Dual Regulation of Gluconeogenesis by Insulin and Glucose in the … · 2017-08-16 ·...

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Dual Regulation of Gluconeogenesis by Insulin and Glucose in the Proximal Tubules of the Kidney Motohiro Sasaki, 1,2 Takayoshi Sasako, 1,3,4 Naoto Kubota, 1,3,5,6,7 Yoshitaka Sakurai, 1 Iseki Takamoto, 1 Tetsuya Kubota, 1,6,7,8 Reiko Inagi, 9 George Seki, 10 Moritaka Goto, 2 Kohjiro Ueki, 1,3,4 Masaomi Nangaku, 9 Takahito Jomori, 2 and Takashi Kadowaki 1,3 Diabetes 2017;66:23392350 | https://doi.org/10.2337/db16-1602 Growing attention has been focused on the roles of the proximal tubules (PTs) of the kidney in glucose metabolism, including the mechanism of regulation of gluconeogenesis. In this study, we found that PT-specic insulin receptor substrate 1/2 double-knockout mice, established by using the newly generated sodiumglucose cotransporter 2 (SGLT2)-Cre transgenic mice, exhibited impaired insulin signaling and upregulated gluconeogenic gene expression and renal gluconeogenesis, resulting in systemic insulin resistance. In contrast, in streptozotocin-treated mice, al- though insulin action was impaired in the PTs, the gluconeo- genic gene expression was unexpectedly downregulated in the renal cortex, which was restored by administration of an SGLT1/2 inhibitor. In the HK-2 cells, the gluconeogenic gene expression was suppressed by insulin, accompanied by phosphorylation and inactivation of forkhead box transcrip- tion factor 1 (FoxO1). In contrast, glucose deacetylated per- oxisome proliferatoractivated receptor g coactivator 1-a (PGC1a), a coactivator of FoxO1, via sirtuin 1, suppressing the gluconeogenic gene expression, which was reversed by inhibition of glucose reabsorption. These data suggest that both insulin signaling and glucose reabsorption sup- press the gluconeogenic gene expression by inactivation of FoxO1 and PGC1a, respectively, providing insight into novel mechanisms underlying the regulation of gluconeo- genesis in the PTs. The kidney plays a pivotal role in systemic glucose metab- olism by regulation of glucose reabsorption, glycolysis, and gluconeogenesis. Gluconeogenesis occurs exclusively in the liver and the kidney (1). In the absorptive state, the kidney accounts for only 10% of the systemic gluconeogenesis, whereas in the prolonged fasting state, the rate rises to as much as 40% (2). Among precursors of gluconeogenesis, glutamine is used as the major glucogenic amino acid in the kidney, whereas alanine is used as the major amino acid in the liver (3,4). Gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), are expressed mainly in the proximal tubules (PTs) of the kidney and in the liver (5,6). In the process of gluconeogen- esis in the PTs, glucose diffuses outward through glucose transporter 2 (GLUT2) in the basolateral membrane (7,8). On the contrary, GLUT2 plays an important role in glucose uptake in the liver, and sodiumglucose cotransporters (SGLTs) in the luminal membrane are involved instead in inward efux of glucose in the PTs (9,10). It is well known that gluconeogenic gene expression in the liver is mainly regulated by insulin, especially via sup- pression of forkhead box transcription factor 1 (FoxO1). FoxO1 is a major transcription factor that binds to the promoter regions of PEPCK and G6Pase to induce the gene expression (1113). Insulin signaling activated after feeding, 1 Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 2 Mie Research Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., Mie, Japan 3 Translational Systems Biology and Medicine Initiative, The University of Tokyo, Tokyo, Japan 4 Department of Molecular Diabetic Medicine, Diabetes Research Center, National Center for Global Health and Medicine, Tokyo, Japan 5 Department of Clinical Nutrition Therapy, The University of Tokyo Hospital, The University of Tokyo, Tokyo, Japan 6 Clinical Nutrition Program, National Institute of Health and Nutrition, Tokyo, Japan 7 Laboratory for Metabolic Homeostasis, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan 8 Division of Cardiovascular Medicine, Toho University Ohashi Medical Center, Tokyo, Japan 9 Department of Nephrology and Endocrinology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 10 Yaizu City Hospital, Shizuoka, Japan Corresponding authors: Takashi Kadowaki, [email protected], and Naoto Kubota, [email protected]. Received 15 January 2017 and accepted 13 June 2017. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1602/-/DC1. M.S. and T.S. contributed equally to this work. © 2017 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for prot, and the work is not altered. More information is available at http://www.diabetesjournals .org/content/license. Diabetes Volume 66, September 2017 2339 METABOLISM

Transcript of Dual Regulation of Gluconeogenesis by Insulin and Glucose in the … · 2017-08-16 ·...

Dual Regulation of Gluconeogenesis by Insulin andGlucose in the Proximal Tubules of the KidneyMotohiro Sasaki,1,2 Takayoshi Sasako,1,3,4 Naoto Kubota,1,3,5,6,7 Yoshitaka Sakurai,1 Iseki Takamoto,1

Tetsuya Kubota,1,6,7,8 Reiko Inagi,9 George Seki,10 Moritaka Goto,2 Kohjiro Ueki,1,3,4 Masaomi Nangaku,9

