Discussion & Outlook

This thesis aimed to elucidate the role of miR92a and p53 as regulators in BAT activity.

Based on previous findings, fasted mice maintained at mild cold stress showed p53 signaling as top upregulated pathway and miRNA-92a-1-5p as most upregulated miRNA in BAT. This, together with a predicted p53 binding site in the mouse miRNA-92a-1-5p locus, led to the hypothesis that p53 directly activates miR92a transcription.

Further bioinformatics predictions revealed a potential miR92a binding site on the 3’UTR of the mRNA of fructose transporter Slc2a5. Because downregulation of the fructose transporter would result in a decreased amount of intracellular fructose, fructose was hypothesized to be a potential energy source in BAT.

Since the organism needs to conserve energy during nutrient deprivation, fructose reduction could be an important factor in reducing/ switching off NST in BAT, which would be consistent with our hypothesis. Therefore, a ChIP-qPCR assay was performed in order to elucidate a possible interaction between p53 and the miR92a locus. Subsequently, the putative interaction of miR92a with Slc2a5 was investigated using a luciferase assay.

The herein conducted ChIP experiments are insufficient to reliably confirm that p53 directly binds to miR92a locus, since only one of several negative controls used (shown throughout this thesis) for ChIP shows low enrichment for the most part compared to positive control targets, pointing to problems of unspecific binding of the antibodies in this method. Other negative controls used, strongly scatter throughout pulldown experiments and often exceed miRNA- target and positive control loci indicative of unspecific binding. However, using the setups established through this thesis, significant enrichment of the miR92a target locus as well as the positive control locus over one negative control locus could be shown.

However, luciferase assay results suggest an interaction between miR92a and Slc2a5, indicating downregulation of Slc2a5 via miR92a in vivo. Since NMR measurements indicate fructose uptake while ECAR measurements suggests fructose metabolism in brown adipocytes, both results also support the hypothesis that fructose is an energy source in mature brown adipocytes.

4.1. Regulation of miRNA92a by p53: Troubleshooting the ChIP protocol

Since data of our group previously showed increased p53 signaling upon fasting, one of the first adjustments done for pulldown was cultivating iBACs 24 hours with

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starvation medium. It was expected that increased ChIP enrichment of p53 targets would be observed when compared to cells maintained in full medium. However, most experiments done with starvation medium in this thesis showed lower enrichment compared to control.

One explanation might be a different efficiency of the crosslinking process when done in starvation medium compared to maintenance medium, resulting in weak binding of proteins on DNA.

Subsequently, we chose treatment with idasanutlin, a known p53 stabilizer, as alternative to increase p53 on protein level. However, results turned out difficult to reproduce as well. No increased enrichment or reduction of unspecific binding could be observed. A possible explanation could be an oscillatory effect in the p53 response, as shown for many other protein responses (Nelson et al., 2004). Therefore, p53 response might already subside when cells are harvested.

Furthermore, results show different levels of “%Input” enrichment throughout experiments with some experiments showing unexpectedly high levels of enrichment (not shown). Lack of reproducibility and high levels of enrichment might hint to problems with the ChIP procedure.

Since p53’s recognition site of D2H9O antibody is C-terminal, the occurring problems with unspecific binding could also be the result of insufficient antibody-epitope recognition. For instance, the c-terminal epitope might be obstructed due to conformational changes. Thus, testing other p53 antibodies (preferably full-length antibodies) might prove more successful.

Overall, further adjustments to the ChIP protocol might lead to more robust and conclusive results.

4.2. miR92a interaction with Slc2a5

Co-transfection of the 3’UTR of Slc2a5 incorporated in PsiCHECK2, along with miR92a in iBACs shows no significant reduction of relative luciferase unit (RLU) when overexpressed with miR92a. Since the successful electroporation of miR92a was confirmed by overexpression experiments (see Figure 15), we investigated ineffective transfection efficiency by transfecting a GFP expressing construct. As expected, the low transfection efficiency along with the likely unphysiologically high amount of

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transfected DNA was supported by transfecting and checking EGFP expression in iBACs (see Figure 16).

Since, lipid-cationic transfection was shown to be ineffective in adipocytes (Enlund et al., 2014), the cell model was changed to HEK 293 cells. Lipid cationic transfection efficiency proved to be relatively more successful than electroporation in iBACs, resulting in reproducible results. Subsequent luciferase assay showed reduced RLU compared to control indicating degradation of Slc2a5 when miRNA mimic 92a was overexpressed.

4.3. Evaluation of fructose uptake in brown adipocytes

This regulatory mechanism of fructose transporter by miRNA92a depending on environmental cues (nutrients, mild cold stress) suggest fructose might be used as energy source in brown adipocytes, which could be a regulatory node determining BAT activity. NMR measurements of intracellular metabolites in iBACs further corroborates this theory and suggests fructose uptake when iBACs are incubated for 24 hours with 5g/L fructose (Figure 19).

4.4. Fructose as a substrate in mature brown adipocytes

ECAR level shows a slight increase after fructose injection. This may support previous NMR results showing uptake in fructose subsequently hinting to fructose being used as energy source for ATP/NST production. This would be in line with Jang et al., 2018 data, suggesting fructose uptake in multiple other organs besides the liver (Jang et al., 2018). Additionally, fructose was shown to be mainly metabolized by the small intestine and only low amounts of fructose spill over into the bloodstream. However, low uptake of fructose in BAT was observed in mice administered dietary fructose intravenously (Jang et al., 2018).

Nevertheless, further individual ECAR measurements are needed in our experiment to confirm a consistent increase in ECAR upon acute fructose injection.

38 4.5. Conclusion & Outlook

Since increased thermogenic activity in adipocytes correlates with many health benefits like anti-obesity effects, studies in thermo-actived adipocytes might lead to novel therapies targeting the metabolic syndrome. This study contributes to the elucidation of the p53-miRNA92a-Slc2a5 axis in BAT regulation upon mild cold stress while simultaneously nutrient deprived.

Taken together, while providing a first indication of p53 enrichment in the miRNA92 locus, further adjustments in ChIP methodology needs to be performed to corroborate the interaction between p53 and miR92a locus. To address the major issues that occurred during ChIP, switching to full-length antibody might turn out to be beneficial in increasing p53 enrichment in the miRNA92a locus above background. While the role of p53 in this context is still not clear, a possible indirect link between p53 and miR92a should also be mentioned. Most of the suppressive transcriptional effects of p53 are proposed to be by indirect effects through regulation of other transcription factors and coregulators (as reviewed in (Fischer et al., 2014)). Therefore, regulation of miR92a through the expression of a p53 target gene might be a possibility.

Luciferase assays performed in this thesis hint to a possible interaction between miRNA92a and the 3’UTR of Slc2a5 mRNA. This suggests that fructose is a source of energy in BAT, whereas intracellular uptake is prevented by expression of miR92a in response to specific signals, such as fasting/starvation. This is supported by our findings showing that intracellular fructose is principally taken up in iBACs, corroborating (Jang et al., 2018) findings in BAT.

Altogether, the presented data provide first evidence of an intact p53-miRNA92a-Slc2a5 axis in the energy regulation of fasted brown adipocytes.

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