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Am J Physiol Renal Physiol 292: F617-F627, 2007. First published October 3, 2006; doi:10.1152/ajprenal.00278.2006
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A role for AMP-activated protein kinase in diabetes-induced renal hypertrophy

Myung-Ja Lee,1 Denis Feliers,1 Meenalakshmi M. Mariappan,1 Kavithalakshmi Sataranatarajan,1 Lenin Mahimainathan,1 Nicolas Musi,2 Marc Foretz,3 Benoit Viollet,3 Joel M. Weinberg,4 Goutam Ghosh Choudhury,1,5 and Balakuntalam S. Kasinath1

Divisions of 1Nephrology and 2Diabetes, Department of Medicine, O’Brien Kidney Research Center, University of Texas Health Science Center, and 5Geriatric Research, Education, and Clinical Center, South Texas Veterans Healthcare System, San Antonio, Texas; 3Institut Cochin, Departement Endocrinologie Metabolisme et Cancer, Inserm U567, Universite Paris 5, Paris, France; and 4University of Michigan School of Medicine, Ann Arbor, Michigan

Submitted 20 July 2006 ; accepted in final form 25 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested the hypothesis that AMP-activated protein kinase (AMPK), an energy sensor, regulates diabetes-induced renal hypertrophy. In kidney glomerular epithelial cells, high glucose (30 mM), but not equimolar mannitol, stimulated de novo protein synthesis and induced hypertrophy in association with increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2, regulatory events in mRNA translation. These high-glucose-induced changes in protein synthesis were phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR) dependent and transforming growth factor-beta independent. High glucose reduced AMPK {alpha}-subunit theronine (Thr) 172 phosphorylation, which required Akt activation. Changes in AMP and ATP content could not fully account for high-glucose-induced reductions in AMPK phosphorylation. Metformin and 5-aminoimidazole-4-carboxamide-1beta-riboside (AICAR) increased AMPK phosphorylation, inhibited high-glucose stimulation of protein synthesis, and prevented high-glucose-induced changes in phosphorylation of 4E binding protein 1 and eukaryotic elongation factor 2. Expression of kinase-inactive AMPK further increased high-glucose-induced protein synthesis. Renal hypertrophy in rats with Type 1 diabetes was associated with reduction in AMPK phosphorylation and increased mTOR activity. In diabetic rats, metformin and AICAR increased renal AMPK phosphorylation, reversed mTOR activation, and inhibited renal hypertrophy, without affecting hyperglycemia. AMPK is a newly identified regulator of renal hypertrophy in diabetes.

protein synthesis; mRNA translation

THE REPERTOIRE OF KIDNEY CELL response to injury includes cell division, cell hypertrophy, and apoptosis. Hyperplasia and hypertrophy contribute to enlargement of the kidney in physiological or pathological states, including diabetes. The first structural change in the kidney in diabetes is enlargement. Most of the kidney enlargement is accounted for by hypertrophy, i.e., increased cellular content of RNA and protein, rather than proliferation (27, 42). Renal hypertrophy occurs rapidly, with significant increases in kidney weight being evident within days of onset of Type 1 or Type 2 diabetes (48). The importance of hypertrophy lies in the possibility that it may predispose to long-term complications of diabetes (27, 51, 58), and, as such, its underlying mechanisms merit further investigation. Exploration of mechanism of diabetes-associated hypertrophy in renal cells has implicated transforming growth factor-beta (TGF-beta) and ANG II (21, 51, 57). These mediators are also involved in matrix accumulation in later stages of diabetes (7, 43, 52), suggesting a potential link between the early and late events in diabetic renal disease. Regulation of high-glucose-induced renal cell growth also involves cell cycle events (15, 50, 58). There could be additional regulatory pathways yet to be identified, including those regulated by energy status of the cell.

Glucose is a metabolic fuel that regulates energy status of the cell. States of energy depletion lead to the activation of AMP-activated protein kinase (AMPK), resulting in inhibition of energy-consuming pathways (55) and activation of ATP-generating processes (22). Cell hypertrophy entails an increased rate of protein synthesis, which accounts for a significant part of cell energy consumption (29). We hypothesized that AMPK, an energy sensor, regulates diabetes-induced renal hypertrophy. High-glucose-induced hypertrophy was studied in glomerular epithelial cells (GECs). Understanding the mechanisms of injury to GEC is important because, recently, attention has been drawn to decrease in their number and/or density as a determinant in proteinuria and progression of diabetic renal disease (10, 41, 53).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Rat GECs, kindly provided by Dr. Jeffrey Kreisberg (Department of Surgery, University of Texas Health Science Center at San Antonio), were cultured as described previously (34). GECs display several of the characteristics of visceral GECs in vivo, including expression of Heymann nephritis antigen (52a), CD2AP, laminin, fibronectin, and basement membrane heparan sulfate proteoglycan (34) and sensitivity to puromycin aminonucleoside (34a). GECs were grown in DMEM containing 7% FBS, 5 mM glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Cells were used between passages 9 and 15. GECs were incubated with normal glucose (5 mM glucose), high glucose (30 mM glucose), or mannitol (5 mM glucose + 25 mM mannitol) for the indicated time points; medium was changed every 2 days.

Protein synthesis measurement. Protein synthesis measurement was as previously described (49). Serum-starved cells were labeled with 10 µCi/ml of [35S]methionine for the terminal 2 h of incubation. Cells were washed in PBS and lysed in radioimmunoprecipitation assay buffer. Cell protein content was measured with a Bio-Rad reagent (Bio-Rad, Hercules, CA). An equal amount of protein (20 µg) was spotted onto the 3-mm filter paper (Whatman, Maidstone, UK). Filters were washed three times by boiling for 1 min in 10% TCA containing 0.1 g/l methionine before radioactivity was determined.

Immunoblotting. Immunoblotting was as previously described (48). Equal amounts of cell lysate protein (15–30 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membrane was probed with primary antibody overnight at 4°C. After an extensive washing, the membrane was incubated with secondary antibodies linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) or IRDye (Rockland Immunochemicals, Gibertsville, PA). Proteins were visualized by chemiluminescence using enhanced chemiluminescence reagent. Alternatively, Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE) was used to detect fluorochrome-coupled antibody. The membrane was stripped in 0.5 M NaOH and reprobed with indicated antibodies. Signal intensity was assessed by densitometric analysis.

