-1,61*

3,72*

2,29*

2,06*

1,83*

-2,0 -1,0 0,0 1,0 2,0 3,0 4,0

Insulin induces de novo lipogenesis in human skin stem cell-derived hepatic cells

Introduction Methods

Results

Conclusion

Joost Boeckmans, Alessandra Natale, Karolien Buyl, Joery De Kock, Vera Rogiers, Robim M Rodrigues* and Tamara Vanhaecke*

Department of In Vitro Toxicology & Dermato-Cosmetology (IVTD). Vrije Universiteit Brussel, Faculty of Medicine and Pharmacy, Laarbeeklaan 103, 1090 Brussels, Belgium. *Equally contributing senior authors.

Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis tohepatocellular carcinoma. Among 25% of the global population suffers fromNAFLD. A strong correlation with the metabolic syndrome (insulin resistance,obesity, dyslipidemia...) is observed. Due to interspecies differences andethical concerns, the use of laboratory animals to investigate this disorder isdiscouraged. Therefore, human-relevant in vitro NAFLD models are urgentlyneeded by the pharmaceutical industry. Postnatal human skin precursor cells,differentiated towards hepatic cells (hSKP-HPC), proved already their potentialto mimic in vivo toxicological responses to steatosis-inducing drugs. Here, weinvestigate whether insulin is able to induce triglyceride accumulation inhSKP-HPCs and mimic hepatic steatosis as observed in subjects suffering fromthe metabolic syndrome. The mechanisms by which insulin influencesintracellular triglyceride accumulation are summarized in Figure 1.

hSKP-HPCs were generated according to anearlier established in-house protocol [1].Subsequently, the cells were exposed to100 nM insulin for 24h. The expression oflipogenic (ACC1, FASN, PPAR-γ, SCD1 andSREBP-1c) and lipid metabolism-related genes(APOB, CD36, ACADSB, CPT1a, PPAR-α,DGAT1, DGAT2 and GPAT1) was evaluated byRT-qPCR. Additionally, 72h exposure wascarried out to assess intracellular lipidaccumulation. Hereto, the cells were stainedwith LipidTOX green for neutral lipids andexamined by fluorescence microscopy.

CPT1aMalonyl-CoA

Acetyl-CoA

Palmitic Acid

Palmitoeic acid

Glycerol-3-P GPAT 1-4AGPAT 1

LPINDGAT1DGAT2

LXRα

ChREBP

Acetyl-CoA

KREBS CYCLE

FFA in blood stream

SREBP-1c

INSULIN

FFAACC1

Stearic Acid

Pyruvate

GLYCOLYSIS

FAS

SCD1

FAT (CD36)

Oleic AcidSCD1

VLDL in blood stream

APOB

SCD1

FASN

ACC1

DGAT2

APOB

Control

100 nM insulin

Increased intracellular lipid load was confirmed by fluorescence microscopy (Fig. 2). Upregulation of three de novo lipogenic genes (ACC1, FASN and SCD1), upregulation of

DGAT2 which is responsible for the final esterification step and decreased triglyceride export by VLDL (very low-density lipoprotein) due to downregulated APOB (Fig. 3).

Other tested genes (PPAR-γ, SREBP-1c, CD36, ACADSB, CPT1a, PPAR-α, DGAT1 andGPAT1) were not significantly modulated.

* Student t-test; p < 0.05

Contact: Joost Boeckmans E: Joost.Boeckmans@vub.ac.be T: 02/477.45.20 Reference: [1] R. M. Rodrigues et al., “In vitro assessment of drug-induced liver steatosis based on human dermal Sponsors:

stem cell-derived hepatic cells.” Arch. Toxicol., vol. 90, no. 3, pp. 677–689, 2016.

Insulin induces intracellular triglyceride accumulation in hSKP-HPCs by increasing

de novo lipogenesis, increasing triglyceride esterification and decreasing triglyceride export

Therefore, hSKP-HPCs may serve as a promising human-relevant

in vitro model to study hepatic lipid-metabolism related disorders

72h

ACADSB

Figure 1: Influence of insulin on intracellular hepatic triglyceride accumulation

Figure 2: hSKP-HPCs stained for neutral lipids

Figure 3: Gene expression modulation (fold change vs. control; n = 3)