Supplemental Notes:
LCMS proteomics analysis.
Hepatic proteome detection of the 18 hr fasted and refed Iqgap2-/- mice and age-matched control
mice was performed using LCMS, as follows: Equal amounts of peptide samples (2 µg) were
injected and captured onto a trap column packed with reverse phase beads (3 cm x 100 µm i.d.)
packed with 200 Å Magic- C18AQ beads (Michrom Bioresources, Auburn, CA) using a flow
rate of 2 µl/min and a solvent system consisting of 99% solvent A and 1% solvent B (acetonitrile
containing 0.1% formic acid) where they were washed for a total of 10 minutes. Peptides were
then eluted from the trap column and separated by reversed-phase chromatography over a pulled
tip, fused silica analytical column (30 cm x 75 µm i.d.) packed with 100 Å Magic- C18AQ at a
flow rate of 300 nL/min using a linear gradient from 95% solvent A/ 5% solvent B to 60%
solvent A/40 % solvent B over 120 min. Data dependent analysis (DDA) with the Velos-FTICR
mass spectrometer consisted of a high resolution (25,000 RP) MS1 scan followed by low
resolution MS2 analysis on the 10 most intense precursors. MS2 settings included a normalized
collision energy of 35V, isolation width of 4, activation time of 10 ms, activation Q of 0.25 and a
minimum signal threshold of 10,0000. Dynamic exclusion was enabled with settings including
an exclusion window of 0.5 m/z low to 1.5 m/z high, exclusion time of 30s, a list size of 500, and
a repeat count of 1. The standard mass spectrometer tune method settings were as follows: Spray
voltage, 1.8 kV; heated capillary temperature, 325 ºC; Automatic Gain Control (AGC) enabled.
All samples were analyzed by LC-MS/MS in triplicates in a randomized order. An equimolar
(500 fmol/µL) binary peptide standard mix of angiotensin I and neurotensin (Sigma-Aldrich, St.
Louis, MO, USA) was used between the sample runs to check the run-to-run reproducibility of
the LC-MS system.
Peptide and protein identification was achieved by subjecting the mass spectrometry data to
Sequest search engine. Sequest output was utilized for protein abundance calculation by the
Normalized Spectral Indexing (SIN) method (detail description in the Methods and Material
section) A comprehensive list of proteins found changing (P-value ≤ 0.05) in fast Iqgap2-/- mice
compared to the control mice (fast Iqgap2-/- mice/ fast control ; fast Iqgap2-/- mice/ refed control
) or in refed Iqgap2-/- mice compared to the age-matched control mice (refed Iqgap2-/- mice/
refed control mice) can be found in Supplementary Table 1, while only critical proteins from
this list were included into Table 3 and their functional role was discussed in the main
manuscript.
Two mitochondrial proteins from Table 3, namely pyruvate carboxylase and Pyruvate
dehydrogenase protein X component (Uniprot Accession: Q05920 and Q8BKZ9) showed
significant higher levels (4.5- and 3.6-fold) in fast Iqgap2-/- relative to the refed Iqgap2-/- mice.
In all fast control samples (biological replicates =3), corresponding peptides of these proteins
were either not detected or had very low confidence identification scores (PepetideProbability
score ~ 0.07), and therefore not identified and quantified successfully. Therefore, we believed
that both proteins were significantly higher in the Fast Iqgap2-/- mice relative to Fast control
mice.
Another protein from Table 3, namely, pyruvate kinase exists in two isoforms, namely R/L
(Uniprot Accession: P53657) and M1/M2 (Uniprot Accession: P52480) sharing 63% sequence
identity. We identified and quantified 20 peptides specific to the R/L isoform of pyruvate kinase
while only two peptides were identified for the M1/M2 form when protein search was conducted
using Sequest. One additional peptide sequence identified is common between the two isoforms.
The quantitation of R/L form by SIN method was validated by manual peptide-level MS1
intensity calculation for 5 unique peptides specific to this form (Supplementary Table 3).
Extracted ion chromatograms (EIC) for a peptide precursor ion of in the sample LC-MS runs
were selected with a window of ± 0.01 m/z around the monoisotopic mass of the precursor ion,
and with the retention time window of ± 1 minute. Areas under the EIC peaks were
automatically calculated using the Thermo Xcalibur (version 2.2) software. Statistically
significant differences in the areas of the peptide ions between Iqgap2-/- and control samples
were determined by performing one-way ANOVA test (nominal P-value cutoff of 0.05). Peak
areas obtained from Iqgap2-/- samples were divided by the peak areas obtained from control
samples to calculate the peptide-level fold changes. The resulting statistically significant (Pvalue <0.05) peptide-level fold changes were then averaged to obtain protein level fold change
by the MS1 quantitation method. Quantitation of M1/M2 form by SIN method was not performed
since none of the peptides of this protein were detected the 6 out of 9 LC-MS runs of fast control
samples. Peptide-level manual MS1 quantitation of M1/M2 form based showed 2.1-fold
increase(P <0.05 for two peptides specific for M1/M2 form) in the fast Iqgap2-/- compared to the
fast control mice.
