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Home , Glycogen synthase, Sirtuin, Sirtuin 1

CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691

Available online at www.sciencedirect.com Metabolism

www.metabolismjournal.com

Activation of the AMPK/Sirt1 pathway by a leucine– combination increases sensitivity in skeletal muscle, and stimulates and lipid metabolism and increases life span in

Jheelam Banerjee⁎, Antje Bruckbauer, Michael B. Zemel

NuSirt BioPharma Inc., 11020 Solway School Road, Knoxville, TN 37931, USA

ARTICLE INFO ABSTRACT

Article history: Background. We have previously shown leucine (Leu) to activate Sirt1 by lowering its KM for Received 28 January 2016 NAD+, thereby amplifying the effects of other activators and improving insulin Accepted 29 June 2016 sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin Keywords: sensitivity and glycemic control while achieving ~80% dose-reduction in diet-induced obese AMPK mice. Accordingly, we sought here to define the mechanism of this interaction. Sirt1 Methods. Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met–Leu on Insulin sensitivity Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies. Leucine Results. Leu (0.5 mmol/L) + Met (50–100 μmol/L) synergistically activated Sirt1 (p < 0.001) at + Metformin low (≤100 μmol/L) NAD levels while Met exerted no independent effect. This was associated with an increase in AMPK and ACC, phosphorylation, and increased fatty acid oxidation, which was prevented by AMPK or Sirt inhibition or silencing. Met–Leu also increased P-IRS1/IRS1 and P-AKT/AKT and in insulin-independent glucose disposal in myotubes (~50%, p < 0.002) evident

within 30 min as well as a 60% reduction in insulin EC50. In addition, in HepG2 liver cells nuclear CREB regulated coactivator 2 (CRTC2) expression and phosphorylation of synthase was decreased, while phosphorylation was increased indicating decreased gluconeogenesis and glycogen synthesis. We utilized C. elegans to assess the metabolic consequences of this interaction. Exposure to high glucose impaired glucose utilization and shortened life span by ~25%, while addition of Leu + Met to high glucose worms increased median and maximal life span by 29 and 15%, respectively (p = 0.023), restored normal glucose utilization and increased fat oxidation ~two-fold (p < 0.005), while metformin exerted no independent effect at any concentration (0.1–0.5 mmol/L).

Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK, 5′ adenosine monophosphate-activated ; CREB, cAMP-responsive element binding protein; CRTC2, CREB regulated transcription coactivator 2; ECAR, extracellular acidification rate; ERK, extracellular signal regulated kinase; GS, glycogen synthase; GSK, glycogen synthase kinase; IR, ; IRS1, insulin receptor substrate 1; JNK, c-Jun N-terminal ; Leu, leucine; LKB1, liver kinase B1; Met, metformin; NAD, adenine dinucleotide; NAMPT, nicotinamide phosphoribosyltransferase; PEPCK, phosphoenolpyruvate carboxykinase; pGS, phosphorylated glycogen synthase; pGSK, phosphorylated glycogen synthase kinase; PKC-ZETA, protein kinase C zeta; Sirt1, ; TNF-α, alpha. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (J. Banerjee). http://dx.doi.org/10.1016/j.metabol.2016.06.011 0026-0495/© 2016 Elsevier Inc. All rights reserved. 1680 METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691

Conclusion. Thus, Leu and Met synergize to enable Sirt1 activation at low NAD+ concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met–Leu's full action, whichresultinimprovementsinenergymetabolismandinsulinsensitivity. © 2016 Elsevier Inc. All rights reserved.

