetic animals. Controls without primary antibodies confirmed the specificity of the staining.


Figure 2
View larger version (142K):
[in this window]
[in a new window]

  		Fig. 2. Presence of catalytic AMP-activated protein kinase (AMPK) {alpha}1 (A)- and {alpha}2-subunits (D) in renal tissue. Both isoforms are found as bands of 64 kDa in tubules isolated from CTL (A) and STZ rats (D). Actin is shown as a loading control. Band intensities expressed in reference to actin (means ± SE; n = 4) reveal significant differences between tissues of control and diabetic animals (**P < 0.01). Immunofluorescence staining reveals AMPK{alpha}1 (B and C) and AMPK{alpha}2 (E and F) in the apical portion (arrows) of distal tubule and TAL cells from both normal (B and E) and diabetic animals (C and F). Clear cells in tubules (asterisks) of diabetic animals are stained for AMPK{alpha}1 (C) and AMPK{alpha}2 (F) in the apical (arrows) as well as basolateral plasma membranes (arrowheads). Bars, 50 µm.

 
Glycogen-binding AMPK subunit β2. The AMPK subunit β2 possesses a glycogen-binding site, which is involved in the control of glycogen synthesis (32). Western blots on isolated tubules confirmed the presence of this isoform at 34 kDa (Fig. 3A), with the β1-isoform not being expressed in renal tissue (12). Expression of the β2-subunit in tissues of diabetic animals was twice that of normal ones (35.9 ± 3.59 vs. 19.5 ± 4.10 as normalized to β-actin) (Fig. 3A). Immunohistochemistry revealed an abundant staining of distal tubule and TAL cells (Fig. 3B) as well as the cytoplasmic thin rim of the Clear cells (Fig. 3C). Controls without primary antibodies were devoid of staining.


Figure 3
View larger version (61K):
[in this window]
[in a new window]

  		Fig. 3. Presence of regulatory AMPK β2-subunit in renal tubules. This subunit of 34 kDa is present in tubules of CTL and STZ rats (A). Actin is shown as a loading control. Band intensities expressed in reference to actin (means ± SE; n = 4) reveal higher expression in tissues of diabetic rats (**P < 0.01). Immunoperoxidase staining reveals AMPKβ2 in the cytoplasm and apical membrane of distal tubules and TAL cells in tissues of normal (arrows) and diabetic animals (B). In tissues of diabetic animals, tubules with Clear cells (asterisks) display staining at the cell surface and basolateral membranes (arrowheads, C). Controls performed by omitting the anti-AMPKβ2 antibody step resulted in absence of staining (D). PT, proximal tubule; DT, distal tubule; G, glomerulus. Bars, 50 µm.

 
Phosphorylated AMPK. Catalytic AMPK{alpha}1 and AMPK{alpha}2 are activated by phosphorylation of Thr172 in a conserved sequence identical for both isoforms (15). Levels of phosphorylated AMPK were lower in tissues from diabetic animals (2.04 ± 0.71 as normalized to β-actin) compared with those from control animals (12.0 ± 2.43 as normalized to β-actin) (Fig. 4A), demonstrating lower activity of AMPK in the hyperglycemic condition. If we report these levels of phospho-AMPK{alpha} to total AMPK (Fig. 2), the decrease in phospho-AMPK{alpha} becomes even more dramatic (5.28 ± 1.02 for control vs. 0.205 ± 0.068 for diabetic animals). Immunostaining was strong in the cytoplasm of distal tubules and TAL cells of normal animal tissues (Fig. 4B), whereas that of Clear cells present in tissues of diabetic animals was markedly reduced (Fig. 4C).


Figure 4
View larger version (61K):
[in this window]
[in a new window]

  		Fig. 4. Phosphorylated activated AMPK (Pi-AMPK{alpha}) in renal tubules (A). Actin is shown as a loading control. Band intensities expressed in reference to actin (means ± SE; n = 4) reveal significant differences between tissues of CTL and STZ animals (**P < 0.01). Immunoperoxidase staining for phosphorylated AMPK is present in the cytoplasm and apical membrane of distal tubule and TAL cells in control animals (arrows, B). Staining is weak or even absent in Clear cells (asterisks) of diabetic animals (C). Bars, 50 µm.

