PCR was performed with SYBR Green I dye
on a LightCycler™ (Roche Diagnostics,
Mannheim,
Germany).
Primers
were
purchased from TIB MOLBIOL (Berlin,
Germany). Primer sequences and PCR
conditions are given in Supplementary Table
1.
Immunoblotting and quantification of
myotube ANGPTL4. 50µg of total cellular
protein (determined by the Bradford method)
or 50µl of culture supernatant were separated
by SDS-PAGE under reducing conditions.
Protein was transferred onto nitrocellulose
and the membrane was blocked with 10mM
Tris pH 8.0, 150mM NaCl, 0.05% Tween 20
containing 5% skim milk powder. After
incubation with anti-ANGPTL4 antibody
(BioVendor, Heidelberg, Germany, 1:2,000),
immunocomplexes were detected with a horse
radish peroxidase-conjugated anti-rabbit
antibody. Specific signals were visualized by
enhanced chemiluminescence. Intracellular
and secreted ANGPTL4 protein was
quantified
by
ELISA
(BioVendor,
Heidelberg, Germany; for further information
about the ELISA, see paragraph “Blood
analyses”). PPARδ immunoblotting was
performed with the anti-PPARδ antibody H74 (Santa Cruz Biotechnology, Heidelberg,
Germany) according to the manufacturer’s
protocol.
RNA interference (RNAi). C2C12 myocytes
were seeded at a density of 150,000cells/well
in 6-well plates. 24h later, cells were
transfected for 8h with 100pmol/well pooled
siRNA molecules using the CellPhect
Transfection kit from Amersham Biosciences
(Freiburg, Germany). Pools consisted of four
different siRNA molecules (siGENOME
siRNA set) designed by Dharmacon
(Lafayette, CO, USA). After transfection,
siRNA was washed out and cells were treated
for 3min with 15% glycerol in 10mM HEPES
pH 7.5, 150mM NaCl. Then, cells obtained
fresh medium for 16h. In the PPARD and
PPARG knock-down setting, some cultures
SUPPLEMENTARY MATERIALS
RESEARCH DESIGN AND METHODS
Cell culture. Primary human SKM cells were
obtained from needle biopsies of the vastus
lateralis muscle, grown and differentiated as
previously described (1). First-passage cells
were used for experiments. On day 5 of
differentiation, human myotubes were treated
either with NEFA-free bovine serum albumin
(BSA) for control or with BSA-conjugated
LCFA (BSA conjugation of LCFA was
described earlier (2)). The medium glucose
concentration was 1g/l (=5.5mM). Murine
C2C12 myocytes were treated analogously.
Human myotubes were also treated with
troglitazone,
rosiglitazone,
Wy-14,643,
fenofibrate, or GW501516 (DMSO was used
for carrier control).
Microarray analysis. Treated myotubes from
a healthy male German donor were washed
and harvested by trypsinization. RNA was
isolated with RNeasy columns (Qiagen,
Hilden, Germany). Total RNA was further
processed for GeneChip® analysis using
Human Genome U133 Plus 2.0 arrays from
Affymetrix by the customer service program
of the Microarray Facility Tübingen, an
official
Affymetrix
service
provider
(www.microarray-facility.com). The service
also included image processing, quality
control of the arrays, scaling and
normalization of signal values, comparison
between experimental and baseline datasets,
and generation of lists with differentially
regulated genes. Only expression changes
≤0.5-fold and ≥2-fold were considered as
relevant.
Reverse transcriptase (RT)-PCR. Cells
were washed and harvested by trypsinization.
RNA was isolated with RNeasy columns.
Total RNA treated with RNase-free DNase I
was transcribed into cDNA using AMV
reverse transcriptase and the First Strand
cDNA kit from Roche Diagnostics
(Mannheim, Germany). Quantitative real-time
1
were treated for further 20h with BSA or
oleate prior to cell lysis.