1. Introduction then seeded into plates for the final treatments and then induced to differentiate with a DMEM containing 2% horse Metformin is considered the initial drug of choice for treating serum and 1% Pen-Strep. The cells were maintained in this , as it is highly efficacious, exhibits an excellent differentiation medium for 5 days before treatment. HepG2 cells safety profile, does not promote weight gain, does not increase were either grown in low glucose DMEM (5 mmol/L glucose) or the risk for hypoglycemia and has been shown to reduce the risk high glucose DMEM (25 mmol/L glucose) containing 10% fetal of diabetes-related comorbidities and death [1]. However, bovine serum (FBS) and antibiotics (1% penicillin–streptomycin) metformin monotherapy often fails to achieve optimal glycemic at 37 °C in 5% CO2 in air. C2C12 cells were treated with control due to inter-individual variability in response to drug metformin (0.1 mmol/L), leucine (0.5 mmol/L) and a combina- initiation and maintenance, failure to achieve optimal dose tion of both drugs for 2 h, followed by 20 min insulin (10 nmol/L) titration secondary to drug-induced gastrointestinal discomfort, stimulation. were then extracted using NP40 buffer. or drug discontinuation due to the development of adverse HepG2 cells were treated with a combination of Met–Leu for 24 h effects [2]. Although there are multiple other antihyperglycemic to 48 h and then cells were harvested. Protein concentrations drug classes which can be used as second choice monotherapy were quantitated with the BCA kit (Thermo Scientific). or add-on treatments, they still show some disadvantages in comparison to successful metformin monotherapy [3,4] 2.2. siRNA Transfection Accordingly, there is still a need to optimize metformin efficacy at lower doses [5]. C2C12 muscle cells were seeded with 104 cells/well on 96-well The AMP-activated protein kinase (AMPK) and the NAD+- plate or 24-well Seahorse plate (confluence, ~50 to 60%). ~24 h dependent (Sirt1 and Sirt6) are key sensors of energy later, cells were transfected with siRNA against Sirt1 (Ambion status and regulators of glucose and lipid metabolism, activating ID# 174219) or AMPK (Ambion ID# 221537) complexed to each other in a finely tuned network [6,7]. Lipofectamine RNAiMAX reagent (ThermoFisher Scientific, While and diabetes are associated with Cat# 13778-030) according to manufacturer's instructions for impairment of this pathway, activation of the AMPK-Sirt1 24 to 48 h. Then they were differentiated in 2% horse serum axis improves and insulin sensitivity [8,9]. for an additional 4 days prior to treatment. Accordingly, Sirt1 transgenic mice exhibit reduced levels of fasting blood glucose and insulin as well as improved glycemic 2.3. Expression Analysis control, showing anti-diabetic effects, during the [10]. Cells were grown in a 96-well plate. Cell Lysis, reverse transcrip- We previously found the branched-chain amino acid leucine tion and RT-PCR were performed using the TaqMan® Gene (Leu) to activate Sirt1 by lowering the activation energy for ™ Expression Cells-to CT Kit (Life Technologies, Cat #4399002) + NAD , thus mimicking the effects of caloric restriction, and according to manufacturer's instructions. was enabling co-activation and amplification of the effects of other assessed by RT-PCR using StepOnePlus™ PCR system (Thermo AMPK/sirtuin activators at low concentrations [11]. As a result Fisher Scientific) and TaqMan® Gene expression assays for of metformin and leucine converging on this AMPK-Sirt1 axis AMPK (Life Technologies, Cat #Mm01264789) and Sirt1 (Life [1], we found leucine and metformin to exhibit synergistic Technologies, Cat #Mm01168521). effects to improve insulin sensitivity and glycemic control in diet-induced obese mice while achieving a ~80% dose- 2.4. Nuclear and Cytosolic Extraction reduction of metformin [12]. Therefore, this study was designed to further define the mechanism of the interaction of leucine Treated HepG2 cells were scraped from culture flasks and and metformin on AMPK/Sirt1 activation, and to assess the washed with cold PBS twice. Cell pellet was re-suspended metabolic consequences on glucose utilization and life span in in 500 μL of hypotonic buffer (20 mmol/L Tris–HCl, pH 7.4; Caenorhabditis elegans. 10 mmol/L NaCl; 3 mmol/L MgCl2)andincubatedonicefor 15 min. Then 25 μL detergent (10% NP40) was added and vortexed for 10 s. The homogenate was centrifuged for 10 min at 3000 rpm 2. Materials and Methods at 4 °C, and the supernatant containing the cytoplasmic fraction was then aliquoted and stored at −80 °C for further experiments. 2.1. Cell Culture The pellet, containing the nuclear fraction, was re-suspended in 50 μL of Cell Extraction buffer (Life Technologies, Grand Island, MurineC2C12musclecellsweregrownintheabsenceofinsulin NY; Cat #FNN0011) supplemented with 1 mmol/L PMSF and in Dulbecco's modified Eagle's medium (DMEM, 25 mmol/L Protease Inhibitor Cocktail (Sigma, St. Louis, MO; Cat #P-2714, glucose) containing 10% fetal bovine serum (FBS) and antibiotics 1:100 dilution) for 30 min on ice with vortexing at 10 min – (1% penicillin streptomycin) at 37 °C in 5% CO2 in air. They were intervals. Then the homogenate was centrifuged for 30 min at METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691 1681