 
In vitro activation of AMPK by AICAR and globular adiponectin. We subsequently assessed whether globular adiponectin and AICAR activate catalytic AMPK in isolated distal tubules and TAL. Globular adiponectin is the high-affinity ligand for ADIPOR1 (52). AICAR is an adenosine analog that is taken up into cells through adenosine transporters and is then phosphorylated intracellularly by adenosine kinase to form ZMP, which is an AMP analog. This ZMP allosterically increases AMPK activity independently from plasma membrane receptors (45) and reduces its rate of dephosphorylation (46). In vitro incubations were carried out in parallel on renal tubules isolated from control and diabetic animals. Total amounts of AMPK{alpha}1 and AMPK{alpha}2 were not affected by AICAR or adiponectin in our conditions for both groups (Fig. 5). Western blots revealed that AICAR (2 mM) and globular adiponectin (10 µg/ml) potently stimulated phosphorylation of the catalytic AMPK subunits in tubules of control animals (basal: 24.1 ± 5.37; AICAR, 66.9 ± 12.2; and adiponectin, 76.1 ± 8.90; as normalized to total catalytic AMPK) (Fig. 5). In tubules of diabetic animals, levels of phosphorylated AMPK, both basal and stimulated, were much lower (basal, 0.724 ± 0.167; AICAR, 1.38 ± 0.137; and adiponectin, 1.55 ± 0.285; as normalized to total catalytic AMPK) (Fig. 5), demonstrating significant decreases in activities and stimulation of catalytic AMPK in hyperglycemic conditions despite the higher pool of catalytic AMPK. Indeed, levels of basal AMPK in diabetic conditions are ~30 times lower than those in control conditions.


Figure 5
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 5. In vitro phosphorylation of catalytic AMPK by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and globular adiponectin in isolated renal tubules from CTL and STZ rats. Activation of AMPK (Pi-AMPK{alpha}) by AICAR (2 mM) or globular adiponectin (AdipoN; 10 µg/ml) was revealed by Western blot and quantified. Results are expressed in reference to total catalytic AMPK (means ± SE; n = 4). For both groups of animals, Pi-AMPK levels increased significantly in the presence of AICAR and adiponectin (**P < 0.01). Levels of basal AMPK relative to Pi-AMPK were drastically reduced in tissues of diabetic animals.

 
Inhibition of GS by globular adiponectin and AICAR. Once activated, catalytic AMPK activates glycogen synthase kinase-3 (GSK-3), which in turn phosphorylates GS on Ser641 (11). To determine whether the low activity of catalytic AMPK in renal tubules of diabetic animals leads to a decrease of inhibited GS, we incubated tubules isolated from control and diabetic rats with AICAR and globular adiponectin. Western blots revealed that the pool of glycogen synthase (phosphorylated and nonphosphorylated forms) was three times as high in tubules isolated from diabetic rats (21.5 ± 4.63 as normalized to β-actin)(+ 203%) as in tubules from control rats (7.1 ± 2.93 as normalized to β-actin). Phosphorylated inactive GS was also found to be increased in tissues from diabetic animals (8.20 ± 1.41 vs. 5.11 ± 1.08 as normalized to β-actin; + 96%) (Fig. 6). Nevertheless, the ratio of inactive GS to total GS was much lower in the diabetic group (Fig. 7). When incubated with AICAR (2 mM) and globular adiponectin (10 µg/ml), tubules isolated from control rats displayed a potent increase in GS phosphorylation (basal, 18.9 ± 4.47; AICAR, 95.2 ± 26.7; and adiponectin, 162.1 ± 33.9; as normalized to total GS) (Fig. 7). The phosphorylation of GS was much weaker in tubules from diabetic animals than for controls (basal, 12 ± 2.3; AICAR, 21.7 ± 3.67; and adiponectin, 22.9 ± 3.06; as normalized to total GS). Total GS levels were not affected by the incubation conditions (results not shown). These results demonstrate that functional GS (expressed as the ratio of phosphorylated to total GS) is higher in diabetic condition, with regulation by AMPK severely impaired. Finally, glucose-6-phosphate, the allosteric activator of GS (31), was also found in higher concentrations in tubules from diabetic rats compared with controls (88.5 ± 24 vs. 154 ± 31.6 nmol/mg protein) (Fig. 8). Together, these results demonstrate that GS activity is higher in tubules of diabetic rats and that its inhibition by AMPK is impaired. All this contributes to the accumulation of glycogen in renal Clear cells of diabetic animals.


