media.nature.com€¦ · Web viewSupplemental data Supplementary table 1. Diet ary compositions.
Transcript of media.nature.com€¦ · Web viewSupplemental data Supplementary table 1. Diet ary compositions.
Supplemental data
Supplementary table 1. Dietary compositions. Presented are the proportions of certain substances
within the diet mixture in percentages based on mass (w/w= weight/weight, in grams). Chow was
provided by Sniff R/M-H diet V1530, Sniff Spezialdiäten GmbH, Soest, Germany, and HFD
(D12451) and HFDB were produced by Research Diets Inc, New Brunswick, USA.
Chow HFD HFDB% (w/w) kcal % % (w/w) kcal % % (w/w) kcal %
Protein 19.0 33.0 24.0 20.0 24.0 20.0Carbohydrates 54.1 58.0 41.0 35.0 41.0 35.0Crude fat 3.3 9.0 24.0 45.0 24.0 45.0
Total 100 100 100kcal/g 3.0 4.7 4.7
IngredientsCrude fibre 4.9 0 5.8 0 5.8 0Starch 36.5 39.1 8.5 7.2 8.5 7.2Sugar 4.7 5.0 20.1 17.0 20.1 17.0Casein - 23.3 19.7 23.3 19.7Lard - 20.7 39.4 20.7 39.4Soybean oil 3.3 9.0 2.9 5.5 2.9 5.5Dextrose - 11.6 9.8 11.6 9.8
Fatty acids:Butyric acid - - 5.00C14:0 0.01 0.01 0.01C16:0 0.47 0.47 0.47C16:1 0.01 0.01 0.01C18:0 0.08 0.08 0.08C18:1 0.62 0.62 0.62C18:2 1.80 1.80 1.80C18:3 0.23 0.23 0.23C20-C22 0.01 0.01 0.01
Minerals & vitaminsCrude ASH 6.4 - -Mineral mix S10026 1.16 0.0 1.16 0.0Vitamins mixV10001 - 1.16 0.9 1.16 0.9Calcium 1.0 0.64 0.64Phosphorus 0.70 1.92 1.92
Supplementary table 2. List of parameters used in MRI experiments
Imaging sequencesAnatomical T2*w ASL DTI rsfMRI
Imaging method GE FAIR-ASL 4-shot spin-echo PI Spin-echo EPIEcho time (ms) 7.35 10.8 21.0 10.0Repetition time (s) 0.86 12.0 7.75 1.80 Image matrix (pixel x pixel) 512x512 128x96 128x128 96x96Field-of-view (mm) 40x40 25x25 20x20 25x25Spatial resolution (µm/pixel) 78x78x500 195x260x 1000 156x156x500 260x260x500Number of slices 20x3 16 20 20Total acquisition time (min) 8 12 35 10
Supplementary table 3. Forward and reverse sequences of the primers. GAPDH: glyceralehyde-3-
phosphate dehydrogenase; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; IL-1β: interleukin-1β;
ObRA: leptin receptor A; ObRB: leptin receptor B; InsulinR: insulin receptor; PSD95: postsynaptic
density protein-95; FW: forward; REV: reverse; CM: custom made.
