Peer review of: Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin (2020)

 

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The authors of this article claim that ApoJ is a novel “hepatokine” that regulates muscle glucose 
metabolism via effects on insulin action. The physiological evidence for this claim is based on 
studies using a liver-specific ApoJ knockout, and on studies using an muscle-specific LRP2 
knockout. Evidence for the claim that LRP2 is necessary for IR endocytosis comes from studies 
using C2C12 cells. 
 
This is an interesting and novel theory of function for ApoJ that, if true, would certainly have a 
high impact on the field. Liver-specific ApoJ knockouts are shown to have a metabolic phenotype 
of impaired glucose tolerance. To my eye, the insulin sensitivity data shown in Fig. 2D are not 
convincing in suggesting impaired glucose clearance following insulin injection. The authors do 
report differences in the activity of the IR tyrosine kinase and subsequent signaling cascade(s). 
Muscle-specific LRP2 (and liver-specific ApoJ) knockout mice also exhibit evidence of impaired 
insulin action in muscle with respect to glucose uptake (Fig. 3A, B). The authors also claim that 
LRP2 works by actions involving IR endocytosis; the model reported is that ApoJ facilitates 
interaction of LRP2 with the IR, and that this step is required for IR endocytosis. 
 
Is the work convincing, and if not, what further evidence would be required to strengthen the 
conclusions? 
While I am impressed with the data presented, in my opinion the experimental evidence falls short 
of proving the mechanism proposed to explain the observations. I also feel that much has been 
left out of the description of the function of ApoJ in the 2nd paragraph of the Introduction. The 
authors cite a series of studies that support their claim, but don’t discuss other findings that could 
raise red flags for the reviewer. ApoJ is a chaperone that aids the folding of secreted proteins. It 
has three isoforms, which have been differentially implicated in pro- or antiapoptotic processes. 
 
1) ApoJ is a lipoprotein - its role as an apoliprotein has not been adequately addressed. The 
associations between serum ApoJ and insulin resistance in mice and humans could also reflect 
differences in hepatic VLDL output. 
2) ApoJ/Clusterin is also a chaperone involved in protein folding - how has loss of ApoJ affected 
hepatic function? Is VLDL production normal? Is the secretion of other secreted proteins (e.g., 
IGF1, IGFBP etc.) normal? Are the livers “healthy” or steatotic? Does secretion of ApoJ correlate 
with increased VLDL production in obesity? 
3) The claim that IR endocytosis is involved in insulin signaling is not novel (Mol Cell Biol. 1998 
Jul; 18(7): 3862–3870). However, the authors present very little data supporting the concept that 
LRP2 interacts with the IR. I am not actually sure what Fig.3C and D are telling us, as I suspect all 
it shows is less insulin binding in skeletal muscle. More evidence for a direct interaction of the IR 
with LRP2 is needed, and more data on how the lack of LRP2 affect IR internalization is also 
required. 
4) Similarly, there is scant evidence for the claim that muscle-LRP2 knockout mice are “ApoJ 
resistant”. If ApoJ is part of the lipoprotein complex, and its interaction with LRP2 is needed for 
uptake of VLDL in muscle, then one could contemplate reduced clearance. There is also a need for 
some kind of feedback mechanism between muscle and the liver to explain how the liver is sensing 
ApoJ action to compensate. For insulin, failure to adequately increase glucose disposal leads to 
higher glucose, which then leads to more glucose-stimulated insulin secretion. I am neither 
convinced nor impressed by this paragraph. Also, the statement on how crossing muscle-LRP2 and 

liver-ApoJ knockout mice will restore “APoJ sensitivity” is pointless, and invites a reviewer to 
request that specific experiment be done. I am not, as I am not convinced it will address the 
underlying principle. 
5) Treatment of PCOS patients with a glitazone could indirectly affect APoJ by effects on hepatic 
lipoprotein metabolism. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In this manuscript entitled “ApoJ is a novel hepatokine regulating muscle glucose metabolism and 
insulin sensitivity,” the authors investigated the role of apolipoprotein J (ApoJ) on insulin 
resistance and glucose intolerance in a low-density lipoprotein receptor-related protein-2 (LRP2)-
dependent manner. The authors stated the importance of the ApoJ and LRP2-insulin (IR) axis. This 
study includes interesting data; however, there are numerous inadequate description and over-
interpretation, particularly the relation to LRP2 is suspicious. In this manuscript, the authors 
showed neither the protein levels of LRP2 nor the direct bound between ApoJ and LRP2. It is also 
unclear that LRP2 is expressed in skeletal muscle. The authors should show the role of LRP2 in 
vitro experiments using C2C12 myocytes. Previous reports showed that the expression levels of 
LRP2 in skeletal muscle are quite low. Therefore this reviewer felt the possibility that the 
statements include over-interpretation. Other comments are as follows: 
 
Major comments: 
1. The abbreviation “ApoJ” is not suitable in the title. In addition, I could not find this description in 
the abstract. 
2. In the introduction, the description of other hepatokine is lacked. Selenoprotein P is known as a 
hepatokine, and one of its receptor is LRP2. The authors should mention about this hepatokine. 
I'm wondering that the effects of ApoJ are dependent or independent of selenoprotein P. 
3. In the results section, the authors stated “circulating ApoJ is retained in muscle via LRP2 and 
that LRP2 signaling is essential for maintenance of ApoJ homeostasis in circulation.” It is unclear 
whether KO of LRP2 is specific in skeletal muscle. The authors should demonstrate this in the LRP2 
protein levels. In addition, the authors should show the co-localization of ApoJ with LRP2 by using 
immunohistochemical method. It is also important to prove this statement by using in vitro 
experiments. 
4. Western blot analysis of ApoJ is plausible because the molecular weight of several blots is not 
constant. 

Overview of revision: We would like to thank the reviewers for their constructive comments, which have 
contributed to a more mechanistic study of the manuscript.  We addressed most of comments by performing 
additional experiments.  New data are below; 
 
Fig. 4d: Serum IGF1 level  
Fig. 4e: Serum IGFBP-1 level  
Fig. 4f: Serum IGFBP-3 level  
Fig. 4g: Serum TG level      
Fig. 4h: Serum cholesterol level 
Fig. 4i: Hepatic TG level 
Fig. 4j: Hepatic cholesterol level 
Fig. 4k: Hepatic H&E staining 
Fig. 4l: Hepatic VLDL production 
Fig. 5i: Glucose-induced insulin secretion 
Fig. 7a-b: Insulin-stimulated IR internalization in C2C12 cells 
Fig. 7c: Akt phosphorylation in C2C12 cells 
Fig. 7d: Glucose uptake in C2C12 cells 
Fig. 8e: Correlations between ApoJ and HDL, LDL, FFA, or adiponectin in humans  
Table 1: Clinical and metabolic characteristics of normal cycle and PCOS women 
 
Sup Fig. 2f: Selenoprotein P level in serum  
Sup Fig. 2g: Selenoprotein P level in the liver 
Sup Fig. 2h: Hepatic gene expression involved in glucose metabolism 
Sup Fig. 2i: Muscle LRP2 mRNA level 
Sup Fig. 4b: LRP2 gene expression in multiple organs 
Sup Fig. 4d: LRP2 gene expression in C2C12 cells transfected with siLRP2 
Sup Fig. 4e: LRP2 expression in muscle 
Sup Fig. 4f: Co-localization of ApoJ and LRP2 in muscle 
Sup Fig. 4g: Co-localization of ApoJ and LRP2 in in C2C12 cells 
Sup Fig. 5: Correlations between ApoJ and age, BMI, or TG in humans