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. Measurements of total glycogen synthase (GS) pool (active and inactive forms) (A) and its inactive phosphorylated form (Pi-GS; B) in isolated tubules. GS appeared on Western blots as a single band of 84 kDa. Actin is shown as a loading control. Levels of expression for GS and Pi-GS (means ± SE; n = 4) were increased in tissues of STZ compared with CTL animals (**P < 0.01).

 

Figure 7
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 7. Inactivation of GS by AICAR and globular adiponectin. Isolated tubules from CTL and STZ rats were incubated with AICAR (2 mM) or globular AdipoN (10 µg/ml). Levels of total GS and Pi-GS were quantified, and results expressed as the ratio of Pi-GS (inactive) to total GS pool (active and inactive) (means ± SE; n = 4) indicate significant increases compared with basal levels (**P < 0.01).

 

Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 8. Concentrations of glucose-6-phosphate in isolated tubules. Amounts (nmol/mg protein) of the allosteric activator of GS (means ± SE; n = 4) were higher in tubules from STZ rats (*P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Control of GS by adiponectin and AMPK in skeletal muscles and liver is well established (11, 32). We presently report that a similar situation occurs in renal distal tubules, where globular adiponectin through ADIPOR1 activates AMPK, leading to the inhibition of GS. This control is impaired in diabetes, indicating that it might contribute to glycogen nephrosis.

As shown by immunohistochemistry and Western blotting, ADIPOR1 and the {alpha}1-, {alpha}2-, and β2-subunits of AMPK were localized to distal tubules and TAL. ADIPOR1 was present on the luminal membrane of tubular cells, but not on the basolateral membrane. ADIPOR2 was not detected in distal tubules and TAL. On the other hand, we confirmed that leptin receptors, which are also potent activators of AMPK, were only present in the renal medulla (results not shown) (40) and not in distal tubules and TAL. The expression of T-cadherin, another protein that binds the HMW isoform of adiponectin, is limited to the cardiovascular and nervous systems and is absent in kidney (19). Therefore, our results suggest that low-molecular-weight globular adiponectin originating from glomerular filtration is the major activator of AMPK through luminal ADIPOR1 receptors in distal tubules and TAL. In accordance, we did find globular adiponectin in samples of rat urine (results not shown); other groups have also reported its presence in urine from healthy and diabetic patients (24, 42). The absence of adiponectin receptors on the basolateral plasma membrane of distal tubule and TAL cells of normal animals suggests that circulating adiponectin does not participate in AMPK activation in distal tubule cells. In muscle cells, ADIPOR1 is more important than ADIPOR2 in the regulation of glucose metabolism (21, 51), and our results seem to favor similar mechanisms in kidney distal tubules.

ADIPOR1 and AMPK subunits display similar distributions in tissues of control and diabetic animals. However, ADIPOR1, AMPK{alpha}1, and AMPK{alpha}2 expression is greatly enhanced in tubules from diabetic animals, whereas phosphorylated AMPK levels are decreased. Insulin deficiency is known to increase expression of adiponectin receptor in muscle, whereas hyperinsulinemia decreases it (21, 47). Increases in AMPK{alpha}1 and AMPK{alpha}2 expression in kidney homogenates from streptozotocin-treated mice have also been reported (25, 48). Mechanisms similar to those reported for adiponectin receptor may explain these enhances (21, 47). Along the same line, insulin has been shown to modulate AMPK activity, and its absence in diabetic condition may interfere with catalytic AMPK synthesis (25, 48). It is also well known that prolonged hyperglycemia does affect cell signaling and gene expression (22, 25). Moreover, these increases in ADIPOR1 and catalytic AMPK may be an attempt by cells to compensate for the development of adiponectin resistance (4, 47). Finally, decreased levels of activated-AMPK{alpha} have been reported in diabetes-induced renal hypertrophy (25) and is similar to the adiponectin resistance present in other tissues (4).

