Peer review of: The kallikrein–kinin pathway as a mechanism for auto-control of brown adipose tissue (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This study addresses the effects of the kininogen/bradykinin signalling system on brown adipocyte 
functions. The authors show that expression of the Kng2 gene correlates with the level of 
thermogenic stimulation. Surprisingly, inhibition of the signalling pathway by antagonists or 
receptor gene inactivation markedly enhances the thermogenic response, suggesting that release 
of BKNs from BAT may possibly be an auto-regulatory loop. The data presented are of excellent 
quality and the analyses conducted up to this stage are expert and require very little to improve. 
This is therefore a potentially interesting study with interest to a broad readership and a good level 
of novelty as it identified a new pathway to regulate thermogenesis. However, the mechanistic 
evidence provided is to some degree a weakness of the manuscript that should be addressed more 
thoroughly. Specifically, the authors could consider addressing the following items: 
 
a. As far as I can tell, there is no direct experimental link between Bradykinin or KNG2 and B1/B2 
inactivation. This would be of particular interest since the observations from gain- and loss-of-
function models seem to oppose each other, or else suggest a negative feedback. It would be 
important to demonstrate that B1B2R-KO mice or isolated cells from such animals, as shown in 
Figure 9, no longer respond to BK- or HWMK-treatment. 
 
b. In relation to the previous comment: What is the mechanism of activation by BKN2 through 
B1/B2? The authors discuss the possibility of Gq activation, which could be easily addressed 
experimentally. At the very least the authors should provide western blot analysis of the most 
common thermogenesis regulations pathways, such as beta-adrenergic signalling, insulin 
signalling, and lipid metabolism in some of the in vitro or in vivo models. 
 
c. It seems initially counterintuitive that a pro-thermogenic stimulus like cold would also increase 
expression of Batokines that are negative regulators of thermogenesis. On which level does Bkn2 
act to inhibit thermogenesis or do brown (but not white) adipocytes become kininogen/bradykinin-
insensitive during thermogenic activation? This would also help to shed light onto the apparent 
discrepancies the authors discuss when comparing BAT/WAT and in vitro/in vivo. 
 
d. The negative effects of the natural ligand, BK, have only been addressed on the level of UCP1 
expression. In order to determine whether kininogen/bradykinin do impair thermogenesis cell-
autonomously, functional assays, such as lipolysis rates and mitochondrial function should be 
analysed. Moreover, it would be helpful to determine whether there is an interaction with the 
adrenergic system, which some data suggest. To this end, data shown in Figure 9A and 9D should 
also be measured in cells exposed to cAMP or the adrenergic agonist. 
 
e. It is not clear to me what is shown in Figure 5B. Is this UCP1 Protein or all total protein in BAT? 
Please indicate in figure legend. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The work from Marion Peirou et al entitled “The kininogen/bradykinin pathway, a mechanism for 
autocontrol of brown adipose tissue activity” brings a new message regarding the role of the 
kallikrein-kinin system (KKS) as a relevant component of brown adipose tissue (BAT) thermogenic 
regulation. The authors have convincingly demonstrated that the KKS might be regarded as a 
novel, previously unsuspected pathway for controlling BAT thermogenic regulation, acting as an 
autocrine, autoregulatory mechanism in response to a thermogenic stimulus. As pointed out by the 
authors, this finding may contribute to expanding the range of potential pharmacological 
candidates in therapeutic strategies against obesity and associated diseases designed to improve 

energy expenditure. This is a novel, original finding, which will be of interest to the wide 
community of researchers and clinicians. 
The work is well done but this reviewer feels that some flaws are evident in the manuscript and 
should be pointed out, as follows: 
1) Introduction: 
- Please refer to the system as kallikrein-kinin and not as kininogen-bradykinin system. 
- Please also use the correct nomenclature of the receptors as kinin B1 or kinin B2 receptors. 
Bradykinin only binds to the kinin B2 receptor and therefore the receptor should be named kinin 
B2 (or bradykinin B2 receptor). However the B1 receptor binds the des-Arg agonists [des-Arg9]-
bradykinin/[des-Arg10]-kallidin and not bradykinin. Therefore it should be named kinin B1 
receptor. 
- The agonists [des-Arg9]-bradykinin/[des-Arg10]-kallidin are not the final peptides from the kinin 
pathway, as described in the text. 
 