Phosphatidylinositol 3-kinase assay. Equal amounts of cell lysate protein (500 µg) were immunoprecipitated with an antibody against p85 {alpha}-subunit of phosphatidylinositol 3-kinase (PI3-kinase) (14, 47, 48). Immunobeads were incubated with 10 µg of phosphatidylinositol for 10 min at 25°C in the PI3-kinase buffer. [{gamma}-32P]ATP (5 µCi) was added and incubated for another 10 min at 25°C. Reaction was stopped by the addition of a mixture of chloroform-methanol. The reactants were extracted with 100 µl of chloroform, and the organic layer was washed with methanol and 1 N HCl. Reaction products were dried, resuspended in chloroform, separated by TLC, and developed with chloroform-methanol-28% ammonium hydroxide-water in the ratio of 129:114:15:21. The spots were visualized by autoradiography.

AMPK activity assay. Equal amounts of cell lysate protein (500 µg) were immunoprecipitated with an antibody against AMPK{alpha} (Cell Signaling, Danvers, MA) and incubated at 30°C with 100 µM SAMS peptide (Upstate, Lake Placid, NY) in reaction buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 5% glycerol, 1 mM DTT, 0.05% Triton X-100) with 250 µM [{gamma}-32P] ATP for 8 min; 25 µl of reaction mixture were spotted onto the phosphocellulose P81 paper (Upstate) and washed with phosphoric acid. Radioactivity on the filter paper was measured (16).

Transfection. The plasmid containing AMPK carrying K45R mutation of the {alpha}1-subunit (pCAGGS) (12) (kindly provided by Dr. N. Fujii, Joslin Clinic, Boston MA) was employed for transient transfection using lipofectamine and Lipo-plus reagent (Invitrogen) (38).

TGF-beta bioassay. The TGF-beta bioassay was as previously described (1). TGF-beta was measured with a mink lung epithelial cell (MLEC) responder line, stably transfected with a construct containing the TGF-beta-responsive element from the human plasminogen activator inhibitor-1 promoter fused to a luciferase reporter gene. MLECs were treated for 24 h with conditioned medium from control or high-glucose-treated GECs. Recombinant TGF-beta (R&D Systems, Minneapolis, MN) was used as a standard. The cell extracts were prepared and assayed for luciferase activity (Promega, Madison, WI).

Animal study. Animal protocols were approved by the Institutional Animal Care and Use Committee. To induce Type 1 diabetes, male Sprague-Dawley rats weighing 200–220 g (Harlan, Indianapolis, IN) received a single injection of 60 mg/kg streptozotocin in citrate buffer (pH 4.5) into the tail vein. The rats became diabetic the next day as indicated by elevation in blood glucose concentration, which was measured by a glucometer (Ascensia Elite XL, Mishawaka, IN). On day 4 of hyperglycemia, rats were killed. One of the kidneys was immediately frozen in liquid nitrogen-chilled 2-methylbutane and stored at –70°C; the other kidney was weighed and used for glomerular isolation. In additional experiments, metformin was administered by gavage starting on the first day of diabetes for 4 days at 300 mg·kg–1·day–1 (35). 5-Aminoimidazole-4-carboxamide-1beta-riboside (AICAR; 750 mg·kg–1·day–1) was given by intraperitoneal route (37) for the same duration. Body weight and blood glucose concentration were measured daily. C57BL6/KsJ db/db mice, a model of Type 2 diabetes, were maintained on regular laboratory chow. Blood glucose concentration was monitored for emergence of diabetes. The mice were killed at 2 wk of diabetes at which time the kidneys are hypertrophic (48). Renal cortex was dissected out for further analysis.

Measurement of AMP and ATP levels. Samples of GEC suspension were immediately deproteinized in TCA, neutralized with trioctylamine-CFC 113, and stored at –20°C. Purine nucleotides and their metabolites in 20-µl aliquots of the neutralized extracts were separated and quantified by the reverse-phase ion-pairing gradient HPLC method as previously described (13).

Statistics. Data were obtained from at least three independent experiments and are expressed as means ± SE. Statistical comparisons between multiple groups were performed by ANOVA, and Bonferroni’s method was applied to control for multiple testing. Statistical comparisons between two groups were performed by the Student’s t-test with a minimum value of P < 0.05 considered to represent statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
High glucose induces hypertrophy of GEC. Cells were incubated with 5 mM glucose (control), 30 mM glucose (high glucose), or 5 mM glucose + 25 mM mannitol (osmotic control) for 1, 3, or 6 days; blood glucose levels are in the 30 mM range in rodents with diabetes (20). High glucose increased protein synthesis by 30 and 40% on days 3 and 6, respectively; the increment on day 1 was not statistically significant (Fig. 1A; P < 0.001); osmotic control had no effect. High glucose also induced cell hypertrophy, defined as protein content of the GEC per unit cell number, by nearly 20% compared with 5 mM glucose-treated cells (Fig. 1B; P < 0.05, for days 3 and 6). We employed 3-day incubation for further studies.


Figure 1
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  		Fig. 1. Regulation of protein synthesis and phosphorylation of 4E binding protein 1 (4E-BP1) and eukaryotic elongation factor 2 (eEF2) by high glucose (Glc). A: glomerular epithelial cells (GECs) were incubated with 5 mM Glc [control (Con)], 30 mM Glc, or 5 mM Glc + 25 mM mannitol (Man) for 1, 3, or 6 days. Protein synthesis was measured by incorporation of [35S]methionine (Met) into TCA-precipitable protein. Composite data from 3 experiments are shown (*P < 0.001 high Glc vs. control by ANOVA). B: cells were treated as described in A. At end of incubation for 3 or 6 days, protein content was measured and expressed per 105 cells. Composite data from 3 experiments are shown (*P < 0.05 high Glc vs. control by ANOVA). C and D: after 3-day incubation with 5 mM Glc (control), 30 mM Glc, or 5 mM Glc + 25 mM Man, equal amounts of cell lysate protein were separated by SDS-PAGE and immunoblotted with an antibody specific for 4E-BP1 phosphorylated on Thr37/46 (C) or for eEF2 phosphorylated on Thr56 (D). The blot was stripped and probed with an antibody against 4E-BP1 or eEF2 to assess loading. Representative blots from 3 experiments are shown. C and D, bottom: composite densitometric data from 3 experiments (*P < 0.001 high Glc vs. control by ANOVA).