In vitro incubation of isolated distal tubules and TAL with globular adiponectin and AICAR potently stimulates phosphorylation of catalytic AMPK in a way similar to other tissues (11, 41, 45). Interestingly, in tubules from diabetic animals, basal and stimulated levels of active AMPK{alpha} were six times lower than controls, despite the strong increase in expression of total catalytic AMPK {alpha}1- and {alpha}2-subunits. Several hypotheses may explain this decrease in AMPK activation. First, tubules made of glycogen-filled Clear cells may be unresponsive to adiponectin and AICAR stimulation; the small increase that we observed may just arise from those tubular cells not yet loaded with glycogen but still present in the tubules. Second, glucose may stimulate AMPK dephosphorylation, as proposed by Lee et al. (25). High concentrations of glucose have been shown to activate the phosphatase PP2A, which dephosphorylates AMPK in pancreatic β-cells and other tissues (9, 22). In addition, PP2A prevents association between AMPK subunits (14). Third, in the diabetic condition, the large volume of intracellular glycogen may cause the sequestration of AMPK, making it unavailable for its receptors (32). Indeed, AMPK β2-subunit possesses a glycogen-binding site, which could render the trimeric AMPK unavailable for activation by receptors (12, 32, 50). These hypotheses do not rule out each other and do not rule out other mechanisms interfering with ADIPOR1 signaling, which includes SOCS proteins (suppressor of cytokine signaling 3), the phosphatase PTP-1B (phospho-tyrosine phosphatase 1B), or an inhibition of LKB-1, the upstream kinase of AMPK that controls Thr172 phosphorylation (23, 46). Finally, it was recently reported that ketoacidosis influences AMPK and decreases its activity in heart muscle (30). Our animals do present strong ketonuria; however, interestingly, Zucker fa/fa rats, which are hyperglycemic without ketoacidosis (29), present numerous renal tubular Clear cells as well as decreased phosphorylated AMPK levels similar to those observed in our streptozotocin-treated rats. Further studies are needed to determine the exact mechanism responsible for the disruption of AMPK activity.

Once activated, AMPK leads to the phosphorylation of GS, resulting in its inactivation (11). In isolated tubules, expression of total GS is three times higher in tissues of diabetic animals than in those of control ones. On the other hand, the inactive phosphorylated form is twofold higher in tubules of diabetic animals. Yet, the ratio of inactive to total GS was lower in diabetic animals, which indicates greater GS activity. In vitro, adiponectin and AICAR potently stimulated phosphorylation of GS in tubules from controls, whereas they only weakly did so in those from diabetic animals, probably reflecting the activity of catalytic AMPK in each respective group. To be emphasized is the fact that glycogen phosphorylase kinase, a key enzyme for the breakdown of glycogen, is not controlled by AMPK (2).

Other mechanisms regulating GS activity must be taken into account. Concentrations of the allosteric activator of GS (31), the glucose-6-phosphate, are higher in tubules from diabetic animals and may contribute to increase GS activity. Moreover, luminal glucose is absorbed through the apical membrane of distal and TAL cells by glucose/sodium transporters and pumped toward the basal extracellular space via GLUT 1 and GLUT 4 (16). AMPK regulates the translocation of glucose transporters (GLUT family) from the cytosol to the membrane in muscle (4, 42); the low AMPK activity in distal tubules of diabetic rats may thus contribute to the decreased translocation of GLUT transporters to the basal membrane, leading to glucose accumulation inside cells and stimulation of glycogen synthesis (16).

In summary, we have demonstrated that globular adiponectin, through the luminal receptor ADIPOR1, potently activates catalytic AMPK in renal distal tubule and TAL cells, similarly to situations in other tissues. In diabetes, this activation is reduced despite an increase in the expression of catalytic AMPK subunits. Given that AMPK blocks glycogen synthesis, its depressed activity may explain, at least in part, the accumulation of glycogen and subsequent apoptosis observed in renal tubular cells in hyperglycemic conditions. However, we should keep in mind that regulation of AMPK and GS activities are complex, and other intracellular pathways and factors may also be involved.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Canadian Institutes of Health Research, the Fonds de la Recherche en Santé du Québec, and Diabète-Québec.


