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Insulin receptor signaling molecules and their phosphorylation levels were measured by Western blot [Streckel et al., 2015, Hinrichs et al., 2018], peformed by PD Dr. Maik Dahlhoff. SDS-PAGE was used to separate protein from liver tissue homogenized in Laemmli buffer. Proteins were transferred to PVDF membranes (Millipore) by electro-blotting. After processed as described above, bound antibodies were detected using the ECL Advance Western Blotting Detection Kit (GE Healthcare). The Imagequant software (GE Healthcare) was used to quantify band intensities. A square root transformation of the densiometric data approximated a normal distribution wich was assesed for differences using Student’s t-test.

Chapter 4 Results

4.1 RNA Sequencing of MIDY liver by NuGEN En-core complete

As the first transcriptome sequencing was performed with the NuGEN Encore Complete library preparation kit, the mRNA enrichment procedure was based on rRNA depletion.

rRNA levels in NuGEN samples were still fairly high at around 38.7% (Table 4.2). This is likely due to the non-random priming process not being intended for pigs as it was originally derived from humans and mice. Due to this, extra depth for the NuGEN experiment was generated and combined with previous reads, allowing for an average read depth of 31.9 million reads per sample.

4.1.1 Removal of outlier sample 742

One transgenic sow, pig 742, was discovered to have an unusual insulin expression, reaching several magnitudes higher than other pigs of both conditions (Figure 4.1). The pig was an outlier in several datasets, including the metabolomics (Figure 4.2) and proteomics data, although it generally clustered with the other transgenic sows. The glucagon expression of 742 was almost identical to that of WT pigs, while beta-hydroxybutyrate levels were only slightly increased. As glucose levels were still moderately increased, this indicated that the animal had mainly an increase in levels of misfolded non-functional insulin, possibly functional enough to have a minor effect on insulin signaling. As the MIDY pig model organism is based on total insulin deficiency, the animal was removed from further analysis.

Even with the decrease in power from a 4vs5 design compared to a balanced 5vs5 design,

Sample Sex Pregnancy status Genotype Weight (kg)

S736 female + WT 244

S737 female - TG 215

S738 female - WT 224.5

S739 female + TG 185

S740 female + TG 184

S741 female + WT 237.5

S742 female + TG 210

S743 female + WT 247

S744 female + TG 217

S745 female - WT 238

Table 4.1: Table describing the MIDY samples of the biobank. Sample S742 was removed from the biobank due to aberrant behaviour.

Sample mapped (M) rRNA (M) % rRNA mapped to genes (M)

S736 44.51 17.22 38.70 27.28

S737 52.53 21.33 40.61 31.19

S738 60.19 25.04 41.59 35.16

S739 50.37 22.20 44.08 28.16

S740 54.57 18.95 34.72 35.62

S741 56.97 25.35 44.50 31.62

S743 47.35 18.09 38.19 29.27

S744 50.98 15.86 31.10 35.13

S745 50.38 17.07 33.88 33.31

Mean 51.98 20.12 38.71 31.86

Table 4.2: Sequencing statistics for the MIDY biobank livers using NuGEN Encore Com-plete. Reads are counted in million reads (M).

4.1 RNA Sequencing of MIDY liver by NuGEN Encore complete 35

Figure 4.1: The concentrations of blood glucose, insulin, glucagon and beta-hydroxybutyrate during the lifetime of the pig. Means and standard deviations for each group is shown. MIDY animal 742 is displayed as grey and not grouped with other MIDY animals in order to show its aberrant phenotype. Significance is indicated between groups excluding 742 (*p <0.05; **p <0.01; ***p <0.001). Borderline significance (p <0.08) is indicated by °.

the number of significantly differentially expressed genes grew on the transcriptomics level, suggesting that pig 742 was more similar to the WT pigs. While the suggestion that the pig was suffering from insulinoma was made, no evidence of said growth could be detected.