Primer Sequence ReferencesGAPDH FW 5'-AGGTCGGTGTGAACGGATTTG-3' CM
REV 5'-TGTAGACCATGTAGTTGAGGTCA-3'TNF-α FW 5'-CAGACCCTCACACTCAGATCATCT-3' CM
REV 5'-CCTCCACTTGGTGGTTTGCTA-3'IL-6 FW 5'-CAAGTCGGAGGCTTAATTACACATG-3' CM
REV 5'-ATTGCCATTGCACAACTCTTTTCT-3'IL-1β FW 5'-GCAACTGTTCCTGAACTCAACT-3' CM
REV 5'- ATCTTTTGGGGTCCGTCAACT-3'ObRB FW 5'-GTTCCAAACCCCAAGAATTG-3' (1)
REV 5'-TGCTCAAATGTTTCAGGCTTT-3'InsulinR FW 5'-ATGGGCTTCGGGAGAGGAT-3' (2)
REV 5'-GGATGTCCATACCAGGGCAC-3'Synaptophysin FW 5'- TCTTTGTCACCGTGGCTGTGTT-3' (3)
REV 5'-TCCCTCAGTTCCTTGCATGTGT-3'PSD95 FW 5'-CCCCAGGATATGTGAACGGAA-3' CM
REV 5'-AAGATGGATGGGTCGTCACC-3'
Supplementary table 4. Mean ± SEM of several parameters at the end of the experiment
Mid-adulthood, 9 m.o. Late adulthood, 12 m.o.Chow, n=10 HFD, n=10 HFDB, n=10 Chow, n=10 HFD, n=11 HFDB, n=9
Body w. (g)
35.90 ±1.32 46.70 ±1.04*** 40.22 ±1.58 41.75 ±1.17 56.45 ±1.45*** 43.10±1.18 ∆∆∆
Food intake (kcal/day/mouse)
8.69 ±2.24 10.05 ±0.56 7.17 ±0.30 10.41 ±1.98 12.44 ±0.73 12.63 ±2.05
Cholesterol (mM)
14.99 ±0.92 30.97 ±2.05*** 20.49 ±1.91∆∆ 13.03 ±0.88 43.30 ±3.60*** 22.74 ±1.68∆∆
Triglycerides (mmol/L)
3.65±0.40 5.47 ±0.78*** 2.86 ±0.44∆∆ 3.09 ±0.35 6.47 ±0.63*** 3.44 ±0.51#
Glucose¹ (mmol/L)
7.90 ±0.31 8.20 ±0.30 8.40 ±0.64 8.30 ±0.26 7.10 ±0.32 7.50 ±0.38
Insulin(ng/ml)
6.50±0.87 8.41±1.13 4.40±0.39∆ 6.53±1.39 13.98±2.11*** 6.28±0.83∆∆∆
Liver w. (g)
1.31 ±0.11 1.74 ±0.16* 1.42 ±0.12 1.59 ±0.09 2.60 ±0.18*** 1.57 ±0.07∆∆∆
Omental fat w. (g)
1.18 ±0.15 1.66 ±0.10*** 1.10 ±0.07∆∆∆ 1.40 ±0.07 2.07 ±0.08*** 1.30 ±0.07∆∆∆
Inguinal fat w. (g)
1.14 ±0.01 2.29 ±0.16*** 1.68 ±0.09∆∆∆ 1.23 ±0.19 3.12 ±0.19*** 2.22 ±0.17∆∆∆
Epididymal fat w. (g)
1.77 ±0.15 2.08 ±0.22 1.69 ±0.13 2.04 ±0.15 2.39 ±0.12 1.89 ±0.09∆∆∆
Preproinsulin 510 ±92 182 ±40*** 176 ±82 328 ±55 214 ±66 136 ±38
ObRb 681 ±100 617 ±75 530 ±49 450 ±61 525 ±85*** 599 ±106
Synaptophysin 9937 ±1479 18784 ±3334* 10813 ±1979# 1000 ±2449 12559 ±2459 13299 ±1424
<Relative values of mRNA levels (2-∆Ctx100 000) for preproinsulin, leptin receptor B (ObRb) and
synaptophysin in the hippocampus. w.= weight; ¹Data derived from analysis at 7 m.o. and
resepectively 10 m.o.; *P<0.05 compared to Chow; ***P<0.001 compared to Chow; #P<0.08
compared to HFD; ∆P<0.05 compared to HFD; ∆∆∆P<0.001 compared to HFD; Data are represented
in Mean ±SEM>
<Supplementary figure 1. Inflammatory aggregates in the liver. Late adult mice fed a HFD have an
increased levels of inflammatory aggregates within the liver. 9 m.o.= mid adult; 12 m.o.= late adult.
