Temporal dynamics of metabolic dysfunctions in liver, adipose tissue and the gut
during diet-induced NASH development in Ldlr-/-.Leiden miceEveline Gart1,2*, Wim van Duyvenvoorde1, Jessica M. Snabel1, Christa de Ruiter1, Joline Attema1, Martien P.M. Caspers3, Serene Lek4, Bertie Joan van Heuven5, Arjen G.C.L. Speksnijder5, Martin Giera6, Aswin Menke1, Kanita Salic1, Kendra K. Bence7,Gregory J. Tesz7, Jaap Keijer2, Robert Kleemann1, Martine C. Morrison1
1 Department of Metabolic Health Research, The Netherlands Organisation for Applied Scientific Research (TNO). 2 Human and Animal Physiology, Wageningen University. 3 Department of Microbiology and Systems Biology, TNO. 4 Clinnovate Health UK Ltd, Glasgow. 5 Naturalis Biodiversity Center, Leiden. 6 Centerfor Proteomics and Metabolomics, Leiden University Medical Center. 7 Pfizer Worldwide Research, Development & Medical, Internal Medicine Research Unit, Cambridge.

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HDL0481216Fraction number2024168**

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438162838***210chow

HFDLdlr-/-.Leiden mice on a HFD develop (A) obesity, (B)hyperinsulinemia as indicator of insulin resistance, andplasma dyslipidemia (e.g., increased (C) cholesterol and (D)triglycerides, mainly confined to the (E-F) atherogenic apo-B
containing lipoproteins) as observed in NASH patients.

3.4 Increased gut permeability, altered microbiota
composition and gut-derived metabolites precede NASH
and liver fibrosis
A3***** ***2**
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In the gut, HFD significantly increased
permeability (A), induced a shift in microbiota
composition associated with changes incirculating gut-derived metabolites: (B) bile acids
and (C) short-chain fatty acids (SCFA). Temporalgut dysfunctions are summarized in (D).

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5008162838DmWAT inflammation(CLS/1000 adipocytes)

eWAT inflammation(ng/ml)0in plasma (g/ml)

16

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10Total SCFA

HFD

20in plasma (mM)

(Cholesterol mM)

chow

38

In the liver, early metabolic dysfunction manifests as an increase in lipid accumulation (A;steatosis), followed by an increase in (B) inflammation and (C) fibrosis development.
Intrahepatic hits from oxidative stress-induced 4-hydroxynonenal (4HNE) (D) representative
4HNE-stained liver sections with portal veins (PV) and central veins (CV) indicated, (E)quantification of 4HNE staining. Accumulation of specific lipids (F) such as free cholesteroland FFA can promote hepatic inflammation and fibrosis. These hepatic temporal dynamics are
summarized in G.

Total bile acids

2028***(Plasma FITC g/ml)

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Gut permeability(mM in plasma)40

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Lipoprotein profile

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(mg/ml)

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(mM in plasma)

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40

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Triglycerides

60(g)Body weightA

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InsulinGut permeability(Plasma FITC g/ml)

3.1 Diet-induced obesity,
hyperinsulinemia and dyslipidemia
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iWAT weight00*** ***

3iWAT cell size (m2)

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38

34iWAT inflammation

28

54(CLS/1000 adipocytes)

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10mWAT weight8015mWAT cell size (m2)

0****(g)050

20eWAT weight

20*(%Sirius-Red )***40***100+60*

Hepatic collagen*** ***

150

3.3 Visceral white adipose tissue hypertrophy
and inflammation precede NASH and liver
fibrosiseWAT cell size (m2)

The aim of this study was to investigate the sequence of
pathogenic events and organ cross-talk during NAFLD
development. For this high-fat diet (HFD)-fed Ldlr-/-.Leiden
mice were compared with chow-fed controls. At t=0, 8, 16, 28
and 38w mice were euthanized, and liver, WAT depots and gut
were analyzed biochemically, histologically and by lipidomics
and transcriptomics together with circulating factors.

80

C

4-HNE+ immunoreactivity

DB
Inflammatory CLS

2. Study aims and design(% of hepatocytes)

NAFLD progression, from steatosis to inflammation andfibrosis, results from an interplay of intra- and extrahepaticmechanisms. Disease drivers likely include signals from white
adipose tissue (WAT) and gut. However, the temporal
dynamics of disease development remain poorly understood.

AF4/80+ macrophages

3.2 NASH and liver fibrosis development
Total steatosis

1. Introduction

0808162838151050162838

E

HFD feeding increases white adipose tissue (A) weight, (B) cell size and (C)inflammation of different depots (D). More specifically, epididymal WAT (eWAT)
was maximally hypertrophic from t=8w, which coincided with inflammation
development. Mesenteric (mWAT) and subcutaneous WAT (iWAT) hypertrophy
developed slower and did not appear to reach a maximum, with minimal
inflammation. These temporal dynamics are summarized in E.

4. Conclusions
08

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HFD-fed Ldlr-/-.Leiden mice develop obesity, dyslipidemia and insulin
resistance, essentially as observed in obese NAFLD patients, underlining theirtranslational value. We demonstrate that marked epididymal-WAT
inflammation, and gut permeability and dysbiosis precede the development of
NAFLD stressing the importance of a multiple-organ approach in theprevention and treatment of NAFLD.
contact: eveline.gart@tno.nl