   ACKNOWLEDGMENTS
 
We thank Elizabeth Gervais for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Bendayan, Département de Pathologie et Biologie Cellulaire, Université de Montréal, 2900 Edouard-Montpetit, Montréal, Québec, Canada H3T 1J4 (e-mail: moise.bendayan{at}umontreal.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bamri-Ezzine S, Ao ZJ, Londono I, Gingras D, Bendayan M. Apoptosis of tubular epithelial cells in glycogen nephrosis during diabetes. Lab Invest 83: 1069–1080, 2003.[CrossRef][Web of Science][Medline]
  2. Beyer A, Kitzerow A, Crute B, Kemp BE, Witters LA, Heilmeyer LM. Muscle phosphorylase kinase is not a substrate of AMP-activated protein kinase. Biol Chem 381: 457–461, 2000.[CrossRef][Web of Science][Medline]
  3. Biava C, Grossman A, West M. Ultrastructural observations on renal glycogen in normal and pathologic human kidneys. Lab Invest 15: 300–356, 1966.
  4. Bruce CR, Mertz VA, Heigenhauser GJ, Dyck DJ. The stimulatory effect of globular adiponectin on insulin-stimulated glucose uptake and fatty acid oxidation is impaired in skeletal muscle from obese subjects. Diabetes 54: 3154–3160, 2005.[Abstract/Free Full Text]
  5. Carling D, Clarke PR, Zammit VA, Hardie DG. Purification and characterization of the AMP-activated protein kinase. Eur J Biochem 186: 129–136, 1989.[Web of Science][Medline]
  6. Carling D, Zammit VA, Hardie DG. A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett 223: 217–222, 1987.[CrossRef][Web of Science][Medline]
  7. Ceddia RB, Somwar R, Maida A, Fang X, Bikopoulos G, Sweeney G. Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells. Diabetologia 48: 132–139, 2005.[CrossRef][Web of Science][Medline]
  8. Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L. Endogenous glucose production is inhibited by the adipose-derived protein Acrp30. J Clin Invest 108: 1875–1881, 2001.[CrossRef][Web of Science][Medline]
  9. Da Silva XG, Leclerc I, Salt IP, Doiron B, Hardie DG, Kahn A, Rutter GA. Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression. Proc Natl Acad Sci USA 97: 4023–4028, 2000.[Abstract/Free Full Text]
  10. Eveloff J, Haase W, Kinne R. Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop. J Cell Biol 87: 672–681, 1980.[Abstract/Free Full Text]
  11. Fediuc S, Gaidhu MP, Ceddia RB. Inhibition of insulin-stimulated glycogen synthesis by AICAR-induced AMPK activation: interactions with Akt, GSK-3{alpha}/β, and glycogen synthase in isolated rat soleus muscle. Endocrinology 147: 5170–5177, 2006.[Abstract/Free Full Text]
  12. Fraser S, Mount P, Hill R, Levidiotis V, Katsis F, Stapleton D, Kemp BE, Power DA. Regulation of the energy sensor AMP-activated protein kinase in the kidney by dietary salt intake and osmolality. Am J Physiol Renal Physiol 288: F578–F586, 2005.[Abstract/Free Full Text]
  13. Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98: 2005–2010, 2001.[Abstract/Free Full Text]
  14. Gimeno-Alcaniz JV, Sanz P. Glucose and type 2A protein phosphatase regulate the interaction between catalytic and regulatory subunits of AMP-activated protein kinase. J Mol Biol 233: 201–209, 2003.
  15. Hawley SA, Davison MD, Woods A, Davies SP, Beri RK, Carling DG, Hardie D. Characterization of the AMP-activated protein kinase kinase from rat liver and identification threonine-172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271: 27879–27887, 1996.[Abstract/Free Full Text]
  16. Heilig C, Zaloga C, Lee M, Zhao X, Riser B, Brosius F, Cortes P. Immunogold localization of high-affinity glucose transporter isoforms in normal rat kidney. Lab Invest 73: 674–684, 1995.[Web of Science][Medline]
  17. Holck P, Rasch R. Structure and segmental localization of glycogen in the diabetic rat kidney. Diabetes 42: 891–900, 1993.[Abstract]
  18. Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271: 10697–10703, 1996.[Abstract/Free Full Text]