 
High glucose stimulates 4E binding protein 1 phosphorylation. The initiation phase of mRNA translation during which the ribosome binds to mRNA is the rate-limiting step in proteinsynthesis (40, 54). This process is controlled by several eukaryotic initiation factors (eIFs), including the mRNA cap binding protein eIF4E. In the basal state, eIF4E is held inactive by its binding protein, 4E binding protein 1 (4E-BP1). Phosphorylation of 4E-BP1 dissolves the complex and releases eIF4E, which, in association with eIF4G and eIF4A, promotes the initiation phase of translation. In renal epithelial cells, phosphorylation of 4E-BP1 is essential for protein synthesis induced by insulin, ANG II, IGF-I, and VEGF (2, 47–49). In contrast to the growth factors, glucose regulation of 4E-BP1 phosphorylation has not been well studied. We examined phosphorylation of Thr37/46 because it is a priming event that leads to phosphorylation of other sites in 4E-BP1 (18, 25). High glucose increased 4E-BP1 phosphorylation at early time points of incubation (30 and 60 min, data not shown) and at 3 days (Fig. 1C; P < 0.001).

High glucose regulates eukaryotic elongation factor 2 phosphorylation. During the elongation phase of mRNA translation, amino acids are sequentially added to the nascent peptide chain by the participation of aminoacyl tRNA (6, 54). Movement of the ribosome in the 5' to 3' direction along the mRNA, resulting in translocation of aminoacyl tRNA from the A site to the P site on the ribosome, is regulated by eukaryotic elongation factor 2 (eEF2). Activation of eEF2 occurs following dephosphorylation at Thr56 (45). High glucose reduced eEF2 Thr56 phosphorylation at early time points of incubation (30 and 60 min, data not shown) and at 3 days (Fig. 1D; P < 0.001). These data show that high glucose regulates important steps in the initiation and elongation phases of mRNA translation.

High-glucose-induced protein synthesis is PI3-kinase and mammalian target of rapamycin dependent. We studied PI3-kinase-Akt-mammalian target of rapamycin (mTOR) pathways because these regulate 4E-BP1 phosphorylation (2). High glucose increased PI3-kinase activity in anti-p85{alpha} immunoprecipitates at early time points (30 and 60 min, data not shown) and at 1 and 3 days of incubation, showing a rapid onset and sustained stimulation of the kinase activity (Fig. 2A). Because we used p85{alpha} antibody in immunoprecipitation, these data show that high glucose induces activation of class IA PI3-kinase. LY-294002, a selective PI3-kinase inhibitor, inhibited both basal and high-glucose-induced increases in 4E-BP1 phosphorylation (Fig. 2B; P < 0.001) and protein synthesis (Fig. 2C; P < 0.001). These data suggest that constitutive and high-glucose-induced protein synthesis in the GEC requires PI3-kinase activity.


Figure 2
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  		Fig. 2. High-Glc-induced protein synthesis is phosphatidylinositol 3-kinase (PI3-kinase) and mammalian target of rapamycin (mTOR) dependent. A: GECs were incubated with 30 mM Glc for different durations and processed for PI3-kinase assay. Representative blots from 3 experiments are shown. PI3-P refers to phosphatidylinositol 3-phosphate, a product of PI3-kinase. B: GECs were incubated with 30 mM Glc for 3 days with or without addition of 25 µM LY-294002 for the terminal 2 h of incubation. Top: immunoblotting for 4E-BP1 was done as described in Fig. 1. Bottom: composite data from 3 experiments (*P < 0.001 for high Glc vs. control, **P < 0.001 for high Glc vs. high Glc + LY-294002, #P < 0.001 for control + LY-294002 vs. control, by ANOVA). C: measurement of protein synthesis was done as described in Fig. 1 except for preincubation with or without 25 µM LY-294002 for 2 h before addition of the label. Graph shows composite data from 3 experiments (*P < 0.001 for high Glc vs. control, **P < 0.001 for high Glc vs. high Glc + LY-294002, #P < 0.001 for control + LY-294002 vs. control, by ANOVA). D: cells were treated as described in Fig. 1C, and immunoblotting (top) was done with an antibody against phosphorylated Ser2448 on mTOR. Bottom: composite data from 3 experiments (*P < 0.001 for high Glc vs. control by ANOVA). E: GEC were incubated with 30 mM Glc for 3 days, with or without addition of 22 nM rapamycin for the terminal 2 h of incubation. Top: immunoblotting for 4E-BP1 was done as described in Fig. 1. Bottom: composite data from 3 experiments (*P < 0.01 for high Glc vs. control, **P < 0.001 for high Glc vs. high Glc + rapamycin, #P < 0.001 control vs. control + rapamycin, ANOVA). F: measurement of protein synthesis was done as described in Fig. 1 except for preincubation with or without 22 nM rapamycin for 2 h before addition of the label. Graph shows composite data from 3 experiments (*P < 0.001 for high Glc vs. control, **P < 0.05 for high Glc vs. high Glc + rapamycin, by ANOVA).

 
We next examined whether high glucose stimulated mTOR activity because mTOR is a direct kinase for 4E-BP1 (25). High glucose significantly augmented mTOR Ser2448 phosphorylation at both early and late points of incubation, suggesting activation (Fig. 2D; P < 0.001). Requirement of mTOR in high-glucose-induced protein synthesis was examined with rapamycin, a selective inhibitor of mTOR (25). Rapamycin inhibited both basal and high-glucose-induced increases in 4E-BP1 phosphorylation (Fig. 2E; P < 0.001). Rapamycin partially but significantly inhibited protein synthesis induced by high glucose (Fig. 2F).

Role of Akt in mTOR regulation was explored. High glucose augmented Akt activity at both early and late time points of incubation (Fig. 3A). In GECs infected with a recombinant adenovirus carrying dominant-negative Akt, but not control green fluorescent protein, high glucose failed to stimulate 4E-BP1 phosphorylation (Fig. 3B); increment in 4E-BP1 phosphorylation is a readout of mTOR activation (26). Thus high glucose induction of mTOR activity is Akt dependent.


Figure 3
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  		Fig. 3. High Glc regulates Akt activity. A: GECs were incubated with 30 mM Glc for 3 days and processed for Akt immunokinase assay with myelin basic protein (MBP) substrate. An immunoblot of cell lysates using anti-Akt antibody was performed to assess loading. A representative blot from 3 experiments is shown. B: GECs were quiesced in serum-free medium for 24 h and infected at room temperature for 1 h with a multiplicity of infection of 70 of replication-defective adenovirus vector carrying mouse Akt mutated at its phosphorylation sites (T308A, S473A) with an HA tag (Ad-DN-Akt). Control cells were infected with the same multiplicity of infection of adenovirus carrying green fluorescent protein (Ad-GFP). After a 3-day exposure to 5 mM Glc (control) or 30 mM Glc, immunoblotting was done with the indicated antibodies. A representative blot from 3 experiments is shown.