Another possibility could be alternate regulation of the mutant insulin cassette due to mutation or homologous recombination. The decision was made to not analyze sow 742 at all in other tissues.

4.1.2 Differential expression

In the MIDY vs WT analysis of hepatic tissue 533 significantly differentially expressed genes were detected, 320 upregulated and 213 downregulated (Figure 4.3 A, B), (Table

Figure 4.2: PCA of metabolomics data from the MIDY liver samples showing the seperation of MIDY and WT samples. Animal 742 is marked as grey, showing its clustering with WT samples.

4.3, Table 4.4). Most highly upregulated was ADAMTS17, which encodes ADAM with thrombospondin type 1 motif 17. ADAMTS17 has a relatively low expression in the WT pigs where the TPM is around 0.1, whereas the TPM in the MIDY pig is around 1.4.

With DESeq2 corrected fold changes this gives a log2fold change of 2.7. The most highly downregulated gene was SHROOM3, with a log2fold change of -1.25. As reflected in the number of differentially expressed genes the log2fold changes of upregulated genes were generally higher than that of the downregulated ones (Figure 4.4). A list of all significant genes including their p-values and log2 fold changes discussed in this thesis can be found in (Supplementary Table A.1).

4.1 RNA Sequencing of MIDY liver by NuGEN Encore complete 37 Gene symbol Mean expression (counts) l2fc (MIDY/WT) p-value FDR

ADAMTS17 39.89 2.69 6.37E-31 9.42E-27

SLC25A47 1539.32 1.83 5.88E-23 4.35E-19

BMP8B 50.19 2.02 1.42E-18 6.98E-15

SLX4IP 887.15 1.13 1.03E-17 3.82E-14

CBR4 456.42 1.23 1.46E-15 4.33E-12

GPT2 6504.60 1.47 2.96E-15 7.30E-12

KLHL3 1383.27 0.92 2.83E-14 5.99E-11

RDH11 3019.80 1.00 4.60E-14 8.51E-11

GOT1 1147.73 1.16 5.30E-14 8.72E-11

LOC102165634 68.65 1.89 7.85E-14 1.06E-10

LOC106505246 50.25 1.88 1.03E-13 1.27E-10

CTH 5813.01 1.27 1.82E-13 2.07E-10

HMGCS2 1613.50 1.79 1.37E-12 1.45E-09

GYS2 9890.09 0.93 1.51E-12 1.49E-09

ELOVL6 6439.98 0.94 7.09E-12 6.55E-09

ARFGAP3 1829.70 0.93 1.22E-11 1.06E-08

ASS1 9331.15 0.80 1.31E-11 1.08E-08

MIGA2 220.90 1.01 3.81E-11 2.96E-08

SEC16A 1099.80 0.78 5.62E-11 4.15E-08

LOC106506349 47.33 1.61 2.04E-10 1.44E-07

Table 4.3: The 20 most significantly upregulated genes in the MIDY pig liver. Mean expression and log2 fold change (l2fc) are DESeq2 normalized. LOCXXXXXXXXX genes are unannotated genes.

4.1.3 Amino acid metabolism

The most common theme in the upregulated transcripts is one of amino acid metabolism.

Most amino acids had one or more catabolic enzymes upregulated. Specifically, the increased enzymes were: pyruvic transaminase 2 (GPT2; alanine), glutamic-oxaloacetic transaminase 1 (GOT1; aspartate), glutaminase (GLS2; glutamine), arylfor-mamidase (AFMID; tryptophan), homogentisate 1,2-dioxygenase (HGD; tyrosine, pheny-lalanine), serine dehydratase (SDS; serine), histidine ammonia-lyase (HAL; histidine), aminoadipate-semialdehyde synthase (AASS; lysine), aldehyde dehydrogenase 7 family member A1 (ALDH7A1; lysine), and kynurenine aminotransferase 1 (KYAT1; tryptophan, cysteine conjugates).