  19. Hug C, Wang J, Ahmad NS, Bogan JS, Tsao TS, Lodish HF. T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin. Proc Natl Acad Sci USA 101: 10308–10313, 2004.[Abstract/Free Full Text]
  20. Ido Y, Carling D, Ruderman N. Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51: 159–167, 2002.[Abstract/Free Full Text]
  21. Inukai K, Nakashima Y, Watanabe M, Takata N, Sawa T, Kurihara S, Awata T, Katayama S. Regulation of adiponectin receptor gene expression in diabetic mice. Am J Physiol Endocrinol Metab 288: E876–E882, 2005.[Abstract/Free Full Text]
  22. Jiang R, Carlson M. Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev 10: 3105–3115, 1996.[Abstract/Free Full Text]
  23. Kaszubska W, Falls HD, Schaefer VG, Haasch D, Frost L, Hessler P, Kroeger PE, White DW, Jirousek MR, Trevillyan JM. Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line. Mol Cell Endocrinol 195: 109–118, 2002.[CrossRef][Web of Science][Medline]
  24. Koshimura J, Fujita H, Narita T, Shimotomai T, Hosoba M, Yoshioka N, Kakei M, Fujishima H, Ito S. Urinary adiponectin excretion is increased in patients with overt diabetic nephropathy. Biochem Biophys Res Commun 316: 165–169, 2004.[CrossRef][Web of Science][Medline]
  25. Lee MJ, Feliers D, Mariappan MM, Sataranatarajan K, Mahimainathan L, Musi N, Foretz M, Viollet B, Weinberg JM, Choudhury GG, Kasinath BS. A role for AMP activated protein kinase in diabetes-induced-renal hypertrophy. Am J Physiol Renal Physiol 292: F617–F627, 2007.[Abstract/Free Full Text]
  26. Londoño I, Bamri-Ezzine S, Gingras D, Bendayan M. Redistribution of integrins in tubular epithelial cells during diabetic glycogen nephrosis. Nephron Exp Nephrol 98: e22–e30, 2004.[CrossRef][Medline]
  27. Lowry OH, Passoneau JV. A Flexible System of Enzymatic Analysis. Orlando, FL: Academic, 1972, p. 71–77, 179–182.
  28. Nakano Y, Tajima S, Yoshimi A, Akiyama H, Tsushima M, Tanioka T, Negoro T, Tomita M, Tobe T. A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin. J Lipid Res 47: 1572–1582, 2006.[Abstract/Free Full Text]
  29. Nosadini R, Ursini F, Tessari P, Garotti MC, de Biasi F, Tiengo A. Hormonal and metabolic characteristics of genetically obese Zucker and dietary obese Sprague-Dawley rats. Eur J Clin Invest 10: 113–118, 1980.[CrossRef][Web of Science][Medline]
  30. Pelletier A, Coderre L. Ketone bodies alter dinitrophenol-induced glucose uptake through AMPK inhibition and oxidative stress generation in adult cardiomyocytes. Am J Physiol Endocrinol Metab 292: E1325–E1332, 2007.[Abstract/Free Full Text]
  31. Piras R, Staneloni R. In vivo regulation of rat muscle glycogen synthetase activity. Biochemistry 8: 2153–2160, 1969.[CrossRef][Web of Science][Medline]
  32. Polekhina G, Gupta A, Michell BJ, van Denderen B, Murthy S, Feil SC, Jennings IG, Campbell DJ, Witters LA, Parker MW, Kemp BE, Stapleton D. AMPK β subunit targets metabolic stress sensing to glycogen. Curr Biol 13: 867–871, 2003.[CrossRef][Web of Science][Medline]
  33. Rakatzi I, Mueller H, Ritzeler O, Tennagels N, Eckel J. Adiponectin counteracts cytokine- and fatty acid-induced apoptosis in the pancreatic beta-cell line INS-1. Diabetologia 47: 249–258, 2004.[CrossRef][Web of Science][Medline]
  34. Rasch R. Tubular lesions in streptozotocin-diabetic rats. Diabetologia 27: 32–37, 1984.[Web of Science][Medline]
  35. Ruderman NB, Saha AK, Kraegen EW. Minireview: malonyl CoA, AMP-activated protein kinase, and adiposity. Endocrinology 144: 5166–5171, 2003.[Abstract/Free Full Text]
  36. Russell RR 3rd, Bergeron R, Shulman GI, 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]
  37. Saraheimo M, Forsblom C, Fagerudd J, Teppo AM, Pettersson-Fernholm K, Frystyk J, Flyvbjerg A, Groop PH: FinnDiane study group. Serum adiponectin is increased in type 1 diabetic patients with nephropathy. Diabetes Care 28: 1410–1414, 2005.[Abstract/Free Full Text]
  38. Schalkwijk CG, Chaturvedi N, Schram MT, Fuller JH, Stehouwer CD: EURODIAB Prospective Complications Study Group. Adiponectin is inversely associated with renal function in type 1 diabetic patients. J Clin Endocrinol Metab 91: 129–135, 2006.[Abstract/Free Full Text]