 
High glucose reduces AMPK phosphorylation. AMPK regulates activity of tuberous sclerosis complex (TSC)-2 (tuberin), which, in the basal state, forms a heterodimer with TSC-1 (hamartin) and tonically inhibits mTOR activity, the key controller of 4E-BP1 phosphorylation (19). Activity of AMPK depends on phosphorylation of Thr172 on its {alpha}-catalytic subunit (23). AMPK phosphorylation on Thr172 was significantly reduced by high glucose but not by mannitol (Fig. 4A; P < 0.001). Immunokinase assay showed that high glucose reduced the activity of AMPK by nearly 40% relative to control (Fig. 4B; P < 0.01), confirming that changes in AMPK phosphorylation reflected changes in AMPK activity.


Figure 4
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  		Fig. 4. High Glc reduces AMP-activated protein kinase (AMPK) phosphorylation and activity. A: cells were treated as described in Fig. 1C, and immunoblotting was done with an antibody against phosphorylated Thr172 on AMPK {alpha}-subunit. Top: representative blot from 3 experiments. Bottom: composite densitometric data from 3 experiments (*P < 0.001, high Glc vs. control by ANOVA). B: GECs were incubated with 30 mM Glc for 3 days and processed for AMPK activity using SAMS substrate. Composite data from 6 experiments are shown (*P < 0.01, high Glc vs. control, by t-test). C: GECs were transfected with either control plasmid or pCAGGS containing a K45R mutation in the {alpha}-catalytic subunit of AMPK. After a 3-day exposure to 5 mM Glc (control) or 30 mM Glc, de novo protein synthesis was measured as described in Fig. 1. Composite data from 3 experiments are presented (*P < 0.01, high Glc vs. high Glc + AMPK mutant, by ANOVA). D: GECs were incubated with 30 mM Glc for different durations, and AMP (top) and ATP (bottom) contents were measured by HPLC and corrected for protein content. Composite data (n = 3 at each time point) are shown with control as 100% (*P < 0.05, **P < 0.001, by ANOVA). E: cells were treated as described in Fig. 3B. After a 3-day exposure to 5 mM Glc (control) or 30 mM Glc, immunoblotting was done with the indicated antibodies. A representative blot from 5 experiments is shown.

 
To further explore the role of AMPK in protein synthesis, GECs were transfected with a pCAGGS plasmid containing dominant-negative AMPK carrying a K45R mutation. High glucose stimulated protein synthesis in cells expressing the control vector; expression of dominant-negative AMPK resulted in further increment in high-glucose-induced protein synthesis (Fig. 4C; P < 0.01). Basal protein synthesis increased in cells expressing the mutant but did not reach statistical significance.

Upstream factors that regulate AMPK phosphorylation were examined. Reduced AMP content and increased ATP content are known to decrease AMPK phosphorylation (8). Contents of AMP and ATP were measured after high-glucose exposure. High glucose significantly reduced AMP content at 1 h but not at 24 and 72 h (Fig. 4D). Reduction in AMPK phosphorylation was seen at all time points of incubation with high glucose, but correlation with reduced AMP content was seen only at the 1 h time point. ATP content of the GEC was unchanged at 1 h but was reduced at 24 and 72 h. However, because high glucose decreased AMPK phosphorylation at these time points and because reduced ATP should increase AMPK phosphorylation, ATP content did not correlate with high-glucose-induced changes in AMPK phosphorylation. Role of Akt was examined because it is reported to modulate AMPK and TSC-2 (9, 28). Compared with reduction in AMPK phosphorylation in adenovirus-green fluorescent protein-infected control cells, high glucose barely decreased AMPK phosphorylation in adenovirus-DN-Akt-infected cells (Fig. 4E), identifying a possible role for Akt as one of the upstream regulators of AMPK.

Metformin and AICAR reverse high-glucose effects on AMPK phosphorylation and protein synthesis. We next investigated whether AMPK activators metformin (61) and AICAR could affect high-glucose stimulation of protein synthesis. Metformin and AICAR dose dependently increased Thr172 phosphorylation (Fig. 5A) and prevented high-glucose-induced reduction in AMPK phosphorylation (Fig. 5B). Both metformin and AICAR caused a significant but partial inhibition of high-glucose-induced protein synthesis (Fig. 5C; P < 0.001 and P < 0.05). Metformin and AICAR reversed high-glucose-induced changes in phosphorylation of 4E-BP1 and eEF2 (Fig. 6, A and B). These data show that stimulation of AMPK inhibits important events in high-glucose regulation of initiation and elongation phases of mRNA translation.


Figure 5
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  		Fig. 5. Metformin and 5-aminoimidazole-4-carboxamide-1beta-riboside (AICAR) reverse high-Glc effects on AMPK phosphorylation and protein synthesis. A: GECs were incubated with different doses of metformin (left) or AICAR (right) for 24 h. Equal amounts of cell lysate protein were separated by SDS-PAGE and immunoblotted with AMPK antibodies as described in Fig. 4. Top: representative blot from 3 experiments. Bottom: composite densitometric data from 3 experiments (*P < 0.05, **P < 0.001 for metformin vs. control and #P < 0.05, ##P < 0.01 for AICAR vs. control, by ANOVA). B: after 3-day incubation with 30 mM Glc, metformin (1 mM) or AICAR (0.5 mM) and equal amounts of cell lysate protein were separated by SDS-PAGE and immunoblotted with AMPK antibodies as described in Fig. 4. A representative blot from 3 experiments is shown. C: after 3-day exposure to 30 mM Glc, metformin, or AICAR, de novo protein synthesis was measured as described in Fig. 1. Composite data from 3 experiments are shown (*P < 0.001 for high Glc vs. control, **P < 0.001 for high Glc vs. high Glc + metformin, #P < 0.001 for high Glc vs. control, ##P < 0.05 for high Glc vs. high Glc + AICAR, &P < 0.05 for AICAR vs. control, by ANOVA).

 

Figure 6
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  		Fig. 6. AMPK regulation of initiation and elongation phases of mRNA translation. A: immunoblotting for 4E-BP1 was done as described in Fig. 1. Top: representative blot from 3 experiments. Bottom: composite densitometric data from 3 experiments (*P < 0.001 for high Glc vs. control, #P < 0.05 for high Glc vs. high Glc + metformin, ##P < 0.01 for high Glc vs. high Glc + AICAR, by ANOVA). B: immunoblotting for eEF-2 was done as described in Fig. 1. Top: representative blot from 3 experiments. Bottom: composite densitometric data from 3 experiments (*P < 0.01 high Glc vs. control, #P < 0.01 high Glc vs. high Glc + metformin or high Glc + AICAR, by ANOVA).

