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# Monitoring of *de novo* lipogenesis *in vivo* via palmitic acid labelling with deuterium

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**Océane DELOS<sup>a,b</sup>, Fadila RAYAH<sup>d</sup>, Jean-Philippe PRADERE<sup>c</sup>, Jean-Charles PORTAIS<sup>a,c</sup>, Catherine POSTIC<sup>d</sup>, Pierre-Damien DENECHAUD<sup>b</sup>, Justine BERTRAND-MICHEL<sup>a,b</sup>**

## Context

* *De novo* lipogenesis (DNL) = *de novo* synthesis of fatty acids, can be altered in a large number of human diseases such as obesity, diabetes but also cancer.

* Quantification of DNL = understand and characterize the mechanisms involved in these pathologies.

* Several non-invasive methods using deuterated water (D₂O) to determine lipogenic flux: bolus of D₂O, prolonged administration.

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## Analytical workflow

Fatty acids analysis

* Bligh and Dyer method<fnref n="2" />

* KOH hydrolysis

* Internal standard: C19:0

* Derivatization with PFB-Br

* HP-5MS 30 m * 0,25 mm, 0,25µm

* Negative ionisation with methane

* Mode : full scan

Data processing

IsoCor<fnref n="3" />

Isotopologue distribution
Mean enrichment

corrects raw MS data for the
contribution of all naturally
abundant isotopes

Measurment
of D-labelling
body water<fnref n="4" />

Exchange H/D acetone

GC-EI-MS analysis

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5 µL of standard
Or 5 µL of plasma
+ 4 µL NaOH 10 N
+ 2 µL acetone
4h at RT

* HP-5MS 30 m * 0,25 mm, 0,25µm

* m/z 58 and 59

Determination of D-
labelling body water thanks
to calibration curve

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<figure><img src="image_6.png" /><figcaption>GC-MS EVO 8000 Thermo</figcaption></figure>

## Confirmation of deuterium incorporation

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Mean enrichment in deuterium of
C16:0

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Total fatty acids were extracted from liver,
brown adipose tissue (BAT), white adipose
tissue (WAT) and plasma.

* Successful incorporation of deuterium with mean enrichment from 0,9 to 1,4% in analyzed tissues

* Labelling detected up to M+6 isotopologue (i.e. palmitic acid with 6 atoms of deuterium)

Bolus +
3 days

3 weeks

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Study of mice under D₂O
during 3 days with bolus and
during 3 weeks

Determination of D-labelling body

water (%):

<table><thead><tr><th>Bolus + 3 days</th><th>3 weeks</th></tr></thead><tbody><tr><td>Mean ± sem</td><td>2,5 ± 0,1</td><td>3,0 ± 0,2</td></tr></tbody></table>

C16:0 enrichment evolution
in liver

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* Monitoring of deuterium incorporation shows a progressive increase in labelling over the weeks

* Balance not reached on palmitate but practically same level of D-labelling body water

## Application to mice with a lipogenesis defect

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Chrebp = gene involved in the expression of lipogenesis

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Study of wild type mice (WT) vs
total ChREBP KO mice, for 3
weeks under D₂O

Comparison of relative abundance of natural C16:0 (abundance/mg of tissue) in different tissues (a,c,e,g) and of isotopologue fractions (b,d,f,h) in WT and KO groups:

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(a)

Isotopologue fraction of C16:0

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(c)

Isotopologue fraction of C16:0

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(e)

Isotopologue fraction of C16:0

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(g)

Isotopologue fraction of C16:0

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* Decrease in the relative abundance of C16:0 in ChREBP KO mice: confirmation the involvement of ChREBP gene in expression of lipogenesis

* Study of isotopologue fraction shows the same incorporation amount of deuterium in WT and KO, even with a different amount of C16:0 synthesized

## Application to a physiopathological model of obesity

Study of mice fed with chow diet (CD) vs
**high fat diet** (HFD), study during 3 weeks
under 4% D2O

Obesity conditions

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* Comparison of relative abundance of natural C16:0 in liver (a) and deuterium enrichment in CD and HFD groups (b):

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C16:0 M0

(b)

Enrichment

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* Determination of newly made palmitate relative abundance<fnref n="5" />:

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* In high fat diet conditions, relative abundance of C16:0 is increased as expected

* Results confirm by the determination of newly made palmitate: more palmitate synthesized in HFD mice

## Conclusion and perspectives

* Implementation of a workflow that allows the incorporation of deuterium during the synthesis of palmitate

* First results show an **mean enrichment around 1% under 3 weeks in various tissues** and mice models

* To **deepen the model**, it will be interesting to study the mean enrichment during a bolus of D₂O to see on the **very short** term conditions, as well as on 6 weeks, to determine the time necessary to reach the **equilibrium**.

## References

<footnote><sup>a</sup>MetaboHUB-MetaToul National Infrastructure of Metabolomics and Fluxomics, Toulouse, France, <sup>b</sup> I2MC, Université de Toulouse, Inserm, Université Toulouse III - Paul Sabatier, Toulouse, France, <sup>c</sup> Institut RESTORE, Inserm, CNRS, Université de Toulouse, Toulouse, France, <sup>d</sup>Institut Cochin, Université de Paris, CNRS, Inserm, Paris, France</footnote>

<footnote n="1">¹ Pathway adapted from Fu, X., Deja, S., Fletcher, J.A. *et al*. Measurement of lipogenic flux by deuterium resolved mass spectrometry. *Nat Commun* **12**, 3756 (2021)</footnote>

<footnote n="2">² Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959</footnote>

<footnote n="3">³ Protocol adapted from Daurio et al. Spatial and temporal studies of lipid metabolic activity: contrasting biochemical kinetics in tissues and pathways during fasted and fed states. Am J Physiol Endocrinol Metab. 2019</footnote>

<footnote n="4">⁴ Millard et al, IsoCor: Correcting MS data in isotope labelling experiments, Bioinformatics, 2012</footnote>

<footnote n="5">⁵ Lee WN, et al. Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis. Am J Physiol. 1994</footnote>