14,000g at 4 °C. The supernatant was aliquoted and stored at 2.7. Glucose Utilization in C2C12 Muscle Cells −80 °C for further experiments. Protein content of the cyto- plasmic and nuclear fraction was measured with the BCA kit In the absence of a fatty acid source and oxidative metabo- (Thermo Scientific, Grand Island, NY). lism, glycolysis and subsequent lactate production result in extracellular acidification. Extracellular acidification rate 2.5. ELISA (ECAR) was measured using a Seahorse Bioscience XF24 analyzer (Seahorse Bioscience, Billerica, MA) in 24-well plates at Differentiated C2C12 muscle cells were treated for 2 h with Met 37 °C. C2C12 cells were seeded at 40,000 cells per well, (0.1 mmol/L)–Leu (0.5 mmol/L). 20 min before harvest they were differentiated as described above, treated for 24 h with or stimulated with 100 nmol/L insulin. The ELISA assays (PathScan without metformin (0.1 mmol/L)–leucine (0.5 mmol/L). For P-IRS1 (S307) and PathScan Total IRS1, (Danvers, SH-experiment, media was changed to 11 mmol/L glucose. MA), cat #7287 and #7328, respectively) were performed according After three baseline readings, insulin (0 to 500 nmol/L) was to the manufacturer's instructions. injected. After 20 min incubation, 14 mmol/L glucose was injected and extracellular acidification rate (ECAR) was measured 2.6. Western Blot over a two-hour period.

Phospho-AMPK (Thr172), AMPK, pACC (Ser79), ACC, pIRS1 (S318), 2.8. Sirt1 Activity IRS1, p-AKT (Thr308), AKT, pGSK, GSK, pGS, GS, TORC2/CRTC2 and β-actin were obtained from Cell Signaling The Sirt1 FRET-based screening assay kit (Cayman Chemical (Danvers, MA). Protein levels of cell extracts were measured by Company, Ann Arbor, MI) was used for the measurement of BCA kit (Thermo Scientific, Pittsburgh, PA). For Western blot, Sirt1 activity in a cell-free system as well as in C2C12 muscle 10–50 μgproteinwasresolvedon4–15% gradient polyacrylamide cells. For the cell-free experiment, Leu (0.5 mmol/L), Met gels (Criterion precast gel, Bio-Rad Laboratories, Hercules, CA), (0.1 mmol/L) or combination was incubated with recombinant transferredtoeitherPVDFornitrocellulose membranes, incubat- Sirt1 under different NAD+ concentrations (500 μmol/L, ed in blocking buffer (3% BSA or 5% non-fat dry milk in TBS) and 100 μmol/L, 50 μmol/L and 10 μmol/L)for30minaccordingto then incubated with primary (1:1000 dilution), washed the manufacturer's protocol. The protocol was modified for and incubated with horseradish peroxidase- or fluorescence- the measurements in cells as follows; cell lysate of differen- conjugated secondary antibody (1:10,000 dilution). Visualization tiated C2C12 muscle cells treated with Leu (0.5 mmol/L), Met was conducted using BioRad ChemiDoc instrumentation and (0.1 mmol/L) or combination for 24 h was incubated with software (Bio-Rad Laboratories, Hercules, CA) and band intensity peptide substrate under low NAD+ (100 μmol/L) concentra- was assessed using Image Lab 4.0 with correction for background tions. The fluorescence was measured with excitation and and loading controls. emission wavelengths of 360 nm and 450 nm, respectively.

Fig. 1 – Met–Leu effect on Sirt1 activation. Recombinant human Sirt1 enzyme was incubated with Leucine (0.5 mmol/L) and/or metformin (0.1 mmol/L) for 45 min under different NAD+ concentrations (10 to 500 μmol/L) and fluorescence of deacetylated substrate was measured. (A and B) Representative fluorescence data with incubation of 50 μmol/L and 500 μmol/L NAD+, respectively. Data are expressed as means ± SEM (n = 5 to 6, p ≤ 0.05). (C) Summary of results represented as % change of Sirt1 activity ± SEM (n = 5 to 6, p ≤ 0.05). * indicates significantly different from control (p < 0.05). 1682 METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691

The increase in fluorescence is proportional to the amount overnight in M9 buffer. Synchronized L1 larvae were transferred of deacetylated substrate and thus Sirt1 activity. to Escherichia coli fed NGM plates containing indicated treat- ments for about 35 h to reach L4/young adult stage. All 2.9. C. elegans Maintenance treatments were added to the agar in indicated concentrations.

