Based on our previous data demonstrating significant effects of leucine and its metabolite HMB on Sirt1 activation and fat metabolism, the primary purpose of this study was to investigate whether leucine and/or HMB synergize with resveratrol, as another Sirt1 activator, in Sirt1 activation and downstream effects. In addition, we wanted to explore possible effects on other sirtuins such as Sirt3. This was first done in cell culture and then extended to an in vivo mouse study where we also measured downstream effects of Sirt1 activation on insulin sensitivity, and glucose and palmitate uptake. Since Sirt1 also modulates oxidative and inflammatory stress, we included plasma markers of both as well. In addition, we included metabolic chamber studies to measure overall heat production and oxygen consumption. Depending on the complexity of experiments, we could not include all possible treatment combinations in all of the experiments. For the same reason, we did not incorporate other branched-chain amino acids as controls for non-specific effects of leucine to our experiments, as we have previously demonstrated that these exert no independent effects in these systems[3, 4, 8].
3T3-L1 pre-adipocytes were incubated at a density of 8000 cells/cm2 (10 cm2 dish) and grown in the absence of insulin in Dulbecco’s modified Eagle’s medium (DMEM,25 mM glucose) containing 10% fetal bovine serum (FBS) and antibiotics (1% penicillin-streptomycin)(adipocyte medium) at 37°C in 5% CO2 in air. Confluent pre-adipocytes were induced to differentiate with a standard differentiation medium consisting of DMEM-F10 (1:1, vol/vol) medium supplemented with 10% FBS, 250 nM dexamethasone (DEXA), isobutylmethylxanthine (IBMX) (0.5 mM) and antibiotics. Pre-adipocytes were maintained in this differentiation medium for 3 days and subsequently cultured in adipocyte medium for further 8 to 10 days to allow at least 90% of cells to reach fully differentiation before treatment. Media was changed every 2–3 days, differentiation was determined microscopically via inclusion of fat droplets.
C2C12 muscle cells were incubated at a density of 8000 cells/cm2 (10 cm2 dish) and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (adipocyte medium) at 37°C in 5% CO2 in air. Cells were grown to 100% confluence, changed into differentiation medium (DMEM with 2% horse serum and 1% penicillin– streptomycin), and fed with fresh differentiation medium every day until myotubes were fully formed (6 days).
Treatment concentrations for all experiments were 200 nM Resveratrol, 0.5 mM Leucine, 5 uM HMB in high (25 mM) glucose unless otherwise stated; incubation time was between 4 and 24 h, depending on experiment.
Sirt1 activity was measured by using the Sirt1 Fluorimetric Drug Discovery Kit (BML-AK555, ENZO Life Sciences International, Inc. PA, USA). The sensitivity and specifity of this assay kit was demonstrated by Nin et al.. They showed no detectable activity in Sirt1 knockout embryonic fibroblasts, demonstrating that the enzymatic activity measured by this assay is not present in cellular extracts that lack Sirt1.
In this assay, Sirt1 activity is assessed by the degree of deacetylation of a standardized substrate containing an acetylated lysine side chain. The substrate utilized is a peptide containing amino acids 379–382 of human p53 (Arg-His-Lys-Lys[Ac]), an established target of Sirt1 activity; Sirt1 activity is directly proportional to the degree of deacetylation of Lys-382. Samples were incubated with peptide substrate (25 μM), and NAD+ (500 μM) in a phosphate-buffered saline solution at 37°C on a horizontal shaker for 45 minutes. The reaction was stopped with the addition of 2 mM nicotinamide and a developing solution that binds to the deacetylated lysine to form a fluorophore. Following 10 minutes incubation at 37°C, fluorescence was read in a plate-reading fluorimeter with excitation and emission wavelengths of 360 nm and 450 nm, respectively. Resveratrol (100 mM) served as a Sirt1 activator (positive control) and suramin sodium (25 mM) as a Sirt1 inhibitor (negative control). The endogenous Sirt1 activity in muscle cell and mouse white adipose tissue (WAT) was measured in a modified assay using 5 μl of cell or tissue lysate. The lysates were prepared by homogenizing cells or frozen tissue in ice-cold RIPA buffer plus protease inhibitor mix (MP Biomedicals LLC). After 10 min incubation on ice, the homogenate was centrifuged at 14,000 x g and the supernatant was used for further experiments. Data for endogenous Sirt1 activation were normalized to cellular protein concentration measured via BCA-assay.
For assaying Sirt3 activity, mitochondrial protein was isolated from treated adipocytes using the Mitochondria Isolation Kit from Sigma (Saint Louis, MO, USA) and Sirt3 activity was assessed by fluorometric measurement of deacetylation of a Sirt3 substrate (Sirt3 Fluorimetric Drug Discovery Kit, Enzo Life Sciences International, Inc. PA, USA), as described for Sirt1.