Takahito Jomori,2 and Takashi Kadowaki1,3

Diabetes 2017;66:2339–2350 | https://doi.org/10.2337/db16-1602

Growing attention has been focused on the roles of theproximal tubules (PTs) of the kidney in glucose metabolism,including the mechanism of regulation of gluconeogenesis.In this study, we found that PT-specific insulin receptorsubstrate 1/2 double-knockout mice, established by usingthe newly generated sodium–glucose cotransporter 2(SGLT2)-Cre transgenic mice, exhibited impaired insulinsignaling and upregulated gluconeogenic gene expressionand renal gluconeogenesis, resulting in systemic insulinresistance. In contrast, in streptozotocin-treated mice, al-though insulin action was impaired in the PTs, the gluconeo-genic gene expression was unexpectedly downregulated inthe renal cortex, which was restored by administration of anSGLT1/2 inhibitor. In the HK-2 cells, the gluconeogenic geneexpression was suppressed by insulin, accompanied byphosphorylation and inactivation of forkhead box transcrip-tion factor 1 (FoxO1). In contrast, glucose deacetylated per-oxisome proliferator–activated receptor g coactivator 1-a(PGC1a), a coactivator of FoxO1, via sirtuin 1, suppressingthe gluconeogenic gene expression, which was reversedby inhibition of glucose reabsorption. These data suggestthat both insulin signaling and glucose reabsorption sup-press the gluconeogenic gene expression by inactivationof FoxO1 and PGC1a, respectively, providing insight intonovel mechanisms underlying the regulation of gluconeo-genesis in the PTs.

The kidney plays a pivotal role in systemic glucose metab-olism by regulation of glucose reabsorption, glycolysis, andgluconeogenesis. Gluconeogenesis occurs exclusively in theliver and the kidney (1). In the absorptive state, the kidneyaccounts for only 10% of the systemic gluconeogenesis, whereasin the prolonged fasting state, the rate rises to as much as 40%(2). Among precursors of gluconeogenesis, glutamine is usedas the major glucogenic amino acid in the kidney, whereasalanine is used as the major amino acid in the liver (3,4).

Gluconeogenic enzymes, including phosphoenolpyruvatecarboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase),are expressed mainly in the proximal tubules (PTs) of thekidney and in the liver (5,6). In the process of gluconeogen-esis in the PTs, glucose diffuses outward through glucosetransporter 2 (GLUT2) in the basolateral membrane (7,8).On the contrary, GLUT2 plays an important role in glucoseuptake in the liver, and sodium–glucose cotransporters(SGLTs) in the luminal membrane are involved instead ininward efflux of glucose in the PTs (9,10).

It is well known that gluconeogenic gene expression inthe liver is mainly regulated by insulin, especially via sup-pression of forkhead box transcription factor 1 (FoxO1).FoxO1 is a major transcription factor that binds to thepromoter regions of PEPCK and G6Pase to induce the geneexpression (11–13). Insulin signaling activated after feeding,

1Department of Diabetes and Metabolic Diseases, Graduate School of Medicine,The University of Tokyo, Tokyo, Japan2Mie Research Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., Mie, Japan3Translational Systems Biology and Medicine Initiative, The University of Tokyo,Tokyo, Japan4Department of Molecular Diabetic Medicine, Diabetes Research Center, NationalCenter for Global Health and Medicine, Tokyo, Japan5Department of Clinical Nutrition Therapy, The University of Tokyo Hospital, TheUniversity of Tokyo, Tokyo, Japan6Clinical Nutrition Program, National Institute of Health and Nutrition, Tokyo,Japan7Laboratory for Metabolic Homeostasis, RIKEN Center for Integrative MedicalSciences, Kanagawa, Japan8Division of Cardiovascular Medicine, Toho University Ohashi Medical Center,Tokyo, Japan

9Department of Nephrology and Endocrinology, Graduate School of Medicine, TheUniversity of Tokyo, Tokyo, Japan10Yaizu City Hospital, Shizuoka, Japan

Corresponding authors: Takashi Kadowaki, [email protected], andNaoto Kubota, [email protected].

Received 15 January 2017 and accepted 13 June 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1602/-/DC1.

M.S. and T.S. contributed equally to this work.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 66, September 2017 2339

METABOLISM

however, promotes translocation of FoxO1 to the cytoplasm,consequently suppressing gluconeogenic gene expression viathe insulin receptor substrates (IRSs) and Akt (14,15). Wehave previously reported enhanced hepatic gluconeogenicgene expression in liver-specific IRS1/2 double-knockoutmice, which was reversed by inactivation of FoxO1 (16).

Similarly, it was shown using PT-specific insulin receptor(IR) knockout mice (17) that insulin also downregulatedgluconeogenic gene expression in the PTs of the kidney.Rich expression of the IRs is seen in the renal cortex, in-cluding in the PTs (17,18), and it is assumed that the IRs onthe basolateral side sense plasma insulin and play an impor-tant role in intracellular signaling (19). However, the role ofinsulin in the regulation of gluconeogenesis in the kidney isstill under debate, because suppression as well as elevationof gluconeogenic gene expression has been reported fromexperiments in rodents in which insulin secretion was sup-pressed by treatment with streptozotocin (STZ) (19,20). It isthus suggested that other potential mechanisms could exist.

Another molecule to regulate gluconeogenic gene ex-pression in the liver is sirtuin 1 (Sirt1), known as an NAD-dependent deacetylase, by deacetylating peroxisomeproliferator-activated receptor g coactivator 1a (PGC1a),which plays a pivotal role as a coactivator of FoxO1 in thetranscription of gluconeogenic genes (21,22). In the field ofnephrology, various roles of Sirt1 in the kidney have beenreported (23–26), and, especially in the context of the re-sponses to nutrition, its importance seems to be highlightedin the presence of relatively low glucose concentrations, bothin vitro and in vivo (27). Involvement of Sirt1 in gluconeo-genesis in the PTs, however, remains to be clarified.