Worms (N2 Bristol wild-type) were obtained from the 2.10. Glucose Content in C. elegans Caenorhabditis Genetics Center (CGC) at the University of Minnesota and grown on standard NGM plates with E. coli Glucose content of worms was measured using the Amplex® (OP50) as food source at 21 °C. For treatments, adult worms Red Glucose/Glucose Oxidase Assay Kit (Invitrogen, now were bleached to obtain eggs, which were allowed to hatch ThermoFisher Scientific, Grand Island, NY). METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691 1683

Synchronized L1 worms were maintained in liquid media, free pH 7.4 low glucose (2.5 mmol/L) DMEM containing carnitine as described in Ref. [13], containing 40 mmol/L glucose and (0.5 mmol/L), equilibrated with 600 mL of the same media in a indicated treatments until adulthood. Adult worms were washed non-CO2 incubator for 45 min, and then inserted into the off the plate. The supernatant was removed after centrifugation instrument for 15 min of further equilibration, followed by O2 at 500g for 1 min. The worms were washed three times with M9 consumption measurement. Three successive baseline mea- buffer. After the last wash, supernatant was removed except that sures at five-minute intervals were taken prior to injection of ~150 μLand50μL of Reagent buffer from assay kit were added. palmitate (200 μmol/L final concentration). Three successive 5-

Then worm solution was homogenized and lysate was used for min measurements of O2 consumption were then conducted, assay according to the manufacturer's instructions. followed by 10 min waiting period. This measurement pattern was then repeated over a 2-h period. 2.11. Life Span Study Protocol 2.13. Statistical Analysis 50 synchronized stage L4 worms per group were placed on NGM agar plates seeded with E. coli strain OP50 (= day 1 of study). All All data are expressed as mean ± SEM, with the exception of treatments were added with the indicated concentrations to the Seahorse data, which are shown as mean ± SD. Data were agar plates. The worms were maintained at 21 °C throughout analyzed by one-way ANOVA, and significantly different group the duration of the study. Live worms were placed on new plates means (p < 0.05) were separated by the least significant difference every day to eliminate progeny. Worms were scored as dead if test using GraphPad Prism version 6 (GraphPad Software, La they did not respond to repeated touches with the platinum pick Jolla, CA; www.graphpad.com). For the statistical analysis of the andscoredascensorediftheycrawledofftheplate. life span study with C. elegans, data were analyzed via Kaplan– Meier survival curves and statistical significance between 2.12. Palmitate-Induced Fatty Acid Oxidation curves was determined with the Gehan–Breslow–Wilcoxon test using Prism 6 (GraphPad Software, La Jolla, CA; www. Cellular oxygen consumption was measured using a Seahorse graphpad.com). Bioscience XF24 analyzer (Seahorse Bioscience, Billerica, MA) in 24-well plates with appropriate modifications for C. elegans or for cells, as follows. For the measurement of fatty acid oxidation 3. Results in worms, treated L4/adult worms were washed off the plate with M9 buffer, and then washed (centrifuged at 500g for 1 min) First we tested the effects of the Met–Leu combination on Sirt1 three times. After final wash, the worm suspension was diluted activation in a cell-free system (Fig. 1). Met–Leu synergistically to a worm count of 2 worms/20 μL worm suspension drop. Then activated human recombinant Sirt1 enzyme under low NAD+ 500 μL of the worm suspension was added to each well to reach conditions (50 μmol/L, Fig. 1A) but not under high NAD+ a final worm count of ~50 worms/well with at least 5 replicates conditions (500 μmol/L, Fig. 1B), shifting the activation curve to + per group in each experiment. The Seahorse program was set the left and lowering the Michaelis constant (KM) value for NAD up at 27 °C. Five successive baseline measures were taken prior from 55.05 to 21.45 (Fig. 1C); the individual compounds exerted to injection of palmitate (200 μmol/L final concentration). Three little or no significant independent effect. There was also a successive measurements of O2 consumption were then con- significant increase in the protein expression and activity of Sirt1 ducted, each followed by a 10-min waiting period. This measure- and of the ratio of Phospho-AMPK (Thr172)/AMPK in C2C12 ment pattern was then repeated over a 2-h period. For fatty acid muscle cells treated with the Met–Leu combination (Fig. 2A–C). oxidation measurement in C2C12 muscle cells, cells were seeded Treatment of C2C12 muscle cells with the Sirt1 inhibitor Ex527 with 10,000 cells per well, then treated with Lipofectamine/siRNA prevented the effects of Met–Leu on AMPK phosphorylation. against Sirt1 or AMPK or lipofectamine vehicle for 24 h. Then they However, the ratio of P-AMPK/AMPK was not changed with were differentiated with 2% horse serum for 4 days, and then EX-527 compared to Met–Leu since there were small but treated for 24 h with the indicated treatments. To measure fat statistically insignificant differences in total AMPK between the oxidation, they were washed twice with non-buffered carbonate- groups. AMPK knockdown blunted the effects of Met–Leu on