 
TGF-beta is not involved in high-glucose-induced protein synthesis. We next examined the potential role of TGF-beta because high-glucose-induced hypertrophy is TGF-beta dependent in some renal cells (51, 60). High glucose did not increase TGF-beta expression as measured by luciferase activity linked to TGF-beta-sensitive plasminogen activator inhibitor-1 promoter in MLECs (Fig. 7A). Addition of neutralizing TGF-beta antibody did not affect high-glucose-induced protein synthesis (Fig. 7B), showing high-glucose induction of protein synthesis in GECs is independent of TGF-beta.


Figure 7
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  		Fig. 7. Transforming growth factor-beta (TGF-beta) is not involved in high-glucose-induced hypertrophy in the GEC. A: mink lung epithelial cells, stably transfected with the human plasminogen activator inhibitor-1 promoter containing TGF-beta responsive element fused to a luciferase reporter gene, were treated with conditioned medium from control or high-Glc-treated GEC and human TGF-beta standard (rh) for 24 h. TGF-beta activity was assessed by luciferase activity. Composite data from 3 experiments are presented. B: after 3-day exposure to 5 mM Glc (control) or 30 mM Glc with or without addition of 30 µg/ml neutralizing anti-TGF-beta antibody or irrelevant IgG, de novo protein synthesis was measured as described in Fig. 1. Composite data from 3 experiments are presented (*P < 0.001, high Glc vs. control, by ANOVA).

 
Renal AMPK phosphorylation is reduced in Type 1 and Type 2 diabetes. We explored whether diabetes-induced renal hypertrophy was associated with changes in AMPK phosphorylation. On day 4 of streptozotocin-induced insulin-deficient diabetes, a model of Type 1 diabetes, kidney weight corrected to body weight was significantly increased by 31% (Fig. 8A; P < 0.0001). In rats with Type 1 diabetes, AMPK phosphorylation was reduced by nearly 60% (P < 0.001) and 80% (P < 0.01) in renal cortex (Fig. 8B) and glomeruli (Fig. 8C), respectively. AMPK phosphorylation was also reduced by nearly 70% in renal cortex of db/db mice, a model of Type 2 diabetes, at ~2 wk of hyperglycemia, at which time the kidneys are hypertrophic (48) (Fig. 8D; P < 0.01). These data suggest an association between reduced AMPK phosphorylation and renal hypertrophy in rodents with Type 1 or Type 2 diabetes.


Figure 8
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  		Fig. 8. AMPK phosphorylation is reduced in the kidney in Type 1 and Type 2 diabetes. A, B, and C: Type 1 diabetes was induced in Sprague-Dawley rats by a single injection of 60 mg/kg streptozotocin in citrate buffer (pH 4.5) into the tail vein. On day 4 of hyperglycemia, rats were killed. A: kidney weight-to-body weight ratio (*P < 0.0001, diabetes vs. control, by Student’s t-test). Equal amounts of renal cortical (B) and glomerular (C) homogenates from rats were separated by SDS-PAGE and immunoblotted with AMPK antibodies as described in Fig. 4 (*P < 0.01, **P < 0.001, diabetes vs. control, by Student’s t-test). D: C57BL6/KsJ db/db mice were killed at ~2 wk of diabetes. Equal amounts of renal cortical homogenates from control and diabetic mice were separated by SDS-PAGE and immunoblotted with AMPK antibodies as described in Fig. 4 (*P < 0.01, diabetes vs. control, by Student’s t-test).

 
Metformin and AICAR reverse Type 1 diabetes-induced renal hypertrophy. Preliminary studies were performed to determine the doses of metformin and AICAR that increase AMPK phosphorylation in the renal cortex without affecting plasma glucose (data not shown). Metformin and AICAR did not affect elevation in blood glucose or reduction in body weight in diabetic rats (Table 1). The gain in kidney-to-body weight ratio seen in diabetic rats on day 4 of diabetes was partially but significantly inhibited by metformin or AICAR (Fig. 9A). Metformin and AICAR prevented the reduction in AMPK phosphorylation in renal cortex in diabetic rats compared with untreated diabetic animals (Fig. 9B). These data demonstrate that reduction in AMPK activity contributes to diabetes-induced renal hypertrophy in rats.


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  		Table 1. Physical and metabolic parameters in control and diabetic rats

 

Figure 9
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  		Fig. 9. Metformin and AICAR inhibit diabetes-induced renal hypertrophy. A: on day 1 of streptozotocin-induced hyperglycemia, rats were treated with metformin or AICAR for a total of 4 days, as described in MATERIALS AND METHODS. A: kidney weight-to-body weight (#P < 0.001 for diabetes vs. control, *P < 0.05 for diabetes vs. diabetes + metformin, **P < 0.001 for diabetes vs. diabetes + AICAR, by ANOVA). B, C, and D: equal amounts of renal cortical homogenates from rats were separated by SDS-PAGE and immunoblotted with the indicated antibodies (left: metforin; right: AICAR).

 
Signaling pathways that regulate protein synthesis were investigated in the renal cortex of rats with Type 1 diabetes. At 4 days of diabetes, Akt phosphorylation was increased in the renal cortex (Fig. 9C). Activation of mTOR was reflected in increased phosphorylation of its direct substrates, p70S6 kinase and 4E-BP1 (Fig. 9D). Treatment with metformin and AICAR prevented increases in phosphorylation of 4E-BP1 and p70S6 kinase in diabetic rats (Fig. 9D), although the diabetes-induced increase in phosphorylation of Akt was unaffected (Fig. 9C). These data show that, in the kidney, AMPK regulates mTOR activity, without any effect on Akt activation.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Protein synthesis consumes a significant amount of cellular energy (29). Induction of protein synthesis by glucose, a source of energy, is likely to involve regulation of energy sensors such as AMPK. Accordingly, in association with high-glucose-induced protein synthesis, we found inhibition of AMPK phosphorylation and activity. AMPK is a heterotrimeric protein consisting of a combination of {alpha}1/2-, beta1/2-, and {gamma}1/2/3-subunits (8), with the {alpha}1- rather than {alpha}2-subunit expressed in the kidney (16). The enzymatic activity of AMPK is dependent on phosphorylation of Thr172 of the {alpha}-subunit (23). The COOH terminus of beta-subunit binds to {alpha}- and {gamma}-subunits (30). The {gamma}-subunit contains the cystathionine beta synthase-like domain to which AMP binds allosterically. Increased AMP content stimulates AMPK activity, whereas increased ATP content results in its suppression (8). On activation, AMPK inhibits energy-consuming reactions, such as synthesis of fatty acids and sterols (55), and activates ATP-generating processes, such as fatty acid oxidation (39).