Gene symbol Mean expression (counts) l2fc (MIDY/WT) p-value FDR

SHROOM3 232.91 -1.25 6.16E-14 9.12E-11

IGFALS 641.04 -0.80 2.45E-10 1.65E-07

MEGF9 253.65 -1.03 5.14E-10 2.62E-07

CRP 3637.87 -1.18 6.15E-10 2.93E-07

PFKFB3 80.71 -1.15 7.63E-09 3.05E-06

THRB 837.77 -0.68 2.27E-08 7.63E-06

SFRP1 378.50 -0.99 1.05E-07 3.05E-05

TMSB4X 3052.66 -0.51 3.16E-07 7.53E-05

GIPC2 114.86 -0.97 3.39E-07 7.95E-05

PDE4B 201.77 -0.83 3.65E-07 8.31E-05

MOB3B 167.14 -0.94 3.87E-07 8.68E-05

AMPD3 532.75 -0.77 4.68E-07 9.75E-05

SGK1 2932.51 -0.88 6.24E-07 0.000123991

VIL1 92.67 -1.05 9.56E-07 0.000181266

SETBP1 424.61 -0.77 1.29E-06 0.000235104

LOC110258394 32.74 -1.19 1.41E-06 0.000250803

NREP 405.63 -1.06 1.81E-06 0.000310762

LOC110256685 151.76 -0.81 2.43E-06 0.000386522

LURAP1L 482.52 -0.72 2.79E-06 0.000430362

LOC110255180 191.79 -0.92 3.46E-06 0.000511224

Table 4.4: The 20 most significantly downregulated genes in the MIDY pig liver. Mean expression and log2 fold change (l2fc) are DESeq2 normalized. LOCXXXXXXXXX genes are unannotated genes.

4.1.4 Gene set enrichment analysis

Due to the count-based nature of RNA-Seq, the standard microarray-based GSEA was avoided and pre-ranked GSEA was utilized. Both weighted and unweighted (“classic”) analyses were performed, however the unweighted approach was given more importance as the weighted analysis is not designed for the pre-ranked analysis type. For the ranked approach either signed log-transformed p-values or log2 fold change values for genes can be used as input. Both approaches were found to give similar results, and for compatibility with other approaches using p-values as cutoffs, the preferred input was -log10 transformed p-values signed by the direction of the change. In total, 40 gene sets were found significantly increased in the MIDY liver, while 90 in total were found decreased (Table 4.5, Table 4.6), showing a reversion of the gene-level trend where upregulated transcripts were more common (Figure 4.4, Supplementary Figure B.1). Increased gene sets in the MIDY

4.1 RNA Sequencing of MIDY liver by NuGEN Encore complete 39

−1 0 1 2

0 2 4 6

mean of normalized counts (log10)

log2 fold change (MIDY/WT) 738 741 736 745 743 739 737 740 744

GPT2 BMP8B GOT1 ADAMTS17 CTH BMP8A SDS HMGCS2 LOC110262053 LOC110260953 LOC106505250 LOC106505246 KIAA1211 LOC106506349 CYP8B1 SLC25A47 AGXT LOC102165634 CBR4 SFXN3 CBR1 SYTL5 GIPC2 LOC110256694 PFKFB3 LOC102163250 LOC110256684 CIART SHROOM3 NTRK3 LOC110258705 VIL1 SFRP1 LOC110258394 CRP NREP LOC110255180 TECTB MEGF9 MOB3B

−2

−1 0 1

A B

2

Figure 4.3: A MA-plot showing the distribution of expression and differential expression among MIDY transcripts. Log2 fold change here describes the DESeq2 corrected log2 fold change, while the normalized counts is the DESeq2 normalization. Red circles repre-sent significantly upregulated transcripts, blue circles reprerepre-sent significantly downregulated transcripts (p<0.05). B Heatmap showing the expression of the top 20 most significantly changed genes in each direction. Each gene is normalized in expression. Adapted from [Backman et al., 2019].