Fig. 2 – Met–Leu effects on Sirt1 and AMPK signaling in C2C12 muscle cells. Differentiated C2C12 muscle cells were treated with indicated treatments for 2 h and stimulated with insulin (10 nmol/L) 20 min prior to harvest. Protein expression of Sirt1 (A) and phospho-AMPK (Thr172) and total AMPK (B) were measured. Quantitative data and representative blots are shown. Data are represented as mean ± SEM (n = 3 to 4). (C) Sirt1 activity was measured in differentiated C2C12 muscle cells treated with Met (0.1 mmol/L), Leu (0.5 mmol/L) or the combination for 24 h. Fluorescence values were normalized to protein amount of cell lysate (n = 4 to 6). (D) Protein expression of phospho (Thr172)-AMPK and total AMPK: differentiated C2C12 muscle cells were treated with Met–Leu in presence or absence of the Sirt1 inhibitor EX527. Bar graphs were calculated from pooled densitometry measurements of two different blots (n = 3 to 6) and representative blots are shown. The bar graph of the ratio of P-AMPK/AMPK is shown as an insert (E) Protein expression of Sirt1: C2C12 muscle cells were infected with siRNA against AMPK as described under methods, then treated after differentiation with Met–Leu for 24 h. Bar graph of densitometry measurement and representative blots are shown (n = 2). (F) Protein expression of phospho (Ser79)-ACC and ACC: differentiated C2C12 muscle cells were treated with Met–Leu in presence or absence of the Sirt1 inhibitor EX527 and AMPK inhibitor Compound C. Bar graphs of densitometry measurement and representative blot are shown (n = 2). 1684 METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691

Fig. 3 – AMPK or Sirt1 knockdown blocks the effects of Met–Leu on fatty acid oxidation. C2C12 muscle cells were infected with siRNA against AMPK or Sirt1 as described under methods, then treated after differentiation with Met–Leu for 24 h. (A) Representative figure for AMPK knockdown experiment: oxygen consumption rate (OCR) was measured after 200 μmol/L palmitate injection (time points A and C). The area under the curve (AUC) of the OCR for (B) AMPK knockdown and (C) Sirt1 knockdown experiment. Data are represented as mean ± SEM (n = 4 to 5). Validation of successful knockdown was measured by RT-PCR for (D) AMPK and (E) Sirt1. siRNA (Sirt1) was used as positive control for AMPK knockdown and siRNA (AMPK) as positive control for Sirt1 knockdown.

Sirt1 protein expression, indicating the interactive effects of Sirt1 AMPK and Sirt1 knockdown or inhibition (Figs. 2Fand3AtoC). and AMPK (Fig. 2D and E). To further explore whether AMPK and The level of AMPK or Sirt1 knockdown is shown in Fig. 3DandE. Sirt1 are both required for the full effect of the Met–Leu We next examined the effects of the Met–Leu combination on combination, we measured ACC phosphorylation as a ratio to insulin signaling. Stimulation with 10 nmol/L insulin 20 min total ACC and fatty acid oxidation as downstream targets of prior to harvest produced the expected increase in phosphory- AMPK activation. Both, ACC phosphorylation and palmitate- lated (Ser318)-IRS in C2C12 muscle cells. Met–Leu treatment for induced oxygen consumption rate were increased by ~100% and 2 h in the absence of insulin resulted in a comparable (~25%) ~20%, respectively, by Met–Leu and totally prevented by either increase in IRS phosphorylation, and further significantly METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691 1685 enhanced IRS phosphorylation by an additional ~15% during In liver HepG2 cells we found a trend (p =0.07)forreduction insulin stimulation compared to insulin-treated controls in CRTC2 in the nucleus with Met–Leu treatment, as well as a (Fig. 4A). Similarly, Met–Leu treatment in the absence of significant increase in glycogen synthase kinase (GSK)-3 β insulin resulted in a significant increase in phosphorylated phosphorylation and an associated decrease in glycogen AKT expression in muscle cells comparable to that achieved synthase (GS) phosphorylation treatment (Fig. 6). with insulin, and Met–Leu further augmented insulin stimula- Next we tested the effects on Met–Leu on glucose and fat tion of P-AKT (Fig. 4B). However, IRS phosphorylation on Ser307 metabolism in a simple , C. elegans. Fig. 7A was not further stimulated by Met–Leu treatment when shows the oxygen consumption rate and the calculated area compared to insulin-stimulated control (Fig. 4C). under the curve in worms treated with Met–Leu. There was a Next we measured ECAR, which is an indicator of the cellular significant ~50% increase in fatty acid oxidation in C. elegans. glucose utilization, in C2C12 cells treated with Met–Leu for 2 h in Next,wemeasuredtheglucosecontentofwormskepteither response to different insulin concentrations via the Seahorse under normal (low) glucose conditions or under high (40 mmol/L Bioscience XF24 analyzer. Fig. 5A shows a representative exper- glucose added to liquid media) glucose conditions. The high iment with A, the time point of insulin injection (0.5 nmol/L, glucose conditions significantly increased the glucose content in 5 nmol/L, 50 nmol/L or 500 nmol/L), and C, (20 min after A) the the worms, which was prevented by simultaneous treatment time point of 14 mmol/L glucose injection, to reach a final well with Met–Leu, indicative of higher glucose utilization in the concentration of 25 mmol/L glucose. Fig. 5B shows the calcu- worms (Fig. 7B). To test whether these effects modulate survival, lated area under the curve of that experiment and Fig. 5Cthe we conducted life span studies under normal and high glucose summarized results of all four experiments. There was a shift in conditions (2% glucose added to agar plates) with or without Met, glucose utilization at the tested insulin concentrations with Leu and the combinations (Fig. 8). High glucose shortened Met–Leu treatment. Met–Leu treatment increased glucose utiliza- median survival by 22% compared to low glucose conditions. tion comparable to 500 nmol/L insulin stimulation, and additional Leu combined with Met (0.1 mmol/L (Fig. 8A) and 0.5 mmol/L insulin to the Met–Leu treatment further enhanced this effect. (Fig. 8B)) prolonged median and maximum life span under

