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



Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This is a resubmission of a manuscript that describes experimental results supporting a role for 
liver-secreted ApoJ in maintaining glucose homeostasis. As the authors correctly state, there is a 
lot of interest in how factors secreted from various cell types participate in homeostasis. In this 
paper, the authors present evidence that ApoJ secreted by the liver is required for efficient control 
of glucose levels. 
 
As a resubmission, the authors have made substantial efforts to respond to the reviewers 
comments. The western blots showing plasma ApoJ levels in mice are in general convincing, and 
the paper is stronger. However, I still have a few comments: 
 
* To my eye, the phenotype of ApoJ knockout mice indicates a beta-cell problem. The mice have 
impaired fasting glucose (although the data shown in Fig. 4B is marginal). My reading of Fig 4M is 
that it confirms higher blood glucose levels, but the impact of insulin is similar (in other words, the 
slope of the decline). The mice have impaired glucose tolerance (4N), and unless I am mistaken 
Fig. 4O indicates a failure of glucose-stimulated insulin secretion (at 15min). Part of the problem 
here may be that ITT is simply not sensitive enough as a tool to measure insulin sensitivity. There 
is also considerable variability in the signaling data shown in 4P (see for example the pIRS1 data, 
with 2 WT showing a strong response and 2 showing a very weak response). The authors do report 
a significant reduction in 2DG uptake in Fig. 6, which shows a modest effect in muscle, adipose 
and BAT of L-ApoJ-/- mice and a more selective impairment in muscle of M-Lrp2-/- animals. The 
phenotype shown in Fig. 6A does not however gel with the ITT data. I would suggest removing the 
ITT data altogether, or doing a clamp study. 
 
* Sure the knockdown of LRP2 in muscle reduces mRNA expression (suppl. fig. 4B - was it 
significant?). However, LRP2 expression in kidney is >200-fold higher. This suggests to me that 
the kidney is a primary site of expression, with low/marginal expression in muscle. This makes it 
hard for me to believe that the ApoJ/LRP2 interaction in muscle is a major driver of ApoJ 
clearance. The dependence on western blots to measure serum ApoJ is also a weakness - this is a 
blunt instrument, and is in my opinion not that reliable when trying to measure 10-20% changes. 
A sensitive EIA is needed. 
 
* The phenotype of Liver ApoJ KO and M-LRP2-/- mice is different. M-LRP2-/- have fasting 
hyperinsulinemia but normal glucose (in other words, insulin resistance). They have evidence of 
blunted insulin response (5G), impaired glucose tolerance (5H) but normal insulin levels during the 
GSIS. As mentioned, to me the phenotype of ApoJ knockout mice indicates a beta-cell problem. I 
can buy that both phenotypes indicate dysregulation of glucose homeostasis and that this is 
potentially a quite important finding. However, the phenotypes are to me very different. This 
should be addressed in the Discussion. 
 
* I don't know what to make of the PCOS data. It is important to support mouse data with clinical 
observations that are consistent. In this case, there is a nice correlation between ApoJ levels and 
indices of insulin sensitivity. This is however dependent on using a blunt instrument to measure 
ApoJ levels - I am assuming this was western blotting as I could not find the method used in the 
methods section on pg. 12. The decline in ApoJ with TZD treatment is also interesting, but difficult 
to interpret. This data is, however, important as it support the model. On the other hand, there 
are commercially available EIA for ApoJ from Thermofisher, RayBiotech, Novus Biologicals and 
probably others that the authors could have used (and perhaps validated using there genetic 
models). 
 
 
 

Reviewer #2: 
Remarks to the Author: 
I was fully satisfied with the additional data and improvements. Thus, I would like to recommend 
the publication of this manuscript.