A Leptin Receptor Antagonist Attenuates Adipose Tissue Browning and Muscle Wasting in Infantile Nephropathic Cystinosis-Associated Cachexia

Round 1

Reviewer 1 Report

This paper studies the effect of a leptin antagonist in a mouse with cystinosis, using this as a model of cachexia. PLA treatment caused a 50% increase in food intake with a very small increase in fat and lean and muscle mass. Treatment caused a reduction in Ucp1 protein and reduced indices of beiging.

This paper is an incremental advance, showing the benefits of PLA in cystinosis cachexia, for example compared to ref 18 (2014) from the same lab, which showed the benefits of PLA in a partial nephrectomy mouse model of cachexia.

1. It is hard to understand from the ms what the mechanistic implications are. How does loss of Ctns cause beiging? Is it due to loss of Ctns from adipocytes? Or is it due to loss of Ctns from other cells, such as other cells in adipose tissue that signal to adjacent adipocytes, or from neurons that drive the sympathetic tone to the WAT and BAT? Or is it more indirect, due to increased inflammatory tone from crystal injury elsewhere in the mouse? Or is it due to the modest renal insufficiency (and if so, by what mechanism)?
2. how is the PLA working—is it only central or are there direct peripheral actions here?
3. The pair feeding experiment is awesome. However, Fig 1 data are uninterpretable without knowing the actual body weights—please show them (show the growth curves and effect of PLA with time). Also, please express the food intake and the metabolic rate in units of kcal/mouse/d. It is very difficult to compare metabolic rates of mice with different body weights/compositions. Adjustment using division by body weight (as done in the ms) is wrong (see DOI: 10.1038/nmeth.1806). For example, fig 1F would appear to show that leptin antagonist decreases metabolic rate, which, it true needs to be investigated mechanistically. Please consult with someone who understands this point.

4. The ms uses multiple 2 sample t-Tests with no multiplicity correction in situations where 2-way ANOVA with post hoc tests is a better statistical approach.

5. Fig 2,4,6 are missing error bars; presentation of the actual points in these figs, as in fig 1,3 would greatly improve the figure. Statistics seem to be missing from most of the figures.

6. The mouse model used is the null for cystinosin. Yet the ms does not mention that cystinosin is the lysosomal membrane protein responsible for the transport of cystine out of the lysosome.

Points in the Discussion:

7. The statement “The basal metabolic rate is responsible for up to 80% of the daily caloric expenditure by an individual [42].” Is true for humans. But not for mice. At room temperature BMR is about 1/3 of daily caloric expenditure http://www.ncbi.nlm.nih.gov/pubmed/26042200 The ms would be improved by acknowledging that for brown/beige adipose physiology the mouse may not be predictive of humans.

8. RE “Leptin increases energy expenditure through activation of BAT thermogenesis [43].” This is misleading; it is true for the case where leptin is being added back to the leptin deficient state. But it is not clear when leptin levels are already adequate.

9. RE “Adipose tissue UCP1 is essential for adaptive adrenergic non-shivering thermogenesis” is not true. Ucp1 is clearly not essential. Under some conditions, Ucp1 null mice handle much of thermal biology normally; https://doi.org/10.1074/jbc.M100466200

10. RE “muscle tissue UCP3 regulates body metabolism [44].” Not true; there is no metabolic phenotype in Ucp3 null mice https://www.ncbi.nlm.nih.gov/pubmed/10748196 http://www.ncbi.nlm.nih.gov/pubmed/10748195

11. define Ctns-/- mice in the abstract

12. mg.kg.day should be mg/kg/day throughout the paper

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

In the article ‘Leptin receptor antagonist attenuates adipose tissue browning and muscle wasting in infantile nephropathic cystinosis-associated cachexia’ by Alex Gonzalez et al., the authors test the efficacy of pegylated leptin receptor antagonist in a mouse model of

 infantile nephropathic cystinosis. The disease constitutes a severe clinical issue for the affected and their families, and poor appetite with growth failure belong to its major symptoms. Infantile nephropathic cystinosis is rare, as the yearly incidence is around one in 200,000 live births. This is a potential limitation to the study as it is going to be interested only for a limited number of readers. However, obviously rare diseases also deserve scientific attention. The authors are experts in the field of leptin associated cachexia. The study is well designed and performed, the aims are clear, methodology seems appropriate, results convincing and conclusions justified. Hence, I support publishing the manuscript in Cells.

Author Response

We appreciate your positive comments.

Round 2

Reviewer 1 Report

The ms is improved, but the presentation and analysis issues in point 3 of the original review have not been  addressed.

 

Author Response

July 19, 2021

 

Dear reviewer,

We appreciate your comments. Please see our response below:

The pair feeding experiment is awesome. However, Fig 1 data are uninterpretable without knowing the actual body weights—please show them (show the growth curves and effect of PLA with time). Also, please express the food intake and the metabolic rate in units of kcal/mouse/d. It is very difficult to compare metabolic rates of mice with different body weights/compositions. Adjustment using division by body weight (as done in the ms) is wrong (see DOI: 10.1038/nmeth.1806). For example, fig 1F would appear to show that leptin antagonist decreases metabolic rate, which, it true needs to be investigated mechanistically. Please consult with someone who understands this point.

Reply: We truly appreciate the comments. We have recalculated the data and new information is shown in the new Figure 1. Both ad libitum and pair-fed intake are expressed as kcal/mouse/day (Figure 1A and Figure 1C, respectively). Weight change of each group of mice are shown in Figure 1B and Figure 1D, respectively. Resting metabolic rate for pair-fed study is shown in Figure 1H.   The data suggest that PLA decreases metabolic rate.  This has been shown in our previous study as well.  The mechanism is indeed interesting but its further investigation will be beyond the scope of the present study.

We have a new version of Figure 1 in the edited manuscript which is now uploaded.

Sincerely,

Robert Mak, MD PhD

Author Response File: Author Response.docx