Fig. 4 – Met Leu effects on insulin signaling in C2C12 muscle cells. Differentiated C2C12 muscle cells were treated with indicated treatments for 2 h and stimulated with insulin (10 nmol/L) 20 min prior to harvest. Protein expressions of P-IRS (Ser318) and IRS (A), and P-AKT and AKT (B) were measured. Bar graphs were calculated from pooled densitometry measurements of two different blots and representative blots are shown. Data are represented as mean ± SEM (n = 3 to 5). (C) P-IRS (Ser307) and IRS were measured in cell extract via ELISA. Data are represented as mean ± SEM (n = 8). 1686 METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691

Fig. 5 – Met–Leu effects on glucose utilization in C2C12 muscle cells. The extracellular acidification rate (ECAR) was measured after different insulin (5 to 500 nmol/L) and glucose (14 mmol/L) injection (points A and C) in differentiated C2C12 muscle cells. (A) Representative experiment after 500 nmol/L insulin injection. (B) The area under the curve (AUC) of this experiment. Data are represented as mean ± SD (n = 5). ECAR response to four different insulin concentrations represented as group means of each individual experiment. * indicates significant difference between Met–Leu curve to control insulin curve (p < 0.03).

these high glucose conditions. The individual compounds uptake [4,19]. In addition, metformin appears to increase both exerted small but statistically insignificant effect (Fig. 8C). Sirt1 activity and protein expression via AMPK-dependent and independent pathways [20,21].Wehavepreviouslydemonstrated that leucine amplifies the effects of metformin on AMPK and 4. Discussion Sirt1 activation and enables a substantial dose reduction in vitro and in vivo [12,22]. Our previous data suggest that some of The major findings of this paper are that the Met–Leu the effects of leucine are mediated by acting as a direct + combination increases insulin-independent stimulation of allosteric activator of Sirt1, reducing the KM for NAD and the insulin signaling pathway by activating the AMPK/Sirt1 thereby allowing Sirt1 activation at lower NAD+ concentrations, pathway and increases insulin sensitivity and overall glucose which are characteristic for a metabolic replete state. [11]. and lipid metabolism. Moreover, leucine facilitates the interaction of Sirt1 with other Metabolic sensors such as AMPK and Sirt1 are crucial to the Sirt1 activating compounds such as , which resulted regulatory network for metabolic homeostasis by modulating in a further left-ward shift of the activation curve [11]. Here we pleiotropic effects in key metabolically relevant tissues such as demonstrate that this ability of leucine can be extrapolated liver, muscle and adipose tissue. SIRT1, a NAD+-dependent to metformin, which also has some direct effects on Sirt1 regulator of energy metabolism, has been reported to be activation [23], as the Met–Leu combination further increased activated through AMPK-mediated induction of nicotinamide Sirt1 activity at low NAD+ concentrations (Fig. 1). Therefore, the phosphoribosyltransferase (NAMPT), the rate-limiting enzyme activation of Sirt1 by the combination may be an early effect and for NAD+ biosynthesis or changes in the NAD+/NADH ratio [14]. AMPK independent, which leads to subsequent AMPK activation On the other hand, Sirt1-induced deacetylation of LKB1 causes and further Sirt1 activation at later time points, as suggested by its movement from the nucleus to the cytosol and subsequent Liang et al. [24]. Moreover, it was demonstrated that Sirt1 acts activation of AMPK [14], suggesting reciprocal activation of these upstream of AMPK to regulate hepatocellular lipid metabolism two energy sensing systems. Both systems represent targets for [25]. Consistent with this concept, the Sirt1 inhibitor EX527 therapeutic intervention, as they are down-regulated during blocked the effects of Met–Leu on AMPK phosphorylation, periods of over-nutrition and in obesity, diabetes and metabolic demonstrating that Sirt1 activation is necessary for AMPK syndrome [15–18]. activation (Fig. 2D). In contrast, AMPK knockdown only partially Metformin, acts in part via AMPK activation in liver to reduce blunted the Met–Leu effect on Sirt1, suggesting both AMPK- hepatic gluconeogenesis as well as in muscle to increase glucose dependent and independent activation of Sirt1 (Fig. 2E). However, METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691 1687