In this study, we focused on insulin signaling using aPT-specific knockout mouse model, as well as on glucose re-absorption by SGLTs, and explored the mechanisms un-derlying the regulation of gluconeogenic gene expression inthe PTs of the kidney.

RESEARCH DESIGN AND METHODS

Generation of the Transgenic MiceTo generate SGLT2-cre transgenic (Tg) mice with a con-struct according to a previous report (28), we obtained agenomic fragment of Slc5a2 (encoding SGLT2) including thepromoter region, exon 1, intron 1, and the first part of exon2 from the RP24–178K1 BAC clone (BACPAC Resources)with the BAC Subcloning Kit (Gene Bridges). It was insertedupstream of the Cre recombinase DNA that was flanked byrabbit b-globin and a polyadenylation sequence, as previ-ously described (29), following deletion of the start codonusing the KOD -Plus- Mutagenesis Kit (TOYOBO). The lin-earized construct was injected into the pronuclei of fertil-ized C57BL/6J embryos, and two-cell stage embryos weretransferred into the oviduct of a pseudopregnant mouse.After confirmation of germline transmission, the Tg micewere crossed with ROSA26-LacZ mice (003474; TheJackson Laboratory) to analyze the expression of Crerecombinase. Then, the Tg mice were crossed with

IRS1/2-floxed mice (16) for generating mice with PT-specific knockout of the genes.

AnimalsC57BL/6J and ob/obmice were purchased from CLEA Japan.C57BL/6NCr Slc and Akita/Slc mice were purchased fromSLC Japan. All mice were housed under a 12-h light/12-hdark cycle and had free access to sterile water and regularchow, CE-2 (CLEA Japan). In the experiments conducted inthe fasting and fed states, the mice were denied access topellet food or given access to pellet food after 24-h fastingin individual cages. The animal care and experimental pro-cedures were approved by the Animal Care Committee ofThe University of Tokyo.

Chemical TreatmentSTZ (Sigma-Aldrich) was diluted in citrate sodium bufferand administered intraperitoneally at the dose of 180 mg/kgbody weight (BW) twice at an interval of 4 days. Mice wereused for the experiments 10 days after the treatments.Phlorizin (PHZ; Sigma-Aldrich) was dissolved in a solutioncontaining 10% ethanol, 15% DMSO, and 75% saline andinjected subcutaneously.

Metabolic StudiesThe insulin tolerance test, pyruvate tolerance test, glutaminetolerance test, and alanine tolerance test were performedafter the mice had been denied access to food for 6 h, unlessotherwise indicated. Mice were injected intraperitoneallywith insulin (0.75 units/kg: Humulin R; Eli Lilly Japan),pyruvate (2.0 g/kg; Wako), glutamine (2.0 g/kg; Biowest), oralanine (2.0 g/kg; Wako) (16,30). Blood glucose levels weremeasured using the Glutest sensor (Sanwa Kagaku Kenkyusho)at the indicated time points. The plasma insulin levels weremeasured using an ELISA kit (Morinaga).

Histological Analysisb-Galactosidase staining was performed using the b-GalStaining Set (Roche), according to the manufacturer’s in-structions. For macroscopic analysis, tissues were fixed in4% paraformaldehyde before the staining, and images wereacquired with Optio WG-2 (RICOH). For microscopic anal-ysis, tissues were first embedded in optimal cutting temper-ature compound (Sakura Finetek Japan), frozen on dry ice,and then carved out for fixation with 2% glutaraldehyde/PBSat room temperature. After b-galactosidase staining, thespecimens were re-embedded in paraffin and sectionedfor periodic acid Schiff (PAS) staining, and images wereacquired with BZ-X710 (Keyence).

Laser MicrodissectionFrozen tissues were cut into 15-mm–thick sections andmounted onto a PEN-Membrane (Leica Microsystems). Thesesections were stained with PAS and excised using the LaserMicrodissection DM6000B (Leica Microsystems).

RNA Preparation and Quantitative PCRThe RNeasy Mini Kit (Qiagen) was used to prepare totalRNA from the mouse tissues and cultured cells, and theRNeasy Micro Mini Kit (Qiagen) was used to prepare total

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RNA from the microdissected tissues. Reverse-transcriptionreaction was carried out with a High Capacity cDNA ReverseTranscription Kit (Applied Biosystems) after treatment withDNase (Promega). Quantitative PCR analyses were per-formed using ABI Prism 7900, with Power SYBR Green PCRMaster Mix (Applied Biosystems) (16). The relative expres-sion levels after normalization to the expression level ofcyclophilin were compared.