2) Results: 
- Sometimes the authors normalize protein expression in Western blots with Ponceau, sometimes 
with Coomassie or beta-actin. Why these different normalizers? 
- Why there are two different bands for KNG2 in WBs? Sometimes the upper band is the stronger 
one and sometimes the lower band. Why does this happen? Are these antibodies reliable? 
- How the authors explain the positive response to thermogenic stimulus at the RNA level resulting 
from the alanine to threonine mutation at residue 163 in the KNG2 gene in the Brown Norway 
Katholiek rats? 
- Regarding the down/up regulation in the kinin receptors depending on the temperature, the 
authors should show the effect also at the protein level. The authors did not provide a rationale for 
this effect. Is the system activated in this condition? Is there an increased production of kinins in 
these conditions? Is the activity of the other components of the system (kallikrein, kininases) 
modulated? 
- This reviewer wonders why the authors decide to study the double knockout mice instead of the 
individual B1 and B2 knockout mice. The same question is valid for the use of antagonists: why 
using both instead of blocking separately each receptor? Since both receptors have different 
pharmacology it is important to pinpoint the role of each one in the thermogenic regulation in BAT. 
- Morais et al (reference 17) show that the double B1B2R KO mice present normal glucose levels 
but lower levels of the hormones leptin and insulin at normal conditions, which is in agreement 
with the data published for the single kinin receptor knockouts. As long as there are two different 
strains of double KO gererated at different laboratories, how do the authors explain this 
difference? How does this data impact the findings presented in this manuscript? This discrepancy 
should be discussed in the present manuscript. 
- The authors should provide details of protocol used for the Real-Tine PCR for the kinin receptors. 
The primers and probes supplied by the manufacturer map within a single exon and should also 
detect genomic DNA and could interfere with the data presented. This assumption should be valid 
for the other assays used for the other genes. Therefore the protein expression/functional data 
should be always performed. 
- 
 
 
 
Reviewer #3: 
Remarks to the Author: 
The paper by Peyrou describes the analysis of the Kinoinogen/Bradykinin system in the regulation 
of brown fat activity. Through the use of several models, both in vitro and in vivo they show that 
BAT expresses HMWK and LMWK, furthermore they demonstrate that especially in BAT signaling 
through B1 and B2 receptor modulates the activity of this tissue. This is an interesting finding 
which put another regulator of BAT on the map which seems to act independent of the classical NE 
mediated pathways. However there are several points that need to be addressed: 
1. Fig. 1. Please show the absolute levels of HMWK and LMWK and not just inductions this is 

especially important in relation to the question where circulating HMWK is coming from (see 
below). Furthermore, a measurement of Kng2 and 1 in tissues should be performed to elucidate 
whether the induction is happening on a protein level. Again, this is required to draw any 
conclusion about the origin of the circulating factors. For Fig. 1D please provide the individual data 
points. 
2. Fig. 2. How do the authors explain the differences in the LMWK changes seen in vivo and in 
vitro. Please also provide a quantification of the blots 2B and 2C (similar to 5C). 
3. Fig. 3. Please show 3E at full scale. 
4. I am extremely puzzled by the data in Fig. 6. At 4C Ucp1 is slightly increased in B1/B2 ko mice 
and the effect is much more pronounced at TN. Why then do the authors not observe an effect 
under mild cold stress ? 
5. Fig. 9. A much more careful analysis of B1/B2 cell autonomous differentiation is required. In my 
opinion the two images show a difference and PPARg levels (even though it is difficult to see 
seems to by upregulated by at least 50%. 
6. The cartoon presented in Fig. 9 is in my opinion an overinterpretation. From the current data it 
is difficult to determine whether the HMWK is indeed acting in a cell autonomous manner, 
especially in vivo. To show this the authors would need to do ko experiments in cultured brown 
adipocytes coupled with media transfer to demonstrate that indeed HMWK acts in an autocrine or 
paracrine manner. 
7. The finding of differential effects on WAT vs BAT browning is very intriguing especially since the 
regulation of LMWK seems to be conserved between the two tissues. Could this be due to different 
modes of induction (i.e de novo recruitment of beige cells vs. activation of existing cells ?). This 
should be discussed in more detail.