Metabolomic Analyses
Lipid content Analysis
Lipid content analysis (diacylglycerides and triacylglycerides) was performed using thin layer
chromatography. Briefly, frozen tissues (quadriceps, liver) isolated from animals sacrificed at
the end of 18 hr fast, 5 hr refeeding experiments were weighed, and Bligh-Dryer extraction for
tissue lipid was performed. The extracted lipid was evaporated under N2 in glass tubes to
dryness and re-dissolved in chloroform/methanol/acetic acid/water (25/15/4/2) normalized by
the weight of the tissue taken to 1mg /ml solvent. 10ul (liver) and 50ul (muscle) of the each
sample was spotted to their respective lane on a silica gel plate (Whatman Partisil K6). TLC was
then performed using the conventional three solvent system.
Determination of glycogen content.
Frozen tissue (quadriceps, liver) isolated from animals sacrificed at the end of 18 hr fast, 5 hr refeeding experiments were weighed homogenized in ice-cold 6% perchloric acid. The resultant
lysate was spun at 2000 rpm for 20 minutes and supernatant containing glycogen was saved.
Glycogen was then precipitated with 5 volumes of ice-cold 100% ethanol and left on ice overnight at 4˚c to ensure complete precipitation. The precipitate was collected by spinning at
5000rpm/20minutes. Ethanol precipitation was repeated one more time to get rid of any free
glucose. The resulting clean glycogen, was dissolved in 0.1M sodium acetate buffer (pH 4.5),
and incubated in 50 microliters of amyloglucosidase overnight at 370C. Glycogen hydrolyzed
to glucose by the over night enzyme reaction was quantitated for glucose colorimetrically using
Quantichrom Glucose assay reagent ( cat # DIGL-200).
Immunoblot analysis.
Frozen liver was homogenized in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM
NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.25% sodium
deoxycholate, 1 mM NaF, 1 mM Na3VO4, and 2 mM Na4P2O7) containing a protease inhibitor
mixture (Roche Diagnostics) and also deacetylase inhibiotors for anti-lysine western. The
resultant lysates were centrifuged at 16,000 × g for 60 min at 4 °C, and protein concentrations
were quantified using the BCA (bicinchoninic acid) protein assays (Pierce, Inc.). The protein
samples (30 μg) were separated on a 8% gradient SDS-PAGE gel and transferred to PVDF
membranes using a semidry electroblotter (BioRad). Membranes were immunoblotted
(Antibodies were purchased from cell signalling, Santa crux Biotech Inc and Immunechem
Pharmaceutical Inc. ) and signals were visualized and quantified using the infrared Odyssey
Western Blotting System (Li-Cor, Lincoln, NE).
Derivatization of metabolites for gas chromatography/mass spectrometry (GC/MS)
analysis.
Fifty to 100µl blood plasma were deproteinized, deionized, and dried. The glucose was treated
with hydroxylamine hydrochloride , acetic anhydride and analysed on GCMS as aldonitrile
pentaacetate derivative. Isotopomeric determinations
were performed on a Hewlett-
PackardMass Selective Detector (model 7890A, Hewlett-Packard, Palo Alto,CA) connected to a
Hewlett-Packard gas chromatograph (model 5975c) using chemical ionization with 20% methane
(10). The glucose derivativewas isolated on an HP5 capillary column, 30 m 250µm internal
diameter. GC conditions were helium as carrier gas at a flow rate of 1.0ml/min, and sample
injector temperature of 250 C.
For glucose derivatives, the oven temperature was programmed from 220–250 C at a ramp of
10 C/min, and the retention time was 2.9 min. The glucose aldonitrile pentaacetate derivative
gives the molecular ion (C1–C6) of the glucose molecule at mass to charge ratio (m/z) of 328
chemical ionization using methane as reagent gas. Selected ion monitoring was used to follow
specific ions m/z 327 to m/z 336, with a fragment of m/z 328 corresponding to unlabeled
glucose.
Electron impact ionization of the aldonitrile derivative was used to characterize glucose
positional isotopomers at m/z 187 for C3–C6 andm/z 242 for C1–C4 fragments. Isotope
enrichment in these fragments was used to determine symmetry of the C1–C3 vs. C4–C6 glucose
carbons.
