DOI: 10.5061/dryad.n8pk0p38d
title: Sirtuins may mediate temperature-induced metabolic remodeling in threespine stickleback

description: The molecular pathway governing metabolic remodeling in response to
 temperature in fishes is unknown. We tested the hypothesis that sirtuins,
 a family of NAD+ dependent lysine deacetylases, drives an increase in
 aerobic metabolism in response to cold acclimation in stickleback.
 Stickleback were acclimated to 5, 12 and 20°C and sirtuin activity
 measured in liver, oxidative and glycolytic muscles using a Promega
 SIRT-Glo Assay kit. Protein levels of SIRT1 and SIRT3 were quantified
 using custom antibodies and capillary western blotting. In a second set of
 experiments, sirtuin activity was inhibited by feeding with EX-527 or
 activated by feeding with the NAD+ pre-cursor, nicotinamide mononucleotide
 (NMN). Standard metabolic rate (SMR) was measured using intermittent flow
 respirometry. Mitochondrial respiration rates were measured in
 permeabilized liver tissue using a substrate-uncoupler-inhibitor titration
 protocol . Sirtuin activity significantly increased in response to cold
 acclimation in liver and oxidative and glycolytic muscles but SIRT3
 protein levels declined and SIRT1 protein was undetectable, suggesting one
 or more other SIRT isoforms contribute to an increase in sirtuin activity
 in response to cold acclimation. Inhibiting sirtuin activity with EX-527
 decreased SMR and the activity of complex I in liver, providing further
 support for the role of sirtuins in metabolic remodeling in
 stickleback. 
description: # Sirtuins may mediate temperature-induced metabolic remodeling in
 threespine stickleback Dataset DOI:
 [10.5061/dryad.n8pk0p38d](https://doi.org/10.5061/dryad.n8pk0p38d) ##
 Description of the data and file structure Stickleback (*Gasterosteus
 aculeatus*) were acclimated to 5, 12, and 20°C for 13 weeks. Sirtuin
 activity was then measured in liver, oxidative, and glycolytic muscles
 using a Promega SIRT-Glo assay kit, and protein levels of SIRT1 and SIRT3
 were quantified using custom antibodies and capillary western blotting. In
 a second set of experiments, sticklebacks were acclimated to 20°C for 12
 weeks. A subset of fish were then moved into an environmental chamber
 where the temperature in the room was decreased by 3°C per day over 2 days
 and then by 4°C on day 3 to reach 10°C. After 14 days at 10ºC, bloodworms
 were supplemented with either the sirtuin inhibitor EX-527 or the sirtuin
 activator, NMN, mixed with 7% agar for 14 days. Standard metabolic rate
 was then measured using intermittent respirometry, and mitochondrial
 function in permeabilized liver tissue was measured using a
 substrate-uncoupler-inhibitor titration protocol. ### Files and variables
 #### File:
 Physical_characteristics_for_sirtuin_activity_and_NAD(H)_levels.csv
 **Description:** Measurements of physical characteristics of stickleback
 used to quantify Sirtuin activity and levels of NAD+ and NADH #####
 Variables * Fish ID: Unique fish identification number * Temp (C):
 temperature animal was acclimated to * Length (cm): standard length of the
 fish * mass (g): body mass of the fish * Condition Factor: = bm/Ls ^3
 X100; bm = body mass (g), and L~s~ = standard length (cm). #### File:
 Sirtuin_activity_and_NAD(H)_levels.csv **Description:** Values for
 measurements of Sirtuin activity and NAD+ and NADH levels liver, oxidative
 skeletal muscle taken from the pectoral adductor muscle, and white,
 glycolytic skeletal muscle.  ##### Variables * Fish ID: Unique fish
 identification number * Tissue: tissue type * Tissue_mass (mg): mass of
 the tissue * Sirtuin activity RxnRate_RL/min/ug: activity of sirtuin
 measured as reaction rate per min per ug protein * NAD Total (pmol/mg
 tissue): total nicotinamide adenine dinucleotide plus reduced nicotinamide
 adenine dinucleotide  in pmol per milligram tissue * NADH (pmol per mg
 tissue): reduced nicotinamide adenine dinucleotide * NAD (pmol per mg
 tissue): nicotinamide adenine dinucleotide measured in pmol per milligram
 tissue * NAD:NADH: ratio of ncotinamide adenine dinucleotide -to-
 reduced nicotinamide adenine dinucleotide #### File:
 Sirtuin_protein_levels.csv **Description:** Measurements of protein levels
 of Sirtuin 3 short and long isoforms in liver, oxidative skeletal muscle
 taken from the pectoral adductor muscle, and white, glycolytic skeletal
 muscle (WM). Sirtuin protein levels were normalized to total protein in
 each sample. Protein levels were quantified using the Compass software
 (ProteinSimple). ##### Variables * Fish_ID liver: Unique fish
 identification number for the liver sample * SIRT3_short_normalized liver:
 amount of the sirtuin 3 short isoform in liver * SIRT3_long_normalized
 liver: amount of the sirtuin 3 long isoform in liver * Fish_ID
 pect: Unique fish identification number for the pectoral adductor sample *
 SIRT3_short_normalized pect: amount of the sirtuin 3 short isoform in the
 pectoral adductor * SIRT3_long_normalized pect: amount of the sirtuin 3
 long isoform in pectoral adductor * Fish_ID WM: Unique fish identification
 number for the white muscle sample * SIRT3_short_normalized WM: amount of
 the sirtuin 3 short isoform in white muscle * SIRT3_long_normalized
 WM: amount of the sirtuin 3 long isoform in white muscle #### File:
 SMR_and_mito_respiration.csv **Description:** Measurements of standard
 metabolic rate (SMR) using intermittent flow respirometry and respiration
 rates of mitochondria in permeabilized liver.  ##### Variables * Fish
 ID: Unique fish identification number * Tank: tank number * Respirometry
 Chamber: number of respirometry chamber * Temperature Group: temperature
 acclimation group of the individual * Treatment: 10 degree C control or 10
 degree C treated with NMN or 10 degree C treated with EX-527, or 20 degree
 C * Weight (g) (anesthetized): mass of fish obtained after it was
 anesthetized * Length (cm): standard length of fish * condition
 factor: bm/Ls ^3 X100; bm = body mass (g), and L~s~ = standard length
 (cm). * OXP I: rate of oxidative phosphorylation through complex I * OXP
 II: rate of oxidative phosphorylation through complex II * OXP I,II: rate
 of oxidative phosphorylation through complex I and II * ETS: rate of
 uncoupled respiration * CCO: cytochrome c oxidase activity * Leak
 Omy: rate of leak respiration in the presence of oligomycin. * SMR:
 standard metabolic rate * Method for calculating SMR: means of the lowest
 normal distribution (MLND) or 20% quantile Missing variable indicated with
 blank cell or N/A