Chromatin immunoprecipitation (ChIP)
analysis. C2C12 cells treated for 6h with
BSA or oleate were cross-linked with
formaldehyde and subjected to anti-PPARδ
ChIP using the antibody supplier’s standard
protocol
(Santa
Cruz
Biotechnology,
Heidelberg, Germany). Genomic DNA coimmunoprecipitated with the anti-PPARδ
antibody K-20 was analysed for the presence
of ANPTL4 DNA using PCR amplification of
a 310-bp fragment harbouring the PPRE in
intron
3
(forward
primer
–
5’CCGATTGGATGAGAGGAAAG-3’; reverse
primer
–
5’GTATCCCACACACACACCCA-3’;
annealing temperature – 58°C; 50 cycles;
20% enhancer solution). PCR products were
visualised by PAGE (5%) and silver staining.
Subjects and metabolic characterization.
The 38 myotube donors were young, normalweight, and non-diabetic participants of the
TÜbingen Family study for type 2 diabetes
(TÜF) and were metabolically characterized
by oral glucose tolerance test (OGTT) and
hyperinsulinemic-euglycemic clamp (HEC),
as previously described in detail (3). For
clinical characteristics, see (4). In addition, a
cohort of 108 subjects at an increased risk for
type 2 diabetes was recruited from the
TUebingen Lifestyle Intervention Program
(TULIP) for plasma ANGPTL4 measurement.
The individuals were characterized by OGTT
and a subgroup of 91 subjects also by HEC.
The cohort’s clinical characteristics are given
in Supplementary Table 2. All TÜF and
TULIP participants gave informed written
consent to the study, and the protocol was
approved by the Ethics Committee of the
University of Tübingen.
Determination of body fat distribution and
ectopic lipids. Body fat content was
measured by bioelectrical impedance. BMI
was calculated as weight divided by squared
height (kg/m²). Waist-hip ratio was calculated
as an index of abdominal body fat
distribution. In addition, total, visceral, and
non-visceral fat contents were determined
with an axial T1-weighted fast-spin echo
technique with a 1.5-T whole-body imager
(Magnetom Sonata, Siemens Medical
Solutions, München, Germany), as described
earlier (5). Intramyocellular (tibialis anterior
and soleus muscle) and intrahepatic lipids
were measured by localized 1H-magnetic
resonance spectroscopy, as formerly reported
(3).
Blood analyses. Glucose was determined
using a bedside glucose analyzer (Yellow
Springs Instruments, Yellow Springs, CO,
USA). Insulin and C-peptide were determined
by microparticle EIA (Abbott Laboratories,
Tokyo, Japan) and RIA (Byk-Sangtec,
Dietzenbach, Germany), respectively. NEFA
and glycerol were measured using enzymatic
assays from WAKO Chemicals (Neuss,
Germany)
and
Sigma
(Deisenhofen,
Germany), respectively. Triglycerides were
measured with a standard colorimetric method
using a Roche/Hitachi analyzer (Roche
Diagnostics,
Mannheim,
Germany).
ANGPTL4 was quantified by ELISA
(BioVendor,
Heidelberg,
Germany).
According to the supplier’s information,
specificity of the human ANGPTL4 ELISA
kit was tested by assessing the assay’s crossreactivity with human ANGPTL3, the
ANGPTL family member with the highest
homology to human ANGPTL4 (27% amino
acid identity). No signal was detected with up
to 100ng/ml ANGPTL3 (6). The polyclonal
antibody used for the ELISA was raised in
rabbits by immunization with recombinant
human ANGPTL4 (amino acids 26-229 fused
to an N-terminal His-tag) and purified by
immunoaffinity chromatography. The intraassay coefficient of variation ranged from 4.37.9% (four serum samples; 8 replicates), the
inter-assay coefficient of variation from 5.810.1% (four serum samples in duplicates; 6
replicate assays).
2
Calculations. Areas under the curve (AUC)
of plasma metabolite levels during OGTT
were calculated using the trapezoidal method.