PrimersThe primers used for the RT-PCR were as follows: mousePEPCK forward, 59-CCCCTTGTCTATGAAGCCCTCA-39 re-verse, 59-GCCCTTGTGTTCTGCAGCAG-39; mouse G6Paseforward, 59-TTACCAAGACTCCCAGGACTG-39 and reverse,59-GAGCTGTTGCTGTAGTAGTCG-39; mouse Podocin forward,59-TGAAAGAGTAATTATATTCCGACTGG-39 and reverse,59-TGATAGGTGTCCAGACAGGGTAA-39; mouse Megalinforward, 59-AGGCCACCAGTTCACTTGCT-39 and reverse,59-AGGACACGCCCATTCTCTTG-39; mouse aquaporin 2forward, 59-TAGCCCTGCTCTCTCCATTG-39 and reverse,59-GAGCAGCCGGTGAAATAGAT-39; mouse IRS1 forward,59-CTATGCCAGCATCAGCTTCC-39 and reverse, 59-TTGCT-GAGGTCATTTAGGTCTTC-39; mouse IRS2 forward, 59-TC-CAGGCACTGGAGCTTT-39 and reverse, 59-GGCTGGTAGC-GCTTCACT-39; mouse Hk1 forward, 59-CGGAATGGGG-AGCCTTTGG-39 and reverse, 59-GCCTTCCTTATCCGTTTC-AATGG-39; mouse Hk2 forward, 59-TGATCGCCTGCTTAT-TCACGG-39 and reverse, 59-AACCGCCTAGAAATCTCCAGA-39; mouse Gck forward, 59- GTGGCCACAATGATCTCCTGC-39 and reverse, 59- TCGGCGACAGAGGGTCGAAGGC-39;mouse SGLT1 forward, 59-TCTGTAGTGGCAAGGGGAAG-39and reverse, 59-ACAGGGCTTCTGTGTCTTGG-39; mouseSGLT2 forward, 59- TATTGGTGCAGCGATCAGG-39 and re-verse, 59- CCCAGCTTTGATGTGAGTCAG-39; mouse GLUT1forward, 59-CGTGCTTATGGGTTTCTCCAAA-39 and reverse,59-GACACCTCCCCCACATACATG-39; mouse GLUT2 for-ward, 59-TTTGCAGTAGGCGGAATGG-39 and reverse, 59-GCCAACATGGCTTTGATCCTT-39; mouse GLUT4 forward,59-CAACTGGACCTGTAACTTCATCGT-39 and reverse, 59-ACGGCAAATAGAAGGAAGACGTA-39; mouse cyclophilinforward, 59-GAGCTGTTTGCAGACAAAGTTC-39 and reverse,59-CCCTGGCACATGAATCCTGG-39; human PEPCK for-ward, 59-TGAGCTGTGTCAGCCTGATCAC-39 and reverse,59-ACCGTCTTGCTTTCGACCTG-39; human cyclophilin for-ward, 59- ATGCTGGACCCAACACAAAT-39 and reverse, 59-TCTTTCACTTTGCCAAACACC-39.

Immunoprecipitation and Western Blot AnalysisTo prepare the lysates, mouse tissues and cells were homog-enized in buffer A (25 mmol/L Tris-HCl [pH 7.4], 10 mmol/Lsodium orthovanadate, 10 mmol/L sodium pyrophosphate,100 mmol/L sodium fluoride, 10 mmol/L EDTA, 10 mmol/LEGTA, and protease inhibitor). For immunoprecipitation (IP)of IR, IRS1, IRS2, and PGC1a, 4 mg lysates was incubatedwith specific antibodies, respectively, overnight at 4°C.Then, protein G-Sepharose was added, followed by incuba-tion for 1 h at 4°C. After washing three times with buffer A,the target proteins were eluted with sample loading buffer.

The lysates were resolved on SDS-PAGE and transferred topolyvinylidene difluoride membranes using the Trans-BlotTurbo Transfer System (Bio-Rad) (16).

AntibodiesAnti-IRS1 (IP and immunoblotting [IB]: 06–248), anti-IRS2(IB: MABS15), and anti-Sirt1 antibodies (05–1243) were pur-chased from Merck Millipore. Anti-IRS2 (IP: 3089), anti-FoxO1 (9454), anti-phosphorylated (p-)FoxO1 (Ser256)(9461), anti-Akt (9272), anti–p-Akt (Ser473) (9271), andanti-PGC1a antibodies (IB: 2178) were purchased fromCell Signaling Technology. b-Actin (sc-1616), insulin Rb(IP and IB: sc-711), p-Tyr (pY99) (sc-7020), and anti-PGC1 antibodies (IP: sc-13067) were purchased fromSanta Cruz Biotechnology.

Urine Electrolyte AnalysisMice were housed in metabolic cages (Shinano Seisakusho)for 24 h to collect the urine samples for analysis of thesodium, potassium, chloride levels, and osmolality usingPVA-EX II (A&T) and an osmometer (model 3320; Ad-vanced Instruments, Inc.), respectively.

Cell CultureHuman kidney PT (HK-2) cells were purchased from Lonzaand maintained at 37°C in a humidified 5% CO2 incubator.The cells were grown in DMEM/Hams F12 medium (Gibco)supplemented with 10% FBS, 5% antibiotics, and 2.5 mmol/Lglutamine and subcultured to 80% confluence using 0.05%trypsin-EDTA (Gibco).

In experiments, HK-2 cells were cultured in DMEM/F12(Biowest) with low or high glucose concentrations (5 or30 mmol/L, respectively), with or without 50 mmol/L PHZovernight. The cells were harvested after stimulation with100 mmol/L 8CPT-cAMP (Abcam) and 100 nmol/L dexa-methasone (Wako), with or without 100 nmol/L insulinor supplemented with or without 1 mmol/L glutamine,for 4 h (31,32). The NADH/NAD+ ratio in the HK-2 cellsand the glucose concentrations in the medium were mea-sured using the NAD+/NADH Quantification ColorimetricKit (BioVision) and the Glucose Assay Kit (Cell Biolabs,Inc.), respectively.

RNA InterferenceSmall interfering RNAs targeting murine FoxO1, Sirt1, andPGC1a were purchased from Ambion (Silencer Select Pre-designed siRNA: ASO224JQ, ASO224JW, and ASO224JR,respectively; Thermo Fisher Scientific) and transfected intocultured cells using Lipofectamine RNAiMax Reagent (LifeTechnologies) according to the manufacturer’s instructions.