Fig. 6 – Met–Leu effects on enzyme of hepatic glucose metabolism in HepG2 liver cells. HepG2 liver cells were treated with indicated treatments for 24 h. Protein expression of P-GSK and GSK (A), and P-GS and GS (B) were measured. Quantitative data and representative blots are shown. Data are represented as mean ± SEM (n = 3 to 4). (C) Nuclear and cytosolic fraction of cell extract was separated as described under Materials and Methods. CRTC2 protein expression was measured in the cytosolic and nuclear fraction and normalized to β-actin. Data are represented as mean ± SEM (n = 3). 1688 METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691

Fig. 7 – Met–Leu effects on glucose and fat metabolism in C. elegans. C. elegans L1 larvae were maintained on agar plates or liquid media with indicated treatments until adulthood. (A) Oxygen consumption rate (OCR) in adult worms was measured after 200 μmol/L palmitate injection (time points A and C) and the area under the curve (AUC) was calculated. Data are represented as mean ± SD (n = 10). * indicates significantly different from control (p < 0.05). (B) Glucose content of worms was measured as described under Methods and normalized to protein content. Different letter superscripts indicate statistically significant difference between groups (p < 0.05). Treatments: control low gluc (no glucose or treatments added), control high gluc (40 mmol/L glucose), Met (metformin 0.1 mmol/L)–Leu (leucine 0.5 mmol/L).

knockdown of either Sirt1 or AMPK prevented the effects of Met– feedback mechanism [26], there is also evidence that Ser 318 Leu on downstream events such as ACC phosphorylation and phosphorylation also occurs in the early phase of insulin palmitate-induced fatty acid oxidation (Fig. 2F and 3), implying signaling increasing AKT phosphorylation and glucose uptake that both are necessary for Met–Leu's full action. and is required for an enhanced induction of insulin action [29]. Since the Met–Leu combination increased AMPK and Sirt1 However, in the late phase of insulin action, this phosphorylation protein expression in C2C12 muscle cells (Fig. 2), we wanted to site attenuates insulin-stimulated and sub- determine if this results in an insulin-independent increase in sequently decreases glucose uptake in skeletal muscle. There- glucose muscle uptake. IRS-1 protein is a key player in the insulin fore, this Ser318 phosphorylation may be necessary to maintain signal transduction pathway to stimulate glucose uptake and is the physiological balance of cellular energy homeostasis [29].In regulated by various kinases in response to metabolic stimuli at contrast, the Ser307 phosphorylation site is assumed to be an multiple serine, threonine and tyrosine phosphorylation sites inhibitory phosphorylation site stimulated by factors such as free [26]. AMPK and Sirt1 also regulate IRS-1 phosphorylation and fatty acids, TNF-α and hyperinsulinemia and is involved in the muscle glucose uptake, although this may be a secondary development of insulin resistance [26,30,31]. In agreement with response to inhibition of inflammatory mediators. For example, this concept, our data indicate that Met–Leu treatment did not knockdown of Sirt1 led to increased inflammatory response affect the inhibitory Ser307 phosphorylation site. However, the accompanied by impaired IRS-1 signaling and decreased insulin- increased Ser318 phosphorylation was associated with increased stimulated glucose transport in 3T3-L1 adipocytes whereas Sirt1 AKT phosphorylation and an increased glucose utilization in activators suppressed the inflammatory pathway and increased muscle (Figs. 4 and 5). insulin sensitivity [27]. Similarly, treatment of 3T3-L1 adipocytes Diabetes and hyperglycemia are associated with increased with AICAR, an AMPK activator, reduced TNF-α induced serine gluconeogenesis and glycogen synthesis in the liver. Phospho- phosphorylation of IRS-1 through ERK and JNK and improved enolpyruvate carboxykinase (PEPCK) and glycogen synthase, insulin resistance [28]. We found an upregulation of Ser 318 key rate limiting of gluconeogenesis and glycogen phosphorylation of IRS-1 with Met–Leu treatment comparable synthesis, respectively, are tightly controlled by transcriptional to insulin alone, and a further augmentation of insulin-induced regulation, phosphorylation and allosteric effectors in response to IRS-1 phosphorylation. Although this phosphorylation site can be energetic signals [32,33]. Glycogen synthase kinase-3-β activates activated by PKC-zeta, JNK and other kinases which presumably GS and is also negatively regulated by phosphorylation. AMPK disrupt the interaction of IR and IRS-1 and provide a negative activation reduces hepatic glucose production and glycogen METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691 1689