First-phase insulin secretion (in pM) was
estimated from plasma insulin and glucose
concentrations during OGTT using validated
equations, as described formerly (7). Insulin
sensitivity from OGTT was estimated, as
proposed by Matsuda and DeFronzo (8):
10,000/(Glc0·Ins0·Glcmean·Insmean)½. Data are
given in units. Clamp-derived insulin
sensitivity (in units) was calculated as glucose
infusion rate necessary to maintain
euglycemia during the last 60min (steadystate) of the clamp (in µmol·kg-1·min-1)
divided by the steady-state insulin
concentration.
Statistics. Unless otherwise indicated, all data
are presented as means±SE. Analysis of
differences between multiple treatment
groups was carried out using ANOVA. Post
hoc comparisons were performed by all-pair
Student’s
t-test.
Differences
between
treatment groups over time were tested using
repeated measures MANOVA. Two-group
comparisons were performed using unpaired
Student’s t-test. Simple and multiple linear
regression analyses were carried out after logtransformation of data. For multiple linear
regression analysis, the standard least squares
method was used. A p-value <0.05 was
considered statistically significant. The
statistical software package JMP 4.0 (SAS
Institue, Cary, NC, USA) was used.
REFERENCES
1. Krutzfeldt,J, Kausch,C, Volk,A, Klein,HH, Rett,K, Haring,HU, Stumvoll,M: Insulin
signaling and action in cultured skeletal muscle cells from lean healthy humans with high
and low insulin sensitivity. Diabetes 49:992-998, 2000
2. Staiger,H, Staiger,K, Haas,C, Weisser,M, Machicao,F, Haring,HU: Fatty acid-induced
differential regulation of the genes encoding peroxisome proliferator-activated receptorgamma coactivator-1alpha and -1beta in human skeletal muscle cells that have been
differentiated in vitro. Diabetologia 48:2115-2118, 2005
3. Stefan,N, Machicao,F, Staiger,H, Machann,J, Schick,F, Tschritter,O, Spieth,C, Weigert,C,
Fritsche,A, Stumvoll,M, Haring,HU: Polymorphisms in the gene encoding adiponectin
receptor 1 are associated with insulin resistance and high liver fat. Diabetologia 48:22822291, 2005
4. Staiger,H, Kaltenbach,S, Staiger,K, Stefan,N, Fritsche,A, Guirguis,A, Peterfi,C,
Weisser,M, Machicao,F, Stumvoll,M, Haring,HU: Expression of adiponectin receptor
mRNA in human skeletal muscle cells is related to in vivo parameters of glucose and lipid
metabolism. Diabetes 53:2195-2201, 2004
5. Machann,J, Thamer,C, Schnoedt,B, Haap,M, Haring,HU, Claussen,CD, Stumvoll,M,
Fritsche,A, Schick,F: Standardized assessment of whole body adipose tissue topography by
MRI. J.Magn Reson.Imaging 21:455-462, 2005
6. Stejskal,D, Karpisek,M, Reutova,H, Humenanska,V, Petzel,M, Kusnierova,P, Vareka,I,
Varekova,R, Stejskal,P: Angiopoietin-like protein 4: development, analytical
characterization, and clinical testing of a new ELISA. Gen.Physiol Biophys. 27:59-63, 2008
7. Stumvoll,M, Mitrakou,A, Pimenta,W, Jenssen,T, Yki-Jarvinen,H, Van Haeften,T, Renn,W,
Gerich,J: Use of the oral glucose tolerance test to assess insulin release and insulin
sensitivity. Diabetes Care 23:295-301, 2000
8. Matsuda,M, DeFronzo,RA: Insulin sensitivity indices obtained from oral glucose tolerance
testing: comparison with the euglycemic insulin clamp. Diabetes Care 22:1462-1470, 1999
3
Supplementary Table 1. Conditions for RT-PCR quantification of mRNAs and 28S-rRNA.