Data are expressed in mean ± SEM. **P<0.01>
<Figure 2. Morris water maze. A & B) The mean latency during the acquisition trials to find and
reach the hidden platform of mid- (A; 9 m.o.) and late (B; 12 m.o.) adult mice. The four trials per day
were averaged. C) Time spend in the NE quadrant during the first 30 seconds of the probe trial. D)
Percentage of used hippocampal dependent search strategies during the first 30 seconds of the probe
trial. Data are presented in mean ± SEM. *P<0.05>
<Supplementary figure 3. resting state fMRI (rsfMRI) of mid- (9 m.o.) and late (12 m.o.) adult
mice. A, D and G) Total correlation matrixes of mid-adult mice (9 m.o.). B, E and H) Total correlation
matrixes of of late adult mice (12 m.o.). A and B) represents functional connectivity of Chow fed mice,,
D and E) shows HFD fed mice and G and H) represents the batch of mice receiving a butyrate
intervention, respectively 9 or 12 m.o. The selected brain regions, dorsal hippocampus (DH), ventral
hippocampus (VH), auditory cortex (AC), motor cortex (MC), somatosensory cortex (SSC) and visual
cortex (VC), are subdivided in left hemisphere region (first row) and right hemisphere region (second
row). In general the DH and VH are strongly connected bilateral, and AC, MC and SSC are highly
interconnected. A higher Z-score (red) indicates a stronger functional connectivity. C, F and I)
Statistical analyses of functional connectivity to reveal a significant age effect, which are represented
as a matrix. Yellow: P<0.050. More specific, Chow and butyrate fed mice of 9 m.o. have an improved
functional connectivity when compared to mice fed Chow and HFDB of 12 m.o. (A-C and G-I) within
the somatosensory and visual cortex. In addition, the functional connectivity of 12 m.o. mice is
comparable with mice of 9 m.o. fed a HFD (D-B,E,H). Mid-adult: Chow, n=10; HFD, n=10 and
HFDB, n=9. Late adult: Chow, n=7; HFD, n=11 and HFDB, n=9>
<Supplementary figure 4. Diffusion tensor imaging (DTI). A) Mean levels of a DTI parameter,
fractional anisotropy (FA), in the hippocampus. Decreased FA levels indicate a decreased white
matter integrity in this brain area. In the 12 m.o. group HFD mice had a decreased FA in the
hippocampus, which indicates a loss of white matter integrity. B) Mean levels of mean diffusivity (MD)
in the hippocampus. An increased MD level indicates a loss of grey matter integrity within the
hippocampus. MD levels were increased in 12 m.o. mice fed a HFD compared to Chow fed 12 m.o.
mice indicating a loss of grey matter integrity. Data are expressed in mean ± SEM. *P<0.050>
<Supplementary figure 5. Relative values of mRNA levels in the hippocampus for A)
preproinsulin, B) leptin receptor B (Ob-Rb) and C) synaptophysin. Data are expressed in
mean ± SEM. **P<0.001, *P<0.050 and #P<0.08>
<Figure 6. Microbiota composition. A) Clustering of fecal microbial variation based on distances
between groups determined with Bray-Curtis dissimilarity index at the genus level. B) Relative
abundance of top 25 minimum entropy decompositions (MEDs) across fecal microbiota of treatment
groups taking HFD fed mid-aged mice as a reference. C) Relative abundance of top 25 MEDs across
fecal microbiota of treatment groups taking HFD fed late-aged mice as a reference. 9 m.o. indicates
mid-adult aged mice fed a Chow, HFD or HFDB diet. 12 m.o. describes late adult aged mice fed
either Chow, HFD or HFDB>
Supplementary table 4. Overview of the results and the related P and F values of all described significant parameters