Statistical AnalysisData are expressed as means 6 SEM, and statistical signif-icance was set at P , 0.05 (one asterisk) or P , 0.01 (twoasterisks). Differences between two groups were assessed byunpaired two-tailed t tests, unless otherwise indicated,whereas those among three or more groups were assessedby one-way ANOVA with post hoc Tukey honest significantdifference in EZ-R (33).

diabetes.diabetesjournals.org Sasaki and Associates 2341

RESULTS

Insulin Signaling and Its Related Gene Expression in theRenal Cortex in the Fasting and Fed StatesFirst, we investigated insulin signaling in the renal cortex inthe fed state in wild-type mice. As the serum insulin levelsas well as blood glucose levels became higher (Fig. 1A),tyrosine phosphorylation of the IRSs, as well as phosphor-ylation of downstream molecules, including Akt and FoxO1,was enhanced (Fig. 1B). Feeding and treatment with anexcess dose of insulin enhanced phosphorylation of the in-sulin signal cascade to an equivalent degree (Fig. 1B). Theexpression of the gluconeogenic genes, including PEPCKand G6Pase, was downregulated in the renal cortex (Fig.1C). Similar changes were seen in the liver, as previouslyreported by us (Supplementary Fig. 1A and B) (16). Thesedata suggest that the expression levels of the gluconeogenic

gene expression could be inversely associated with insulinsignaling in the renal cortex in the fed state.

Expression of the IRSs and Gluconeogenic Enzymesin the PTsTo explore the insulin actions in the kidney, we used lasermicrodissection (LMD) to investigate the precise distribu-tion of the insulin signaling-related genes in the kidney,which was poorly understood, even though the IRs hadbeen reported to be abundantly expressed in the renalcortex (17–19). We identified and isolated the glomeruli,PTs, and distal tubules (DTs) morphologically in PAS-stained sections prepared from wild-type mice (Fig. 2A),and successfully confirmed rich expression of the distribu-tion markers, namely, of podocin in the glomeruli, megalinin the PTs, and aquaporin 2 in the DTs (Fig. 2B) (34).Among the insulin signal-related genes IRS1 and IRS2, as

Figure 1—Insulin (Ins) signaling and its related gene expression in the renal cortex depending on the feeding condition of the animals. C57BL/6J-type mice were allowed access to food ad libitum for 6 h after denial of access to food for 24 h. Blood glucose and serum insulin levels (n = 4) (A),insulin signaling analyzed by Western blotting (n = 4) (B), and gluconeogenic gene expression analyzed by RT-PCR (n = 4) (C). Values are themean 6 SE. *P < 0.05; **P < 0.01 compared with fasting for 24 h (refed 0 h).

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well as IR, were expressed in all three segments at expres-sion levels that were roughly equivalent to those found inthe liver. Gluconeogenic enzymes, SGLTs and GLUT2 amongthe GLUTs, were abundantly expressed in the PTs (Fig. 2C).

Insulin Mediates Gluconeogenesis in the PTsThese data prompted us to investigate the roles of insulinsignaling in the regulation of gluconeogenesis in the PTsusing genetically modified mouse models. We generatedmice with IRS1/2 knockout specifically in the PTs, becausewe intended to explore the roles of insulin signaling byfocusing on IRS1 and IRS2 as key molecules of the insulin-signaling cascade. First, we generated SGLT2-cre Tg mice toexpress Cre recombinase in the PTs, with the construct

prepared according to a previous report (SupplementaryFig. 2A) (28). The SGLT2-cre mice appeared to show effi-cient expression of the Cre recombinase in the PTs, where-as no expression was detected in any of the other tissues(Supplementary Fig. 2B and C). We then crossed the Tgmice with IRS1/2-floxed mice, which yielded PT-specificIRS1/2 double-knockout (SIRS1/2DKO) mice. Both theIRS1 and IRS2 mRNA levels were significantly lower in thePTs, but not in the glomeruli or the DTs, as isolated byLMD, of the SIRS1/2DKO mice (Fig. 3A). In the renal cor-tex, at the protein level, expression of IRS1 and IRS2 wasalso reduced (Fig. 3B). Besides, insulin-mediated tyrosinephosphorylation of the IRS1 and IRS2 proteins and, conse-quently, phosphorylation of downstream molecules Akt and

Figure 2—Expression of the IRSs and gluconeogenic enzymes in the PTs. A representative PAS-stained section of the kidney for identificationof the glomeruli (red circles), PTs (blue boxes), and DTs (green frames) (A) and analysis by RT-PCR of gene expression in segments isolated byLMD (B and C) using C57BL/6J mice allowed access to food ad libitum for 6 h after denial of access to food for 24 h (n = 7–8). Values are themean 6 SE. **P < 0.01 compared with fasting.

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FoxO1 were markedly attenuated (Fig. 3B), suggesting theexistence of an insulin signaling defect in the PTs of theSIRS1/2DKO mice.

The SIRS1/2DKO mice showed scarce differences in the BW(Fig. 3C), blood glucose levels (Fig. 3D), urine electrolytes

(Supplementary Fig. 3A), and histological findings (Sup-plementary Fig. 3B). Although these mice failed to ex-hibit glucose intolerance (Supplementary Fig. 3C), theyshowed elevated blood glucose levels after administrationof glutamine (Fig. 3E) and pyruvate (Fig. 3F), but not after

Figure 3—Insulin mediates gluconeogenic gene expression in the PTs. Validation of SIRS1/2DKO mice after denial of access to food for 16 h:analysis by RT-PCR of gene expression of the IRSs in segments isolated by LMD (n = 8) (A) and analysis by Western blotting of insulin signalingin the renal cortex 10 min after insulin injection via the inferior vena cava at the dose of 0.1 units/kg BW (B). Metabolic phenotypes ofSIRS1/2DKO mice denied access to food for 6 h: BW (C); blood glucose levels (D); blood glucose levels after glutamine challenge (E), pyruvatechallenge (F), alanine challenge (G), and insulin challenge (H); and gluconeogenic gene expression in microdissected segments were analyzedby RT-PCR (n = 8–11) (I). Values are the mean 6 SE. *P < 0.05; **P < 0.01 compared with control mice.