Tannealing
No. of
MgCl2
[°C]
cycles
[mM]
RNA
Forward primer (5’→3’)
Reverse primer (5’→3’)
human ANGPTL4 mRNA
AGCATCTGCAAAGCCAGTTT
GCGCCTCTGAATTACTGTCC
68
45
4
murine ANGPTL4 mRNA
CAAAACAGCAAGATCCAGCA
TTGGAAGAGTTCCTGGCAGT
66
45
3
human PPARA mRNA*
CCATCGGCGAGGATAGTTCT
CTGCGGTCGCACTTGTCATA
67
45
4
murine PPARA mRNA
TCCCTGTGAACTGACGTTTG
TCACCGATGGACTGAGAAATC
64
45
5
human PPARG mRNA
AGAACAGATCCAGTGGTTGC
GCCAACAGCTTCTCCTTCTC
64
40
4
murine PPARG mRNA
CTGGCCTCCCTGATGAATAA
GGCGGTCTCCAGTGAGAATA
61
45
5
human PPARD mRNA*
AAGAGGAAGTGGCAGAGGCA
TGCCACCAGCTTCTTCTTCT
67
40
4
murine PPARD mRNA
GCCATCATTCTGTGTGGAGA
CCGTCTTCTTTAGCCACTGC
66
45
3
human CD36 mRNA
CTAATGCCAGTTGGAGACCT
ACTGTGAAGTTGTCAGCCTC
64
45
4
human UCP3 mRNA
ATGGACGCCTACAGAACCAT
CTGGGCCACCATCTTTATCA
62
45
4
human PDK4 mRNA
CATGAAGCAGCTACTGGACT
GGTTCATCAGCATCCGAGTA
62
45
3
murine LIPE mRNA
AGACACCAGCCAACGGATAC
ATCACCCTCGAAGAAGAGCA
68
45
4
murine PNPLA2 mRNA
TCCGAGAGATGTGCAAACAG
CTCCAGCGGCAGAGTATAGG
68
45
3
human 28S-rRNA
ACGGCGGGAGTAACTATGACT
CTTGGCTGTGGTTTCGCT
63
50
4
murine 28S-rRNA
CCAGTACTTCACTCCTGTCT
TCTAAGAGTGAGCAACGACG
61
45
3
*Reactions contained 5% DMSO
6
Supplementary Table 2. Clinical characteristics of the TULIP cohort.
Quantitative trait data are given as means±SE (range). Unadjusted data are shown.
N (women/men)
56/52
age [y]
43±1 (19-65)
BMI [kg/m²]
29.6±0.5 (19.5-48.4)
body fat content [%]
32±1 (16-52)
waist-hip ratio
0.91±0.01 (0.61-1.13)
glucose, fasting [mM]
5.10±0.04 (4.16-6.11)
glucose, 120 min OGTT [mM]
6.36±0.12 (3.61-11.00)
insulin sensitivity, OGTT [units]
14.04±0.75 (2.58-32.51)
1st-phase insulin secretion, OGTT [pM]
1386±77 (339-4385)
triglycerides, fasting [mg/dl]
120±9 (26-857)
NEFA, fasting [µM]
606±23 (226-1315)
NEFA, 120 min OGTT [µM]
86±8 (25-730)
7
Supplementary Table 3. Genes repressed by both palmitate and linoleate in human
myotubes. Cells were treated for 20 h with 1.25% BSA for control or 0.5mM LCFA (changes
≤0.5-fold, LCFA vs. BSA).
Gene title
Gene symbol
UniGene ID
fold-change
by palmitate
fold-change
by linoleate
hypothetical protein MGC33371
dolichyl-phosphate β-glucosyltransferase
PLEKHH1 protein
DREV1 protein
LOC442530
MGC33371
ALG5
PLEKHH1
DREV1
-
Hs.288304
Hs.507769
Hs.546414
Hs.279583
Hs.304253
0.14
0.34
0.34
0.35
0.40
0.31
0.46
0.43
0.47
0.44
8