9 m.o. Mid-adulthood 12 m.o. Late adulthoodHFD vs Chow HFD vs HFDB HFD vs Chow HFD vs HFDBP and F values P and F values P and F values P and F values
Body w. ↑ P<0.001 F(1,18)=18.71 n.s. ↑ P<0.001 F(1,19)=45.62 ↓ P<0.001 F(1,18)=20.00
Food intake ↓ P<0.05 F(1,3)=21.80 n.s. ↓ P<0.01 F(1,2)=84,58 n.s.Cholesterol ↑ P<0.001 F(1,18)=50.46 ↓ P<0.01 F(1,18)=13.93 ↑ P<0.001 F(1,19)=60.22 ↓ P<0.001 F(1,18)=22.68
Triglycerides ↑ P=0.050 F(1,18)=4.37 ↓ P<0.01 F(1,18)=8.54 ↑ P<0.001 F(1,19)=20.95 ↓ P<0.01 F(1,18)=13.14
Insulin n.s. ↓ P<0.01 F(1,18)=11.21 ↑ P<0.01 F(1,19)=8.34 ↓ P<0.01 F(1,18)=9.77
Liver w. ↑ P<0.05 F(1,18)=4.99 n.s. ↑ P<0.001 F(1,18)=24.15 ↓ P<0.001 F(1,18)=24.15
Omental fat w. ↑ P<0.05 F(1,18)=7.05 ↓ P<0.001 F(1,18)=21.00 ↑ P<0.001 F(1,18)=32.02 ↓ P<0.001 F(1,18)=50.10
Inguinal fat w. ↑ P<0.001 F(1,18)=28.12 ↓ P<0.001 F(1,18)=10.51 ↑ P<0.001 F(1,18)=48.78 ↓ P<0.001 F(1,18)=11.66
Epididymal fat w. n.s. n.s. n.s. P<0.001 F(1,18)=10.60
LiverMircovesicular steatosis ↑ P<0.001 F(1,18)=109.84 P<0.001 F(1,19)=103.31
Macrovesicular steatosis ↑ P<0.01 F(1,18)=13.14 ↓ P<0.001 F(1,18)=20.67 ↑ P<0.001 F(1,19)=38.83 ↓ P<0.001 F(1,19)=67.39
Hypertrophy ↑ P<0.01 F(1,18)=14.97 ↓ P<0.01 F(1,18)=11,36 ↑ P<0.001 F(1,19)=71.54 ↓ P<0.001 F(1,19)=57.47
Inflammation n.s. n.s. ↑ P<0.01 F(1,19)=12,14 n.s.Fibrosis ↑ P<0.049 F(1,18)=4.47 n.s. ↑ P<0.01 F(1,19)=11.07 ↓ P<0.05 F(1,17)=7.07
MWMAcquisition n.s. ↑ P<0.001 F(1,18)=10.55 n.s. n.s.
Probe, NE quadrant n.s. ↑ P<0.05 F(1,19)=4.30 n.s. n.s.Probe, search strategy n.s. ↑ P=0.05 F(1,19)=4.30 n.s. n.s.
SBP ↑ P<0.05 F(1,17)=6.83 n.s. n.s. n.s.CBF ↓ P<0.05 F(1,17)=5.74 ↑ P<0.05 F(1,17)=6.92 n.s. n.s.DTI
FA n.s. n.s. ↓ P<0.05 F(1,13)=5.85 n.s.MD n.s. n.s. ↑ P<0.05 F(1,13)=5.80 n.s.
Functional connectivity ↓ P<0.05 ↑ P<0.05 n.s. n.s.IBA-1
Hippocampus ↑ P<0.05 F(1,17)=6.19 ↓ P<0.05 F(1,17)=5.51 n.s. n.s.CA1 n.s. ↓ P<0.05 F(1,17)=6.38 n.s. n.s.
Thalamus ↑ P<0.05 F(1,17)=8.02 ↓ P<0.05 F(1,17)=7.23 n.s. n.s.qRT-PCRs
Preproinsulin ↓ P<0.001 F(1,12)=12.74 n.s. n.s. n.s.ObRb n.s. n.s. ↑ P<0.00 F(1,17)=6.34 n.s.
Synaptophysin ↑ P<0.05 F(1,14)=4.83 ↓ P<0.08 F(1,14)=3.63 n.s. n.s.<↑=significant increase; ↓=significant decrease; w.= weight; n.s.= non significant; MWM= Morris water maze; NE= North-East; SBP= systolic blood pressure; CBF=
cerebral blood flow; DTI= diffusion tensor imaging; FA= fractional anisotropy; MD= mean diffusity; IBA-1= ionized calcium-binding adapter molecule 1; CA1= cornu
ammonis 1; qRT-PCRs= quantitative real-time polymerase chain reaction; ObRb= leptin receptor B>
References
1. Schroyen B, Guimaraes EL, Dolle L, Coulon S, Empsen C, Nyssen M, Geerts A, Colle I, Geerts A, van Grunsven LA: Leptin-mediated reactive oxygen species production does not significantly affect primary mouse hepatocyte functions in vitro. Eur J Gastroenterol Hepatol 2012;24:1370-13802. Lofqvist C, Willett KL, Aspegren O, Smith AC, Aderman CM, Connor KM, Chen J, Hellstrom A, Smith LE: Quantification and localization of the IGF/insulin system expression in retinal blood vessels and neurons during oxygen-induced retinopathy in mice. Investigative ophthalmology & visual science 2009;50:1831-18373. Gilabert-Juan J, Castillo-Gomez E, Perez-Rando M, Molto MD, Nacher J: Chronic stress induces changes in the structure of interneurons and in the expression of molecules related to neuronal structural plasticity and inhibitory neurotransmission in the amygdala of adult mice. Exp Neurol 2011;232:33-40