The signal in diabetic kidney that alters activity of AMPK is not known but may include changes in levels of AMP and ATP. An increase in proximal tubular ATP content and mitochondrial enlargement has been observed in diabetes (32). However, infusion of high glucose into an isolated kidney has been reported to reduce ATP content (33). Careful measurement of ATP and AMP content in the GEC at various time points of incubation with high glucose revealed a single time point of 1-h exposure to high glucose when reduced AMP content correlated with decreased AMPK phosphorylation. Reduced AMPK phosphorylation did not correlate with AMP content at other time points or with ATP content at any time point. These data suggest that, in addition to adenine nucleotides, other factors may be involved in high-glucose regulation of AMPK in GECs. High osmolality, known to augment AMPK activity (17), is unlikely to be involved in GECs because mannitol did not affect AMPK phosphorylation. Ca2+/calmodulin-dependent kinase kinase-beta also stimulates AMPK activity (24). Because Ca2+/calmodulin-dependent kinase kinase-beta is expressed mostly in the central nervous system, other factors yet undiscovered may be involved (3). High-glucose-induced reduction in AMPK activity could increase fatty acid synthesis, which, in turn, could affect mRNA translation. Arachidonic acid derivatives PGF2{alpha} and HETE have been reported to stimulate the initiation phase of mRNA translation (44, 59). In addition to modulation of fatty acid synthesis, recent investigations have implicated AMPK in protein synthesis (46), which is regulated at the level of mRNA translation.

Important events in mRNA translation, e.g., phosphorylation of 4E-BP1 and eEF2, are partly under the control of the PI3-kinase-Akt-mTOR signaling pathway. Reversal of high-glucose-induced changes in 4E-BP1 and eEF2 phosphorylation by stimulation of AMPK activity suggests that AMPK interacts with this pathway. High-glucose-induced increases in 4E-BP1 phosphorylation and protein synthesis in the GECs were found to be dependent on PI3-kinase and mTOR activation. Inhibition of high-glucose-induced Akt activation by expression of dominant-negative Akt construct partly restored AMPK phosphorylation, suggesting AMPK is downstream of Akt. Because stimulation of AMPK also inhibited high-glucose-induced mTOR activity, as shown by inhibition of phosphorylation of 4E-BP1 and p70S6 kinase, AMPK appears to be interposed between Akt and mTOR (Fig. 10). Inhibition of AMPK by Akt has also been observed in cardiac myocytes (36). In addition, Akt induces phosphorylation of TSC-2 and inhibits the activity of the TSC-1/TSC-2 dimer (28), which would contribute to increase in mTOR activity. Akt could also stimulate the activity of a phosphatase or inhibit upstream kinase for AMPK such as LKB-1, which could result in reduction in AMPK phosphorylation. There could be additional mechanisms by which high glucose may suppress AMPK activity in the GEC.


Figure 10
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  		Fig. 10. Schematic of AMPK regulation in diabetes-induced renal hypertrophy.

 
mTOR plays a central role in regulation of initiation and elongation phases of mRNA translation. 4E-BP1, an important controller of eIF4E activity in the initiation phase, is a direct substrate of mTOR. Increase in activity of p70S6 kinase, another important mTOR substrate, promotes phosphorylation of eEF2 kinase on Ser366 (56), resulting in inhibition of its activity. eEF2 kinase directly phosphorylates eEF2 on Thr56, leading to its inactivation (45). Thus phosphorylation and inactivation of eEF2 kinase contribute to decreased eEF2 phosphorylation. Recently, AMPK has been reported to regulate eEF2 kinase by phosphorylation of Ser398, which increases activity of eEF2 kinase, resulting in inhibition of the elongation phase (5). Thus high-glucose-induced reduction in AMPK activity in the GEC appears to reduce activity of eEF2 kinase, resulting in decreased phosphorylation of eEF2 and facilitation of the elongation phase of protein synthesis.

Observations with dominant-negative AMPK suggest that AMPK normally inhibits protein synthesis and that its suppression appears to mediate high-glucose-induced protein synthesis. That inhibition of AMPK is not a parallel event but is actually required for high-glucose-induced protein synthesis is suggested by abrogation of the high-glucose effect with metformin and AICAR, two distinct activators of AMPK. Studies in cardiomyocytes have shown a similar reduction in AMPK phosphorylation in hypertrophy induced by phenylephrine and its reversal by metformin and AICAR (9). Furthermore, mutations in the {gamma}2-subunit of AMPK are associated with cardiac hypertrophy (4). Administration of metformin and AICAR partly or fully prevented renal hypertrophy seen in early stages of insulin-deficient diabetes in association with increased AMPK phosphorylation in the renal cortex without affecting the degree of hyperglycemia. These observations support a definitive role for AMPK in control of renal protein synthesis in diabetes.

Findings in this report extend previous observations in which AMPK was found to be involved in high-glucose regulation of L-type pyruvate kinase gene expression in islet cells and in glucose regulation of its own uptake in skeletal muscle (11, 31). Identification of AMPK as a regulating factor in diabetes-induced renal hypertrophy has implications for management. Hypertrophy of GEC and the rest of the kidney cells may be linked to subsequent events that result in demise of kidney function (27, 58). Preliminary data indicate that AMPK may be also involved in abnormal extracellular matrix protein synthesis induced by high glucose in the GEC (Lee and Kasinath, unpublished observations). Accumulation of matrix proteins has been directly linked to loss of kidney function in diabetes. Ready availability of agents such as metformin and thiazolidinediones, which increase AMPK activity, make AMPK an attractive therapeutic target for reversing diabetes-induced renal injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1 DK-50190 and DK-55815 (G. G. Choudhury), a National Institutes of Health O’Brien Kidney Center grant (B. S. Kasinath), the American Diabetes Association (B. S. Kasinath), a Veterans Affairs Research Service Merit Review Grant (B. S. Kasinath and G. G. Choudhury), the National Kidney Foundation of South and Central Texas (M. M. Mariappan and B. S. Kasinath), the Juvenile Diabetes Research Foundation (D. Feliers, B. S. Kasinath, G. G. Choudhury), and the European Union FP6 program EXGENESIS (M. Foretz and B. Viollet). G. G. Choudhury is recipient of Veterans Affairs Research Career Scientist Award.