AMPK activity in 3T3-L1 adipocytes was measured via the AMPK Kinase Assay Kit (CycLex Co., Ltd., Nagano, Japan) according to manufacture’s instruction. This assay provides a non-isotopic, sensitive and specific method in form of an ELISA and uses anti-phospho-mouse IRS-1 S789 monoclonal antibody and peroxidase coupled anti-mouse IgG antibody as a reporter molecule. The amount of phosphorylated substrate is determined by measuring absorbance at 450 nm. Differentiated cells were incubated with indicated treatments for 24 h. Cells were washed three times with ice-cold PBS, then lysed in Cell Lysis Buffer for 90 minutes on ice, centrifuged at 3,500 rpm for 15 min at 40C. Then 10 μl of clear supernatant was used for each assay experiment. Purified recombinant AMPK active enzyme was included as a positive control for phosphorylation. To calculate the relative AMPK activity of the samples, an inhibitor control with Compound C for each sample was included once and inhibitor control absorbance values were subtracted from test sample absorbance values.
Fatty acid oxidation
Fatty acid oxidation was measured using 3H]-palmitate, as previously described. Briefly, cells were rinsed twice with phosphate-buffered saline (PBS) and incubated in substrate mixture containing 22 uM unlabeled palmitate plus 5 uCi 3H]-palmitate in Hank’s basic salt solution containing 0.5 mg/ml BSA for 2 h. The reaction medium was then collected and treated with 0.2 ml 10% trichloracetic acid. The protein precipitate was removed by centrifugation while supernatants were treated with 6 N NaOH and then applied to a poly-prep chromatography column with 1 ml Dowex-1. The 3H2O passed through the column and the following 1 ml of water wash was collected and radioactivity was measured with a liquid scintillation counter. Protein content of the cell monolayer was measured using Bradford protein assay reagents and used for normalization.
Animals and Diet
Six-week-old male c57/BL6 mice (Harlan Laboratories, Indianapolis, IN) were fed a high-fat diet with fat increased to 45% of energy (Research Diets D12451) for 6 weeks to induce obesity. At the end of this obesity induction period, animals were either maintained on the control diet or randomly divided into one of the diet treatment groups (10 animals per group) which were supplemented with resveratrol (low dose (12.5 mg/kg diet) or high dose (225 mg/kg diet), calcium salt of hydroxymethylbutyrate (Ca-HMB: low dose (2 g/kg diet) or high dose (10 g/kg diet)) or leucine (increased to 24 g/kg diet), alone or in combination. All diets were isocaloric (4.7 kcal/g).
The animals (two/cage) were housed in polypropylene cages at a room temperature of 22°C ± 2°C and regime of 12 h light/dark cycle. The animals had free access to water and their experimental food throughout the experiment. All animals were checked daily for any signs of disease or death and moribund animals (as defined by the facility veterinarian) were humanely euthanized. Overall, one animal died before start of intervention, and three moribund animals were euthanized (two before start of intervention, one from low Resv/low HMB group). Weight was measured to the nearest gram at the beginning of the experiment and then weekly until the end of the study. At the end of the treatment period (6 weeks) all animals were humanely euthanized with isoflurane overdose, followed by cervical dislocation to assure death. Blood was immediately collected by cardiac puncture. The excised tissues were immediately weighed and used for further studies. All sacrifices were done in the morning with 7 animals per day with a minimum of 4 to 5 hours fast.
The University of Tennessee Graduate School of Medicine is a AAALAC-I-accredited institution. This study and all animal procedures were performed under the auspices of an Institutional Animal Care and Use Committee-approved protocol and in accordance with PHS policy and recommendations of the Guide.
Oxygen consumption/substrate utilization
At the end of the obesity induction period (day 0 of treatment) and at 2 and 6 weeks of treatment, oxygen consumption and substrate utilization was measured via metabolic chambers using the Comprehensive Lab Animal Monitoring Systems (CLAMS, Columbus Instruments, Columbus, OH) in subgroups of each treatment group. Each animal was placed in individual cages without bedding that allow automated, non-invasive data collection. Each cage is an indirect open circuit calorimeter that provides measurement of oxygen consumption (VO2), carbon dioxide production (VCO2), and concurrent measurement of food intake. All mice were acclimatized to the chambers for 24 h prior to the experiment and maintained under the regular 12:12 light:dark cycle with free access to water and food. All experiments were started in the morning and data were collected for 24 h. Each chamber was passed with 0.6 l of air/min and was sampled for 2 min at 32-minute intervals. Exhaust O2 and CO2 content from each chamber was compared with ambient O2 and CO2 content. Food consumption was measured by electronic scales. The respiratory exchange ratio (RER) was calculated from the ratio between carbon dioxide production and oxygen consumption (RER = VCO2/VO2) before weight normalization or mass correction. Heat production (kcal/h) was derived from a calculated calorific value based on the observed RER which was then multiplied by the observed VO2 (heat (kcal/h) = [3.815 + (1.232 x RER)] x VO2.
