Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis

PNAS, 2013, Vol. 110, No.40 16109–16114

Yan Wanga, Fabiana Quagliarinib, Viktoria Gusarovac, Jesper Gromadac, David M. Valenzuelac, Jonathan C. Cohend, and Helen H. Hobbsa

Introduction

C

TT

C

T

C

Intestine 1

VLDLCT

T C

CHM

T C

C

CT

VLDL

T

C

LDL

CLDL

CCCE

CE

CE

CE

Capillary

Liver

Tissues

HDL

FA

T : triglycerideC : cholesterolCHM : chylomicronLPL : lipoprotein lipaseVLDL : very low density lipoproteinHL : hepatic lipaseLDL : low density lipoproteinCE : cholesteryl esterss

LPL CT

FA

LPLC

HL

Adpose

A very briefly view of Lipid metabolism

muscle

Adpose

muscle

T

T

T C

CHM

CT

VLDL

C

LDL

Capillary

LPL CT

LPLC

HL

Angptl3Overexpression hypetriglyceridemiaDefection hypotriglyceridemia

Liver

CHMR

Angptl8Overexpression hypetriglyceridemia (Angptl3 exist)Defection ?

Co-expression of Angptl3 & Angptl8 raised a more higher plasma triglyceride level (10 folds)

Shizugawa et al, 2002 ; Gee H et al, 2005 ; Zhang R et al, 2012 ; Quagliarili F wt al, 2012

To stimulate beta cell proliferationTo promote insulin secretion

The functions of Angptl8 may related to tryglceride and glucose homeostasis

Materials, methods, and results

Angptl8 knockout (KO) mice development and characterization

L LF F

WT Angptl8 KO

Fat storage alternation in Angptl8 KO mice

L : lean body massF : fat mass

Adipose tissue accumulation was reduced in Angptl8 KO mice

518 bp

357 bp

Biological parameters have no significant differences between WT and Angptl8 KO mice

Angptl8 KO induced plasma triglyceride (TG) level decline was accompanied with reducing of VLDL-TG content

Enzymatic assay

FPLC

Plasma VLDL-TG content of Angptl8 KO mice was decreased in refeeding state

7 am7 am 7 pm

fast feed

FPLC

kill

Hepatic Angptl8 mRNA level was dramatic increased after refeeding

RNA-seq

FPKM : Fragments Per Kilobase of exon per Million fragments mapped

Plasma TG level decreased in refeeding but not in fasting Angptl8 mice

Glucose homeostasis in Angptl8 KO mice

Plasma level of glucose and insulin were not altered in Angptl8 KO mice

Glucometer ELISA

Fast group

7 pm 7 am 7 pm 7 am

Refed groupOO

XX

XO

Plasma level of glucose and insulin were not altered after glucose administration in Angptl8 KO mice

16 h

Fast for 16 h

0 h

1.5g/kg glucose i.p.

Harvest

Insulin sensitivity and gluconeogenesis ability have no differences between WT and Angptl8 KO mice

Fast7 am

0.75U/kg insulin i.p.

Harvest11 am 7 am

2mg/kg pyruvate i.p.

Harvest

Insulin sensitivity and glucose tolerance was changed in a same pattern when WT and Angptl8

KO mice was fed with a high-fat diet

W11

chow or HFD

W0

Harvest (plasma)

HFD : 60 % fat

Fast(HFD fed mice)

0h 5h

Harvest (plasma)

1.5g/kg glucose i.p.

The reasons of TG decreased in Angptl8 KO mice

VLDL secretion and postheparin LPL activity in Angptl8 KO mice

TG secretion rate decreased in Angptl8 KO mice

0h

Triton WR-1339

Harvest (plasma)

VLDL secretion rate decreased was not due to defection of the machinery required to

assemble and secret VLDL in Angptl8 KO mice

Dysfunction of VLDL secretion is associated with TG accumulation

The translation level of hepatic TG metabolism related genes were not changed in Angptl8 KO mice

Angptl8 KO was associated with LPL activity enhancement(fed already) 0 mins 15 mins

preheparin plasma → heparin, i.v. (1U/g) postheparin plasma

Angptl8 KO mice showed an increasing dietary fat clearance rate compare to WT mice

7 am

200 ml corn oil, gavage

Harvest

AUC : area under the curve

VLDL failed to transport GT to adipose tissue in Angptl8 KO mice

WAT : White adipose tissue

0 mins

15 mins

120 ug [3H] palmitate labeled VLDL, i.v. WAT and heart

N SAngptl3 (cleavage)

Angptl3

Angptl8

Overexpression BOTH

N (extracellular)

Angptl8 is not essential for Angptl3 cleavage

Angptl8 is not essential for Angptl3 cleavage

Discussion

L LF F

WT Angptl8 KO

Fat storage alternation in Angptl8 KO mice

L : lean body massF : fat mass

While Angptl8 was knocked out …

(3) secretion ↓

(2) clearance ↑

(4) storage ↓

(1) activity ↑

Function of Angptl8

• LPL inhibition• Promoting VLDL secretion → TG trafficking → fat storage

Highly oxidative tissues neglected LPLinhibition effect caused by Angptl8 KO which

may contribute to fat accumulation failure

CT

VLDL

FA

LPL C

Adpose

muscle

heart

Angptl8

CT

VLDL

FA

LPL C

Plasma TG : < 50 % compare to WT mice

SREBP1c may be the possible transcription regulator to Angptl8

Oxidative tissue(ex : muscle)

adipose tissue

Excessive carbohydrate

Fat

refeeding

SREBP1c

ChERBP

Ins

Angptl8

Quagliarili F wt al, 2012

Angptl8 and Angptl3 may form a functional complex

Evidences :

(1) Express together [WB]

(2) Angptl8 is necessary for Angptl3 cleaving [Angptl8 overexpression / WB]

(3) The addition effect occurred while co-overexpressing Angptl8 and Angptl3

Angptl8 is not required for beta cell function and development maintenance

Angptl8overexpression

Angptl8Knockout

• Beta cell mass [↑]• Insulin secretion [↑]

• Glucose tolerance [ = ]• Insulin tolerance [ = ]

Supra-concentration of Angptl8 using for beta cell expansionwas expected to cause hypertriglyceridemia 

Thanks for your attention

Con FFA Ins FFA + Ins

Secreted betatrophin   

(2) production ↓

(1) clearance ↑

(4) storage ↓

(5) activity ↑