Fig. 8 – Met–Leu effects on survival in C. elegans. (A and B) Survival curves of synchronized L4 worms maintained on NGM agar plates with indicated treatments under high glucose conditions (2% glucose added to agar plates) until death. (C) Life span table summarizing median survival, maximum life span and statistical significance. Treatments: control low (no glucose or treatments added to NGM agar plates), control high (2% glucose added to NGM agar plates), Met (metformin 0.1 or 0.5 mmol/L added to NGM agar plates), Leu (leucine 0.25 mmol/L added to NGM agar plates).

synthesis through phosphorylation of GSK-3β and of TORC2 This concentration shortened both median and maximum life (transducer of regulated CREB activity 2 or CRTC2), a co-activator span [38,39]. In another study [40], 2% glucose added to the agar of cAMP-responsive element binding protein (CREB) [34].Phos- plates shortened life span of C. elegans by ~20%, similar to data phorylation of CRTC2 leads to its translocation from the nucleus from the present study. Addition of the Met–Leu combination to to the cytosol and consequently to a reduced transcriptional the agar plates reversed the high glucose-induced impairment activity of CREB, an important transcriptional regulator of hepatic in fat and glucose metabolism in the worms and produced a PEPCK. CREB, in turn, is also directly phosphorylated by GSK-3β corresponding increase in life span (Figs. 7 and 8). [34,35]. Therefore, AMPK-induced reduction of gluconeogenesis is The concentrations of metformin and leucine, used in the mediated both by phosphorylation of GSK-3β and consequent cell studies, were derived from our previous work for optimal reduction in CREB activity, and by decreased nuclear presence of Sirt1 activation and are pharmacologically relevant, as TORC2 (CRTC2), which together reduce transcription of PEPCK. follows [11,12,36]. The fasting plasma concentration of leucine Since we have recently demonstrated in mice treated with a Met– in humans is ~0.1 mmol/L and a plasma concentration Leu combination that hepatic AMPK was activated associated of 0.5 mmol/L can be reached after ingestion of ~300 mg with increased fat oxidation and decreased liver triglyceride leucine/kg/day [41]. The peak systemic plasma concentration accumulation [36], we wanted to explore the effects of AMPK reached after a therapeutic dose of metformin (1000 mg) is activation with our treatment on hepatic glucose metabolism. ~2.4 mg/L (=18 μmol/L). However, it is higher in the portal Consistent with downstream effects of AMPK activation, we vein where it reaches between 40 and 70 μmol/L [42,43]. found trend for reduction (20%) of CRTC2 in the nucleus with Met– Therefore, the 0.1 mmol/L concentration used in our cell studies Leu associated with a significant increase in GSK-3β phosphory- is close to a pharmacologically achievable concentration, and is lation and an accompanying decrease in GS phosphorylation in about 10 to 100 times lower than the metformin concentration liver HepG2 cells (Fig. 6). used in typical cell studies, which usually range from 1 to The nematode C. elegans conserves 65% of the human 10 mmol/L [44–48]. In fact, it was demonstrated that metformin disease-related , including pathways for fat and glucose at low concentration (≤80 μmol/L) increases AMPK activity metabolism, and is used as a well-defined simple model system and suppresses dibutyryl-cAMP-stimulated glucose production for obesity and insulin resistance [37]. Worms kept on high while the inhibition of the respiratory chain complex 1 happens glucose (40 mmol/L) agar plates reached an internal glucose only at high concentrations (~5 mmol/L) [43]. concentration of 14 mmol/L, comparable to plasma glucose There are some limitations to this study. While we mainly concentrations under poorly controlled diabetic conditions. focus on the downstream targets of Sirt1 and AMPK, as they 1690 METABOLISM CLINICAL AND EXPERIMENTAL 65 (2016) 1679– 1691

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