  39. Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF. A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270: 26746–26749, 1995.[Abstract/Free Full Text]
  40. Serradeil-Le Gal C, Raufaste D, Brossard G, Pouzet B, Marty E, Maffrand JP, Le Fur G. Characterization and localization of leptin receptors in the rat kidney. FEBS Lett 404: 185–191, 1997.[CrossRef][Web of Science][Medline]
  41. Shibata R, Sato K, Pimentel DR, Takemura Y, Kihara S, Ohashi K, Funahashi T, Ouchi N, Walsh K. Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms. Nat Med 11: 1096–1103, 2005.[CrossRef][Web of Science][Medline]
  42. Shimotomai T, Kakei M, Narita T, Koshimura J, Hosoba M, Kato M, Komatsuda A, Ito S. Enhanced urinary adiponectin excretion in IgA-nephropathy patients with proteinuria. Ren Fail 27: 323–328, 2005.[CrossRef][Web of Science][Medline]
  43. Sikri KL, Alexander DP, Foster CL. Localization of Tamm-Horsfall glycoprotein in the normal rat kidney and the effect of adrenalectomy on its localization in the hamster and rat kidney. J Anat 135: 29–45, 1982.[Web of Science][Medline]
  44. Stapleton D, Michellhill KI, Gao G, Wilmer J, Michell BJ, The T, House CM, Fernandez CS, Cox T, Witters LA, Kemp BE. Mammalian AMP-activated protein kinase subfamily. J Biol Chem 271: 611–614, 1996.[Abstract/Free Full Text]
  45. Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, Beri RK. Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase. FEBS Lett 353: 33–36, 1994.[CrossRef][Web of Science][Medline]
  46. Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D. Dissecting the role of 5'-AMP for allosteric stimulation, activation and deactivation of AMP-activated protein kinase. J Biol Chem 281: 32207–32216, 2006.[Abstract/Free Full Text]
  47. Tsuchida A, Yamauchi T, Ito Y, Hada Y, Maki T, Takekawa S, Kamon J, Kobayashi M, Suzuki R, Hara K, Kubota N, Terauchi Y, Froguel P, Nakae J, Kasuga M, Accili D, Tobe K, Ueki K, Nagai R, Kadowaki T. Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity. J Biol Chem 279: 30817–30822, 2004.[Abstract/Free Full Text]
  48. Wada J, Zhang H, Tsuchiyama Y, Hiragushi K, Hida K, Shikata K, Kanwar YS, Makino H. Gene expression profile in streptozotocin-induced diabetic mice kidneys undergoing glomerulosclerosis. Kidney Int 59: 1363–1373, 2001.[CrossRef][Web of Science][Medline]
  49. Williams R, Airey M. Epidemiology and public health consequences of diabetes. Curr Med Res Opin 18: S1–S12, 2002.[Medline]
  50. Wojtaszewski JFP, Nielsen JN, Jorgensen SB, Frosig C, Birk JB, Richter EA. Transgenic models—a scientific tool to understand exercise-induced metabolism: the regulatory role of AMPK (5'-AMP-activated protein kinase) in glucose transport and glycogen synthase activity in skeletal muscle. Biochem Soc Trans 31: 1290–1294, 2003.[Web of Science][Medline]
  51. Yamauchi T, Nio Y, Maki T, Kobayashi M, Takazawa T, Iwabu M, Okada-Iwabu M, Kawamoto S, Kubota N, Kubota T, Ito Y, Kamon J, Tsuchida A, Kumagai K, Kozono H, Hada Y, Ogata H, Tokuyama K, Tsunoda M, Ide T, Murakami K, Awazawa M, Takamoto I, Froguel P, Hara K, Tobe K, Nagai R, Ueki K, Kadowaki T. Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nat Med 13: 332–339, 2007.[CrossRef][Web of Science][Medline]
  52. Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423: 762–769, 2003.[CrossRef][Medline]
  53. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7: 941–946, 2001.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/F881    most recent
00373.2007v1
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 ISI 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 Google Scholar
Google Scholar
Right arrow Articles by Cammisotto, P. G.
Right arrow Articles by Bendayan, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cammisotto, P. G.
Right arrow Articles by Bendayan, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.