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administration of alanine, a major substrate in the liver(Fig. 3G). The SIRS1/2DKO mice also showed systemic in-sulin resistance (Fig. 3H) and consistently elevated gluco-neogenic gene expression in the PTs (Fig. 3I). Thesephenotypes were not seen in the SGLT2-cre Tg mice (Sup-plementary Fig. 2E–G) or PT-specific IRS1 or IRS2 single-knockout mice (Supplementary Fig. 4). These data suggestthat insulin signaling, mediated by both IRS1 and IRS2,suppresses gluconeogenesis in the PTs and affects systemicglucose metabolism.

SGLT Inhibition Modulates Gluconeogenic GeneExpressionNext, we investigated the gluconeogenic gene expressionin other mouse models: ob/obmice, a model of hyperglycemiaand hyperinsulinemia (Fig. 4A and B), and mice treated withSTZ, a model of hyperglycemia and hypoinsulinemia (Fig.4C and D). In the ob/obmice, gluconeogenic gene expressionwas downregulated in the renal cortex in the fed state,although they were upregulated rather than downregulatedin the liver (Fig. 4B). Moreover, we analyzed the latter modeljust 10 days after the treatment to eliminate secondary effectsand found that, contrary to the findings in the SIRS1/2DKOmice, the gluconeogenic gene expression was downregulated inthe renal cortex in the fed state, although they were upregulatedin the liver (Fig. 4D). These data suggest the existence ofmechanisms other than insulin signaling that regulate thegluconeogenic gene expression profile unique to the PTs.

We then focused on glucose, another potential regulatorof gluconeogenesis, whose uptake is mediated by theSGLTs in the PTs, and analyzed the primary impact ofSGLT inhibition by a single administration of an SGLT1/2inhibitor, PHZ. The administration to the STZ-treatedmice reduced the blood glucose levels to almost thosefound in the control mice, without elevating the seruminsulin levels (Fig. 4E). Interestingly, treatment with PHZrestored the gluconeogenic gene expression to levels similarto those seen in the untreated control mice (Fig. 4F). Sim-ilar results were observed in Akita mice, a model of chronichypoinsulinemia and hyperglycemia (Supplementary Fig. 5).

We further explored the impact of SGLT1/2 inhibition inthe SIRS1/2DKO mice, a model of suppressed insulin actionsin the PTs and normoglycemia. A single administration ofPHZ lowered the blood glucose and plasma insulin levels in asimilar manner in both the SIRS1/2DKO mice and floxedmice (Fig. 4G). As expected, the enhanced gluconeogenicgene expression in the SIRS1/2DKO mice was further en-hanced by the PHZ treatment (Fig. 4H). These data suggestthat the gluconeogenic gene expression in the PTs can bemodulated not only by insulin signaling, but also by thereabsorbed glucose via the SGLTs, and also that the impactof the latter could be larger than that of the former undercertain conditions.

Insulin and Glucose Suppress Gluconeogenic GeneExpression via FoxO1 and Sirt1/PGC1aWe then explored the molecular mechanisms underlyingthe regulation of the gluconeogenic gene expression by

insulin and glucose in vitro. In HK-2 cells, a cell line derivedfrom human PT cells, insulin promoted Akt phosphoryla-tion (Fig. 5A) and suppressed PEPCK expression (Fig. 5B).Knocking down of FoxO1 mimicked the suppressed expres-sion of PEPCK seen under the condition of insulin stimu-lation (Fig. 5A and B).

We also found that high glucose levels suppressedPEPCK expression in the HK-2 cells, which was reversedby treatment with PHZ (Fig. 5C). Moreover, the glucoselevels were elevated in the medium supplemented with glu-tamine under a low-glucose condition, but not a high-glucosecondition, suggesting that the gluconeogenesis in the PTs isglucose dependent (Fig. 5D). We focused on Sirt1, because ithas been reported to promote gluconeogenesis in the liverby deacetylation of PGC1a under the condition of a reducedNADH/NAD+ ratio (22). In the HK-2 cells, the NADH/NAD+ ratio was elevated in the presence of high glucose levels,which was reversed by PHZ treatment (Fig. 5E). Althoughhigh glucose levels did not alter the expression levels ofSirt1, they enhanced acetylation of PGC1a, suggesting sup-pression of the activity of Sirt1 as a deacetylase, which wasabrogated by PHZ treatment (Fig. 5F). Consistent withthese data, acetylation of PGC1a was enhanced in the renalcortex of the STZ-treated mice, which was abrogated byPHZ treatment (Fig. 5G). Knockdown of either Sirt1 orPGC1a replicated the suppressed expression of PEPCK seenunder high glucose conditions (Fig. 5H and I). These datasuggest that gluconeogenic gene expression was regulatedby not only insulin but also glucose via suppression of theactivities of FoxO1 and Sirt1/PGC1a, respectively.