   ACKNOWLEDGMENTS
 
The authors thank Dr. H. E. Abboud for critical reading of the manuscript and Dr. M. A. Venkatachalam for help with animal experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. S. Kasinath, Dept. of Medicine, Mail Code 7882, Univ. of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229 (e-mail: kasinath{at}uthscsa.edu)

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. Abe M, Harpel JG, Metz CN, Nunes I, Loskutoff DJ, Rifkin DB. An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal Biochem 216: 276–284, 1994.[CrossRef][ISI][Medline]
  2. Bhandari BK, Feliers D, Duraisamy S, Stewart JL, Gingras AC, Abboud HE, Choudhury GG, Sonenberg N, Kasinath BS. Insulin regulation of protein translation repressor 4E-BP1, an eIF4E-binding protein, in renal epithelial cells. Kidney Int 59: 866–875, 2001.[CrossRef][ISI][Medline]
  3. Birnbaum MJ. Activating AMP-activated protein kinase without AMP. Mol Cell 19: 289–290, 2005.[CrossRef][ISI][Medline]
  4. Blair E, Redwood C, Ashrafian H, Oliveira M, Broxholme J, Kerr B, Salmon A, Ostman-Smith I, Watkins H. Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 10: 1215–1220, 2001.[Abstract/Free Full Text]
  5. Browne GJ, Finn SG, Proud CG. Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398. J Biol Chem 279: 12220–12231, 2004.[Abstract/Free Full Text]
  6. Browne GJ, Proud CG. Regulation of peptide-chain elongation in mammalian cells. Eur J Biochem 269: 5360–5368, 2002.[ISI][Medline]
  7. Burns KD. Angiotensin II and its receptors in the diabetic kidney. Am J Kidney Dis 36: 449–467, 2000.[ISI][Medline]
  8. Carling D. The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem Sci 29: 18–24, 2004.[CrossRef][ISI][Medline]
  9. Chan AY, Soltys CL, Young ME, Proud CG, Dyck JR. Activation of AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte. J Biol Chem 279: 32771–32779, 2004.[Abstract/Free Full Text]
  10. Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, Fioretto P. Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes. Diabetes 52: 1031–1035, 2003.[Abstract/Free Full Text]
  11. Da Silva Xavier G, 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]
  12. Dyck JR, Gao G, Widmer J, Stapleton D, Fernandez CS, Kemp BE, Witters LA. Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits. J Biol Chem 271: 17798–17803, 1996.[Abstract/Free Full Text]
  13. Feldkamp T, Kribben A, Roeser NF, Senter RA, Kemner S, Venkatachalam MA, Nissim I, Weinberg JM. Preservation of complex I function during hypoxia-reoxygenation-induced mitochondrial injury in proximal tubules. Am J Physiol Renal Physiol 286: F749–F759, 2004.[Abstract/Free Full Text]
  14. Feliers D, Duraisamy S, Faulkner JL, Duch J, Lee AV, Abboud HE, Choudhury GG, Kasinath BS. Activation of renal signaling pathways in db/db mice with type 2 diabetes. Kidney Int 60: 495–504, 2001.[CrossRef][ISI][Medline]
  15. Feliers D, Frank MA, Riley DJ. Activation of cyclin D1-Cdk4 and Cdk4-directed phosphorylation of RB protein in diabetic mesangial hypertrophy. Diabetes 51: 3290–3299, 2002.[Abstract/Free Full Text]
  16. 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]
  17. Fryer LG, Parbu-Patel A, Carling D. The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem 277: 25226–25232, 2002.[Abstract/Free Full Text]
  18. Gingras AC, Gygi SP, Raught B, Polakiewicz RD, Abraham RT, Hoekstra MF, Aebersold R, Sonenberg N. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev 13: 1422–1437, 1999.[Abstract/Free Full Text]
  19. Goncharova EA, Goncharov DA, Eszterhas A, Hunter DS, Glassberg MK, Yeung RS, Walker CL, Noonan D, Kwiatkowski DJ, Chou MM, Panettieri RA Jr, Krymskaya VP. Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). J Biol Chem 277: 30958–30967, 2002.[Abstract/Free Full Text]
  20. Ha TS, Barnes JL, Stewart JL, Ko CW, Miner JH, Abrahamson DR, Sanes JR, Kasinath BS. Regulation of renal laminin in mice with type II diabetes. J Am Soc Nephrol 10: 1931–1939, 1999.[Abstract/Free Full Text]
  21. Hannken T, Schroeder R, Stahl RA, Wolf G. Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals. Kidney Int 54: 1923–1933, 1998.[CrossRef][ISI][Medline]
  22. Hardie DG. The AMP-activated protein kinase pathway—new players upstream and downstream. J Cell Sci 117: 5479–5487, 2004.[Abstract/Free Full Text]
  23. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of 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]
  24. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, Hardie DG. Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2: 9–19, 2005.[CrossRef][ISI][Medline]
  25. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 18: 1926–1945, 2004.[Abstract/Free Full Text]
  26. Holz MK, Ballif BA, Gygi SP, Blenis J. mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell 123: 569–580, 2005.[CrossRef][ISI][Medline]
  27. Hostetter TH. Progression of renal disease and renal hypertrophy. Annu Rev Physiol 57: 263–278, 1995.[CrossRef][ISI][Medline]
  28. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4: 648–657, 2002.[CrossRef][ISI][Medline]
  29. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115: 577–590, 2003.[CrossRef][ISI][Medline]
  30. Iseli TJ, Walter M, van Denderen BJ, Katsis F, Witters LA, Kemp BE, Michell BJ, Stapleton D. AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186–270). J Biol Chem 280: 13395–13400, 2005.[Abstract/Free Full Text]
  31. Itani SI, Saha AK, Kurowski TG, Coffin HR, Tornheim K, Ruderman NB. Glucose autoregulates its uptake in skeletal muscle: involvement of AMP-activated protein kinase. Diabetes 52: 1635–1640, 2003.[Abstract/Free Full Text]
  32. Kaneda K, Iwao J, Sakata N, Takebayashi S. Correlation between mitochondrial enlargement in renal proximal tubules and microalbuminuria in rats with early streptozotocin-induced diabetes. Acta Pathol Jpn 42: 855–860, 1992.[Medline]
  33. Kanwar Y, Yoshinaga Y, Liu Z, Wallner E, Carone F. Biosynthetic regulation of proteoglycans by aldohexoses and ATP. Proc Natl Acad Sci USA 89: 8621–8625, 1992.[Abstract/Free Full Text]