Lactate was analysed as its n-propylamide-heptafluorobutyric ester.
100ul of plasma was
acidifed and extracted with ethyl acetate and dried. Dried lactate sample, was reacted 2,2
Dimethoxypropane, methanolic HCl , n-propylamine. and heptafluro butyric anhydride to form
as n-propylamide-heptafluorobutyric esters.
GC column: HP5, length 30m, 250m, programmed at constant He flow of 1ml/min. Inlet temp.
200oC, split ratio 1:100 adjusted depending on sample concentration.
Oven temperature
programmed as follows: 100oC for 3min, then a 20oC/min ramp to 160oC and hold for 1min.
etention time: 3.96 min Relevant ion clusters for SIM analysis were CI mode (20% methane):
m/z 328 cluster for C1-C3. (ions monitored were 327 to 332).
Fatty acids and cholesterol was analyzed as its trimethylsilyl derivative using gas
chromatography (GC) -electron impact ionization mass spectrometry (MS). C16:0 (m/z 313),
C16:1 ( m/z 311), C18:0 ( m/z 341), C18:1 ( m/z 339), C18:2 ( m/z 337), Cholesterol ( m/z 368).
Hepatic Metabolite Profiling
Metabolite measurements for fast / refed Iqgap2-/- mice and control mice liver samples were
performed using freeze clamped liver samples from fasted/re-fed experiments that were extracted
in ice-cold methanol/water(1:1) spiked with internal standards (U-13C4 succinate and U-13C6
citric acid, (150 n moles / gm liver). The aqueous phase was lyophilized, derivatized as
previously described,
Breifly lyophilized liver extract of was derivatized to its (Meox-)TMS-derivatives through
reaction with 100µL of 20mg/mL methoxyamine hydrochloride solution in pyridine at 37ºC
overnight followed by 1hr reaction at 60ºC with 75µL of Nmethyl-trimethylsilyltrifluoroacetamide (MSTFA), GC-MS analysis performed using an Agilent 7890a GC with a
Gerstel automatic liner exchange cooled injection system CIS 4 PTV injector. Both split (20:1)
and large volume injections (LVI) of the sample were made. A Zebron ZB-5MS guardian
capillary column ( 30x0.25mmIDx0.25µm thickenss). GC- ramp conditions were 60ºC for 1 min,
then 10ºC/min to 320ºC for 5 minutes. PTV inlet with a total flow of 54ml/min, septum purge
flow of 3ml/min and vent flow 150ml/min, vent pressure was 2psi until 0.5 min.
MS transfer line temperature was 250 ºC. Positive EI, MS source was held at 230 ºC and MS
quad was held at 150 ºC.MSD was operated in fullscan mode ( m/z 50-m/z 600), Metabolite
identification using using NIST 11 and Fiehn mass spectral libraries.
For hepatic mass isotopomer analysis freeze clamped liver samples from [U-13C6]glucose infusion experiments was extracted in ice-cold methanol. Extracts were lyophilized and
analysed on GCMS as trimethyl silyl esters under chemical ionization conditions with 20%
methane as reagent gas under GC oven conditions as described above. Positive CI, MS source
was held at 150 ºC and MS quad was held at 150 ºCMSD. Selective ion monitoring of
m/z 218 for alanine (m/z 217-222), m/z350 for aspartate ( m/z 349-354) , m/z348 for glutamate (
m/z 347-353) ions cluster were monitored.
Mass isotopomers of glycogen glucose and RNA: RNA ribose and glycogen glucose were
isolated by acid hydrolysis of cellular RNA after Trizol purification of cell extracts. Ribose and
glycogen glucose were derivatized to their aldonitrile acetate form using hydroxylamine in
pyridine with acetic anhydride. (monitored the ion cluster around the m/z256 (carbons 1-5 of
ribose) (chemical ionization, CI). For glycogen glucose we monitored m/z327-332 using CI.
Acylcarnitine Analysis
Hepatic acylcarnitines in fasted and re-fed liver samples was determined using LC tandem MS.
For sample preparation, pulverized liver samples were extracted in 1:1:3
(water:methanol:acetonitrile) acidified to 0.1% formic acid. Supernatent from spun down
samples were lypophilized, redissolved in 0.1 % formic acid, filtered and used LCMS analysis.
Acetyl CoA measurements
Hepatic acetyl CoA in the fasted and re-fed liver samples was measured according to [36].
Briefly frozen pulverized liver samples were homogenized in phosphate buffered saline (pH 7.2)
and were acidified to 0.1% formic acid. After centrifugation to get rid of cell debris and roteins,
the supernatent was collected and pH was adjusted to 7.5 with 2-3 drops 50% ammonium
hydroxide and was taken for LCMS analysis.