DISCUSSION

Although the kidney has been known to play an importantrole in systemic glucose metabolism, the mechanism ofregulation of gluconeogenesis in the PTs remains poorlyunderstood. In this study, we suggest novel mechanismsunique to the PTs of the kidney: gluconeogenesis could becollaboratively regulated by both insulin signaling andthe reabsorbed glucose in the PTs. In the fasting state,suppressed insulin signaling transduced by the IR/IRSsexpressed on the basolateral side increases FoxO1 activity.In parallel, decreased glucose reabsorption via SGLT2 on theluminal side downregulates the NADH/NAD+ ratio, activat-ing Sirt1 and PGC1a. Therefore, both pathways result inenhanced gluconeogenesis. Inversely, in the fed state, glu-coneogenic gene expression is suppressed by both activatedinsulin signaling and increased glucose reabsorption (Fig. 6).

Just as in the liver, insulin signaling in the PTs isactivated in the fed state: the IRSs and Akt are phosphor-ylated, followed by suppressed gluconeogenic gene expres-sion. Moreover, our findings in the microdissected tissuesindicate that the expression levels of IRS1 and IRS2 in thePTs are similar to those in the liver, suggesting that theycould play important roles in glucose homeostasis. Toexplore their roles in the PTs, we successfully generatedSGLT2-cre Tg mice and then PT-specific IRS1/2 DKO mice.

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Indeed, residual expression of the IRSs and phosphorylationof Akt were detected by Western blotting in the renalcortex, but this finding could be attributable to inclusion ofcells other than the PT cells, as was argued for the case ofthe g-glutamyl transferase–driven PT-specific IR knockoutmice (17). Still, gluconeogenic phenotypes were clearly

observed in the SIRS1/2DKO mice, suggesting that reducedexpression, even if not amounting to complete knockout ofthe IRSs, is sufficient to disrupt physiological glucose me-tabolism in the PTs.

The knockout mice showed renal and systemic insulinresistance and enhanced renal gluconeogenesis, phenotypes

Figure 4—SGLT inhibition modulates gluconeogenic gene expression. ob/ob mice (A and B) and C57BL/6J mice (C and D) treated with STZwere allowed access to food ad libitum for 6 h after 24 h of denial of access to food. A and C: Blood glucose and serum insulin levels. B and D:Gluconeogenic gene expression in the renal cortex and liver analyzed by RT-PCR (n = 4–7). E and F: C57BL/6J mice treated with STZ wereadministered PHZ in a period of ad libitum access to food. Blood glucose and serum insulin levels (E) and gluconeogenic gene expression in therenal cortex analyzed by RT-PCR (n = 7–13) (F). G and H: SIRS1/2DKO mice were administered PHZ in a period of ad libitum access to food.Blood glucose and serum insulin levels (G) and gluconeogenic gene expression in the microdissected PTs analyzed by RT-PCR (n = 6–8) (H).Values are the mean 6 SE. *P < 0.05; **P < 0.01 compared with control mice; †P < 0.05; ††P < 0.01 compared with PHZ-treated mice.

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that were compatible with the phenotypes of g-glutamyltransferase–driven PT-specific IR knockout mice (17), sug-gesting that insulin signaling through IRS1 and IRS2 in thePTs plays important roles in systemic glucose homeostasis.

These phenotypes are not seen in PT-specific IRS1 or IRS2single-knockout mice, suggesting that each of the IRSs cancompensate for the lack of the other in the PTs. This redun-dancy is not seen in insulin-mediated sodium reabsorption in

Figure 5—Insulin and glucose regulate gluconeogenic gene expression via FoxO1 and PGC1a in HK-2 cells. HK-2 cells were treated with insulinand knockdown of FoxO1 (n = 6–8) (A and B), glucose, and PHZ (C, E, and F) (n = 6–12). D: HK-2 cells were treated with low (5 mmol/L; LG) andhigh (30 mmol/L; HG) glucose conditions with or without 1 mmol/L glutamine (Gln). G: C57BL/6J mice treated with STZ were administered PHZin a period of ad libitum access to food. HK-2 cells were treated with glucose and knockdown of Sirt1 and PGC1a (H and I) (n = 6–8). Proteinlevels analyzed by Western blotting (A and F–H), gluconeogenic gene expression analyzed by RT-PCR (B, C, and I), glucose concentrationsof the medium (D), and the NADH/NAD+ ratio (E). Values are the mean 6 SE. *P < 0.05; **P < 0.01 compared untreated low-glucose group;†P < 0.05 compared with untreated high-glucose group.

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the PTs, in which IRS2 plays a pivotal role (35,36), or in theregulation of glucose and lipid metabolism in the liver, inwhich IRS1 and IRS2 show functional differences (16), al-though FoxO1 is shared as the key downstream molecule ofinsulin signaling.

In another model of impaired insulin action in the PTs inthe STZ-treated mice, we analyzed gluconeogenic geneexpression relatively soon after the treatment and foundthat it was suppressed in the fed state, in clear contrast tothe case in the liver, in which it was enhanced in the fedstate. Similar results were obtained also in Akita mice, amodel of chronic impaired insulin action. Existence ofmechanisms other than insulin signaling was suggested,and we focused on the differences in the blood glucoselevels among the three models: SIRS1/2DKO mice showedalmost normoglycemia, whereas the STZ-treated mice andAkita mice showed hyperglycemia. We further hypothesizedthat the gene expression could be affected by the glucosereabsorbed via the SGLTs in the luminal membrane inparallel with primary urine glucose. Acute glucose admin-istration and SGLT1/2 inhibition modulated the geneexpression and activity of the gluconeogenic enzymes, asreported previously (5,37,38). Together with the results ofthe experiments involving a single PHZ treatment of theSIRS1/2DKO mice and those involving HK2 cells treatedwith PHZ, these data show the importance of glucose reab-sorption in the regulation of gluconeogenic gene expression

in the PTs, sharing the downstream molecules Sirt1 andPGC1a in response to the inward efflux of glucose withthe liver.