pig were almost exclusively metabolic in nature. The most enriched gene set was of amino acid metabolism, with the gene sets of carbohydrate metabolism, propanoate metabolism and TCA-cycle following. Several individual pathways of amino acid metabolism were also increased. Non-metabolism connected pathways included folding, sorting and degradation, autophagy, and aging. Insulin signaling and insulin resistance pathways were also signif-icant to a smaller degree. In the gene sets found decreased in the MIDY liver were an abundance of immune response-related gene sets. Infectious Diseases: Parasitic was the most enriched of all gene sets, followed by Hematopoietic Cell Lineage, Development, and Immune Diseases. A focus seemed also to be on Focal Adhesion and ECM interaction.

0 20 40 60

−1 0 1 2

log2 Fold Change (MIDY/WT)

Figure 4.4: Histogram of the distribution of log2fold changes in mRNA between MIDY and WT in the liver showing more upregulated genes than downregulated ones.

Many gene sets here were irrelevant to the liver such as osteoclast differentiation, digestive system, and hypertrophic cardiomyopathy. The Diabetes Type I pathway was also found to be decreased.

4.1.5 Gene network

The alternate approach to the rank-based GSEA is to make an interaction network using only the significant genes. This was done using ClueGO, generating a network where similar trends to the results found in the GSEA approach could be identified (Figure 4.5).

Metabolism pathways were dominant among genes increased in MIDY liver while Immune Response and ECM interaction were featured among genes decreased in the MIDY liver.

Signaling pathways such as PPAR signaling and Sphingolipid signaling pathways served as the link between the two groups of genes.

4.1 RNA Sequencing of MIDY liver by NuGEN Encore complete 41

Figure4.5:ClueGOgenenetworkofthesignificantgenesoftheMIDYliver.Circlesindicategenesetswhilesquaresand diamondsrepresentupanddownregulatedgenesrespectively.Eachlinerepresentsamembershipofageneinaset.Red colorindicatesanincreaseinMIDYwhileblueindicatesadecrease.AdaptedfromBackmanetal.[2019].

Gene set name Size NES p-value FDR

amino acid metabolism 259 4.43 0.00E+00 0.00E+00

carbohydrate metabolism 304 4.01 0.00E+00 0.00E+00

propanoate metabolism 30 3.55 0.00E+00 0.00E+00

citrate cycle (tca cycle) 29 3.45 0.00E+00 0.00E+00

valine, leucine and isoleucine degradation 48 3.28 0.00E+00 0.00E+00 metabolism of cofactors and vitamins 172 3.15 0.00E+00 0.00E+00 folding, sorting and degradation 441 3.09 0.00E+00 0.00E+00

arginine biosynthesis 19 2.78 0.00E+00 2.11E-04

pyruvate metabolism 36 2.50 0.00E+00 1.90E-03

mtor signaling pathway 143 2.48 2.02E-03 1.88E-03

glycine, serine and threonine metabolism 36 2.45 1.98E-03 2.03E-03 protein processing in endoplasmic reticulum 157 2.45 0.00E+00 2.15E-03 cysteine and methionine metabolism 42 2.45 0.00E+00 1.99E-03 metabolism of other amino acids 91 2.43 0.00E+00 2.20E-03

ampk signaling pathway 116 2.37 0.00E+00 3.12E-03

selenocompound metabolism 17 2.34 2.09E-03 3.50E-03

autophagy animal 122 2.34 0.00E+00 3.29E-03

butanoate metabolism 23 2.34 0.00E+00 3.16E-03

autophagy other 31 2.28 0.00E+00 5.25E-03

glycolysis / gluconeogenesis 57 2.27 0.00E+00 5.16E-03 Table 4.5: Upregulated gene sets from the GSEA of the liver tissue in the Munich MIDY Pig biobank.

4.2 Sequencing of MIDY liver by alternate