microPET/CT - measurement of tissue glucose and palmitate uptake
After 6 weeks of treatment intervention, subgroups of each treatment diet group (5 animals/group, 35 animals total) were used to measure whole body glucose and palmitate uptake via PET/CT Imaging. To visualize these compounds using microPET imaging, the glucose or palmitate was labeled with fluorine-18 (18F, 110 min half life). Based on previous experience with palmitate imaging, after fasting overnight, each mouse was injected iv with an average of ~124 μCi of each tracer, then be left for a period of time ~ 1 h to allow the uptake of the tracer[32–34].
After that time, the animals were anesthetized using 1-3% isoflurane delivered by nose cone or in a mouse-sized induction chamber purpose-built for small animal imaging protocols and the PET/CT images were acquired using an Inveon trimodality, PET/SPECT/CT platform (Siemens Medical Solutions, Knoxville, TN). PET data were acquired using an energy window of 350 – 650 keV and histogrammed using a 3D rebinning algorithm using a span of 3 and ring difference of 79. Data were reconstructed using a 2D ordered subset expectation maximization (OSEM) algorithm. CT data were collected using X-ray tube settings of 80 kVp and 0.5 mA. Exposure times were adjusted based on manufacturer recommendations resulting in a 225 ms exposure time per projection. Each image comprised 360 projections acquired at 1° intervals and was reconstructed using a modified Feldkamp algorithm. During image acquisition, the mice were kept warm using a thermostatically controlled heated bed and were treated with ophthalmic ointment prior to scanning. Following the live scan the mice were returned to their cage and revived. Mice were monitored constantly during this time. Each animal received both probes 48 h apart in the same order (first palmitate, second glucose) to ensure complete decay of the previous probe. Following live data acquisition of the last probe the mice were sacrificed by isoflurane overdose and organs harvested for further experiments.
Quantitation of radiotracer uptake in defined regions of adiposity was achieved by determining the standard uptake value (SUV) using the PET image data. This quantitative value is the most commonly used representation of PET data and is often seen in conjunction with 18FDG studies and has been used here to quantify 18F-palmitate also.
PET image data were corrected for radionuclide decay to the acquisition start time. Images were further calibrated using a scaling factor determined, using standard procedures resulting in image data expressed in Bq/ml. Regions of interest were drawn on the subject images to include perirenal, omental or subcutaneous fat depots as well as regions of outer and inner leg muscle using IRW (Siemens Medical Solutions). SUVs were calculated using the following equation
where the injected dose was decay corrected from the time of injection to the image acquisition start time.
Mean and maximum SUVs were calculated for each ROI and the “muscle-to-adipose tissue” ratio for the each were calculated. Three mice deemed to be diabetic by fasting blood glucose were omitted from the analysis of FDG uptake. Additionally, a blinded review of the PET image data was performed (D.O. and E.M) to identify mice in which abnormal FDG distribution was observed and these mice were also removed from the analysis (n = 4).
Calculation of visceral adipose volume (mm3)
The volume of adipose tissue in specific areas was determined by drawing regions of interest (ROIs) on the microCT data using 2D images and interpolated through multiple image slices in order to generate a 3D volume. These measurements were performed by a single operator (to maintain consistency – E.M) using Inveon Research Workplace image analysis software (IRW, ver. 3.0, Siemens Medical Solutions, Knoxville, TN).
The homeostasis model assessment of insulin resistance (HOMAIR) was used as a screening index of changes in insulin sensitivity. HOMAIR is calculated via standard formula from fasting plasma insulin and glucose as follows: HOMAIR = [Insulin (uU/mL) X glucose (mM)]/22.5. The plasma glucose and insulin concentrations were measured using the Glucose Assay Kit from Biovision (Milpitas, CA) and the Insulin kit from Millipore (Billerica, MA), respectively.
Plasma malondialdehyde (MDA) was measured using a fluorometric assay from Zeptrometrix (Buffalo, NY), and plasma 8-isoprostane F2α was measured by using the ELISA kit from Assay Designs (Ann Arbor, MI).
Inflammatory markers and cytokines
IL-6, adiponectin, MCP-1 and CRP levels in plasma were determined by ELISA (IL-6 and MCP-1: Invitrogen, Grand Island, NY; adiponectin: Millipore: Billerica, MA; CRP: Life Diagnostics, West Chester, PA).
Data were analyzed by one-way ANOVA, and significantly different group means (p < 0.05) were then separated by the least significant difference test. Normality of distribution and homogeneity of variance was confirmed for each data set prior to further analysis.