Such dual regulation of gluconeogenesis could havedeveloped because the PTs are a unique tissue that releasesglucose into the general circulation via two energy-consumingmechanisms, namely gluconeogenesis and glucosereabsorption. PTs are different from the liver in that theyreabsorb glucose to prevent external glucose loss in theurine. Theoretically, to release a single molecule of glucoseinto the bloodstream, glucose reabsorption mediated bySGLT1 and SGLT2 requires only two out of three and oneout of three molecules of ATP (39), respectively, whereasgluconeogenesis requires six molecules of ATP. It is reason-able that in the fed state, which is associated with a highrisk of external glucose loss, ATP-consuming gluconeogen-esis is suppressed, not only by insulin signaling, but alsoby glucose reabsorption. It is not until the fasting state isreached that the inability to reabsorb sufficient glucose,together with inactivated insulin signaling, promotes theATP-consuming gluconeogenesis. In this context, the PTsare in clear contrast with the liver, which is equipped withonly a single mechanism of glucose release, namely gluco-neogenesis, which seems to be regulated mainly by insulin.

Another difference between PTs and the liver lies in howto handle ammonia produced in breakdown of glucogenicamino acids for gluconeogenesis. In the PTs, production of

Figure 6—Schematic description of the hypothesis: gluconeogenic gene expression is dually regulated by insulin and the reabsorbed glucosesignaling in the PTs of the kidney.

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ammonia is important in maintenance of acid–base homeo-stasis, and excessive ammonia is discharged directly intourine (40,41), whereas in the liver, ammonia is convertedto urea by consuming additional ATP. That could accountfor the reason why the PTs prefer glutamine, which con-tains an amino group in addition to an amide group andproduces two molecules of ammonia, and the liver prefersalanine, which lacks an amino group and produces a singlemolecule of ammonia. Moreover, the PTs could have ac-quired such preference to cope with a prolonged fasting,which is characterized not only by lowered plasma glucoselevels but also acidosis because of enhanced ketogenesis,because glutamine can be used for efficient production ofammonia to maintain the acid–base balance, as well as glu-coneogenesis. In this sense, the PTs could have played muchlarger roles in survival through the maintenance of homeo-stasis in the prehistoric era when the humans were forcedto cope with severe fasting.

In clinical practice, SGLT2 inhibitors are drawingattention as a new class of antidiabetic agents (42). Ourdata, however, show that SGLT inhibition actually enhancesgluconeogenic gene expression in the PTs via interferingwith the physiological glucose-sensing mechanism of thePTs and generating a discrepancy between the primaryurine glucose and reabsorbed glucose levels. Recently, wereported that administration of an SGLT2 inhibitor en-hanced the gluconeogenic gene expression in the liver(43). Precursors of gluconeogenesis are known to be in-creased in diabetes (32) and possibly further increased bySGLT2 inhibition via hypercatabolism in the skeletal muscle(38). Therefore, these progluconeogenic aspects of SGLT2inhibitors, possibly contributing to prevention of hypogly-cemia, should be kept in mind when using the drug inclinical practice.

Moreover, our data also suggest potential mechanismsunderlying hypoglycemia in chronic renal failure (CRF),which is a clinically important issue. Hyperinsulinemia isknown to be caused in CRF, probably because of loweredrenal insulin clearance, which could suppress the gluconeo-genic gene expression in the PTs. Moreover, Sirt1 was re-ported to be inactivated in renal diseases (44), which couldalso suppress the expression. ATP-consuming gluconeogen-esis might be further impaired by inability of the hypoxicPTs in CRF to produce ATP (45), altogether resulting indecreased insulin requirement and elevated risk of hypogly-cemia in CRF.

Taken together, these findings suggest that gluconeo-genesis is dually regulated in the kidney by both thereabsorbed glucose via the SGLTs in the luminal membraneand insulin signaling transduced from the basolateralmembrane in the PTs. Our findings provide a novel insightinto the unique mechanisms underlying the regulation ofgluconeogenesis in the PTs.

Acknowledgments. The authors thank Ayumi Nagano, Eishin Hirata, AyumiOhuchi, Yuka Kobayashi, Yuko Kanto, Ritsuko Hoshino, Yoshiko Ito, and Naoki

Ishikawa, all of whom are at The University of Tokyo, for excellent technical assistanceand assistance with the animal care and especially Katsuyoshi Kumagai, formerly atThe University of Tokyo and currently at Tokyo Medical University, for generating theTg mice. The authors also thank Prof. Ryuichi Nishinakamura (Kumamoto University)for advice on designing the SGLT2-cre Tg mice and Drs. Yu Ishimoto and MotonobuNakamura (The University of Tokyo) for advice on isolation of segments of the kidney.Funding. This work was supported by a grant for Translational Systems Biologyand Medicine Initiative, Creation of Innovation Centers for Advanced InterdisciplinaryResearch Areas Program of the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT), and a MEXT Grant-in-Aid for Scientific Research (B)(15H04847 to N.K.).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.S. developed the hypothesis, designed andperformed the experiments, analyzed the data, and wrote the manuscript. T.S.developed the hypothesis, designed the experiments, generated and validated theTg mice, analyzed the data, and wrote the manuscript. N.K. developed thehypothesis, designed the experiments, analyzed the data, and wrote the manuscript.Y.S. validated the Tg mice. I.T., T.Ku., R.I., G.S., M.G., K.U., M.N., and T.J. designedthe experiments. T.Ka. developed the hypothesis, designed the experiments,analyzed the data, and reviewed and edited the manuscript. T.Ka. is the guarantorof this work and, as such, had full access to all of the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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