  34. Kasinath BS, Grellier P, Choudhury GG, Abboud SL. Regulation of basement membrane heparan sulfate proteoglycan, perlecan, gene expression in glomerular epithelial cells by high glucose medium. J Cell Physiol 167: 131–136, 1996.[CrossRef][ISI][Medline]
  35. Kasinath BS, Singh AK, Kanwar YS, Lewis EJ. Effect of puromycin aminonucleoside on HSPG core protein content of glomerular epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 255: F590–F596, 1988.[Abstract/Free Full Text]
  36. Katakam PV, Ujhelyi MR, Hoenig M, Miller AW. Metformin improves vascular function in insulin-resistant rats. Hypertension 35: 108–112, 2000.[Abstract/Free Full Text]
  37. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422–39427, 2003.[Abstract/Free Full Text]
  38. Lemieux K, Konrad D, Klip A, Marette A. The AMP-activated protein kinase activator AICAR does not induce GLUT4 translocation to transverse tubules but stimulates glucose uptake and p38 mitogen-activated protein kinases alpha and beta in skeletal muscle. FASEB J 17: 1658–1665, 2003.[Abstract/Free Full Text]
  39. Mariappan MM, Senthil D, Natarajan KS, Choudhury GG, Kasinath BS. Phospholipase C{gamma}-Erk Axis in vascular endothelial growth factor-induced eukaryotic initiation factor 4E phosphorylation and protein synthesis in renal epithelial cells. J Biol Chem 280: 28402–28411, 2005.[Abstract/Free Full Text]
  40. Merrill GF, Kurth EJ, Hardie DG, Winder WW. AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol Endocrinol Metab 273: E1107–E1112, 1997.[Abstract/Free Full Text]
  41. Miron M, Sonenberg N. Regulation of translation via TOR signaling: insights from Drosophila melanogaster. J Nutr 131: 2988S–2993S, 2001.[Abstract/Free Full Text]
  42. Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, Meyer TW. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 99: 342–348, 1997.[ISI][Medline]
  43. Preisig PA, Franch HA. Renal epithelial cell hyperplasia and hypertrophy. Semin Nephrol 15: 327–340, 1995.[ISI][Medline]
  44. Qi Z, Breyer MD. Diabetic nephropathy: big and bad. Kidney Int 68: 1896–1897, 2005.[CrossRef][ISI][Medline]
  45. Rao GN, Madamanchi NR, Lele M, Gadiparthi L, Gingras AC, Eling TE, Sonenberg N. A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells. J Biol Chem 274: 12925–12932, 1999.[Abstract/Free Full Text]
  46. Redpath NT, Price NT, Severinov KV, Proud CG. Regulation of elongation factor-2 by multisite phosphorylation. Eur J Biochem 213: 689–699, 1993.[ISI][Medline]
  47. Reiter AK, Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside. Am J Physiol Endocrinol Metab 288: E980–E988, 2005.[Abstract/Free Full Text]
  48. Senthil D, Choudhury GG, Abboud HE, Sonenberg N, Kasinath BS. Regulation of protein synthesis by IGF-I in proximal tubular epithelial cells. Am J Physiol Renal Physiol 283: F1226–F1236, 2002.[Abstract/Free Full Text]
  49. Senthil D, Choudhury GG, McLaurin C, Kasinath BS. Vascular endothelial growth factor induces protein synthesis in renal epithelial cells: a potential role in diabetic nephropathy. Kidney Int 64: 468–479, 2003.[CrossRef][ISI][Medline]
  50. Senthil D, Faulkner JL, Choudhury GG, Abboud HE, Kasinath BS. Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells. Biochem J 360: 87–95, 2001.[CrossRef][ISI][Medline]
  51. Shankland SJ, Wolf G. Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis. Am J Physiol Renal Physiol 278: F515–F529, 2000.[Abstract/Free Full Text]
  52. Sharma K, Jin Y, Guo J, Ziyadeh FN. Neutralization of TGF-beta by anti-TGF-beta antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 45: 522–530, 1996.[Abstract]
  53. Sharma K, Ziyadeh FN. Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-beta as a key mediator. Diabetes 44: 1139–1146, 1995.[Abstract]
  54. Singh AK, Kasinath BS. Metabolic fate of monovalent multivalent antibodies of Heymann nephritis following formation of surface immune complexes on glomerular epithelial cells. Clin Exp Immunol 94: 403–411, 1993.[ISI][Medline]
  55. Susztak K, Raff AC, Schiffer M, Bottinger EP. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes 55: 225–233, 2006.[Abstract/Free Full Text]
  56. Thornton S, Anand N, Purcell D, Lee J. Not just for housekeeping: protein initiation and elongation factors in cell growth and tumorigenesis. J Mol Med 81: 536–548, 2003.[CrossRef][ISI][Medline]
  57. Viollet B, Andreelli F, Jorgensen SB, Perrin C, Flamez D, Mu J, Wojtaszewski JF, Schuit FC, Birnbaum M, Richter E, Burcelin R, Vaulont S. Physiological role of AMP-activated protein kinase (AMPK): insights from knockout mouse models. Biochem Soc Trans 31: 216–219, 2003.[ISI][Medline]
  58. Wang X, Li W, Williams M, Terada N, Alessi DR, Proud CG. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J 20: 4370–4379, 2001.[CrossRef][ISI][Medline]
  59. Wolf G, Mueller E, Stahl RA, Ziyadeh FN. Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous transforming growth factor-beta. J Clin Invest 92: 1366–1372, 1993.[ISI][Medline]
  60. Wolf G, Schanze A, Stahl RA, Shankland SJ, Amann K. p27(Kip1) Knockout mice are protected from diabetic nephropathy: evidence for p27(Kip1) haplotype insufficiency. Kidney Int 68: 1583–1589, 2005.[CrossRef][ISI][Medline]
  61. Zeng ZZ, Yellaturu CR, Neeli I, Rao GN. 5(S)-hydroxyeicosatetraenoic acid stimulates DNA synthesis in human microvascular endothelial cells via activation of Jak/STAT and phosphatidylinositol 3-kinase/Akt signaling, leading to induction of expression of basic fibroblast growth factor 2. J Biol Chem 277: 41213–41219, 2002.[Abstract/Free Full Text]
  62. Ziyadeh FN, Snipes ER, Watanabe M, Alvarez RJ, Goldfarb S, Haverty TP. High glucose induces cell hypertrophy and stimulates collagen gene transcription in proximal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 259: F704–F714, 1990.[Abstract/Free Full Text]
  63. Zou MH, Kirkpatrick SS, Davis BJ, Nelson JS, Wiles WGt Schlattner U, Neumann D, Brownlee M, Freeman MB, Goldman MH. Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species. J Biol Chem 279: 43940–43951, 2004.[Abstract/Free Full Text]



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