Low-protein/high-carbohydrate diet induces AMPK-dependent canonical and non-canonical thermogenesis in subcutaneous adipose tissue
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Reply to the reviewers
__Reviewer #1 __
1.The stimulatory effect of LPHC diet on browning of some WATs has been previously reported (Nutrition, 42, 37-45 Oct 2017). Also, the activation of AMPK was observed in this study. However, the detailed mechanism responsible for AMPK activation by LPHC diet remains elusive in the present study, which lowers its scientific importance.
Response: OK. We will adequately mention previous study in which the AMPK activation was observed upon LPHC diet and more deeply decipher the molecular mechanisms that lead to AMPK activation by analyzing AMP- and ROS/Ca2+-dependent pathways according to the Reviewer’s suggestions (for more details, see point …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
__Reviewer #1 __
1.The stimulatory effect of LPHC diet on browning of some WATs has been previously reported (Nutrition, 42, 37-45 Oct 2017). Also, the activation of AMPK was observed in this study. However, the detailed mechanism responsible for AMPK activation by LPHC diet remains elusive in the present study, which lowers its scientific importance.
Response: OK. We will adequately mention previous study in which the AMPK activation was observed upon LPHC diet and more deeply decipher the molecular mechanisms that lead to AMPK activation by analyzing AMP- and ROS/Ca2+-dependent pathways according to the Reviewer’s suggestions (for more details, see point 2-4).
2.Different with WAT, LPHC diet increases glucose uptake and FA synthesis in BAT (Nutrition. 30 (4), 473-80 Apr 2014). ____Is it possible that AMPK activation in WAT due to the lowered glucose uptake, which might increase AMP/ATP ratio? It is recommended to determine the uptake of glucose in WAT.
Response: In the article cited by the Reviewer, authors measured glucose uptake only in BAT and found that it was significantly increased. On the contrary, no data are reported regarding glucose metabolism in WAT. Our in vitro data clearly indicate that AMPK activation occurs upon amino acid restriction (AAR) and in the presence of glucose in the culture medium (see Fig. 6C, Suppl. Fig. 5I). Moreover, glucose uptake is increased upon this condition (see Fig. 5G). Hence a decreased glucose uptake by WAT and the activation of AMPK via a decrease of AMP/ATP ratio has to be likely excluded. However, we will test AMP and ATP levels both in vivo and in vitro and this, together with experiments aimed at deciphering the contribution of mitochondrial ROS (mtROS) and CaMKK (see points 3, 4), we will hopefully clarify the mechanisms of AMPK activation upon LPHC diet.
__ The present study indicates that the promotional effect of LPHC diet on WAT browning is dependent on mitochondrial ROS generation. However, it is still unknown why the production of ROS increased and why ROS could activate AMPK. The authors should clarify these critical steps. __
Response: Redox unbalance is widely reported to directly or indirectly stimulate AMPK activation (Shao et al., 2015, Cell Metab; Hinchi et al., 2018, J Biol Chem). Moreover, it has been demonstrated that activation of AMPK could depend on mtROS and be independent of an increase in AMP/ATP ratio (Emerlin et al., 2009, Free Radic Biol Med). Based on this evidence and our results, we believe that, upon AAR or LPHC diet, the recorded increase of mtROS concentration could not derive from an enhanced production but rather to a decrease of intracellular availability of the sulfur amino acid cysteine that represents an efficient ROS scavenger. Actually, by replenishing cysteine through N-acetyl cysteine (NAC) treatment we were able to buffer mtROS increase (see Fig. 6A), as assayed by cytofluorimetric analyses through mitoSox staining, and avoid AMPK phosphorylation (see Fig. 6C and 6J) as well as the downstream upregulation of brown fat and muscular genes (see Fig 6B and 6I). In line with this result, treatment with erastin, a cysteine depleting agent, was able to mimic the effects of AAR and LPHC diet by up-regulating the expression of brown-like and muscular genes (see Fig. 6G). Therefore, to more deeply decipher the mechanisms involved in AMPK activation and to further involve cysteine depletion in mtROS increase and AMPK activation, we could assay mtROS and AMPK levels also following erastin treatment. Of course, to involve cysteine decrease in AMPK activation, measuring intracellular cysteine levels upon AAR and LPHC diet is mandatory and will be carried out. Importantly, we have preliminary data, not included in the present manuscript, indicating that cysteine is decreased both upon AAR and LPHC diet; hence, after increasing the sample size, we will include this result in the revised version.
__4.The relationship between cytosolic calcium and AMPK was not clear. In addition to the fact that AMPK regulates SERCA to increase cytosolic Ca depicted in the present study, AMPK could also be activated by increased Ca via CaMKK. A recent study indicates that the activation of AMPK requires TRPV4-mediated Ca release from ER (Cell Metabolism Volume 30, Issue 3, 3 September 2019, Pages 508-524.e12). This issue should also be clarified. __
Response: Regarding the possible involvement of TRPV4-mediated Ca release from ER, through RNAseq we found that TRPV4 mRNA is slightly expressed in subcutaneous white adipose tissue and changes in its expression were not found upon LPHC diet. Moreover, TRPV4 protein was not detected in our samples by proteomic analysis. Notably, by integrating transcriptomic and proteomic data, it emerged that cell membrane intracellular calcium transporters (i.e. CACNG1, CACNA2D1), which are interconnected to the network of sarcoplasmic reticulum calcium cycle, are upregulated upon LPHC diet (see Fig. 5I). Therefore, we will evaluate the effects of a calcium channel blocker (e.g. Verapamil) and/or extracellular calcium chelator (e.g. BAPTA) on AMPK activation and its downstream gene expression cascade. In parallel, to possibly involve CAMKK in the activation of AMPK, treatment with a CAMKK inhibitor (e.g. Sto-609) will be carried out. Importantly, mtROS are upstream inducers of intracellular calcium raise (Mungai et al., 2011, Mol Cell Biol) and therefore an involvement of mtROS-Ca2+ axis could not be ruled out. In line with this hypothesis, by buffering mtROS through NAC treatment, we were able to abrogate intracellular calcium raise elicited by AAR (see Fig. 6F). Therefore, by performing the above described experiments and by evaluating CAMKK following NAC treatment, we will be hopefully able to establish whether AMPK activation is AMP-(in)dependent and/or relies on mtROS/Ca2+/CAMKK pathway.
__Reviewer #2 __
o Interesting paper but see comments below.
Response: OK, thanks
o __The relevance of the described effects for whole-body energy balance regulation is not shown. Indirect calorimetry could be interesting. The only whole-body effect (slightly improved glucose clearance in oGTT) was very small. __
Response: OK. As suggested by this Reviewer we can include indirect calorimetry to give a more comprehensive view of the effects of LPHC diet on the whole-body energy balance (see also the following point).
o …__1) Indirect calorimetry could be very helpful to show effects on energy metabolism. 2) Can the authors discuss why they didn't conduct the experiment also under thermoneutral conditions? __
Response: OK. As stated above, we will add indirect calorimetry experiments and, as suggested by this Reviewer, we will discuss this issue in the revised version. Importantly, we already have indirect calorimetry data that were not included in the present version of the manuscript and that we will add in the revised version.
__o Maybe an additional collaborator is necessary. __
Response: Yes, collaborators who performed indirect calorimetry will be included as co-authors in the revised version.
o Article numbers of all diets must be added and information if the all diets were purified diets. This could have effects on the gut microbiome.
Response: OK. We will add the article numbers as well as more detailed information about all the diets.
o Sample sizes are very low. The authors should explain why only males were used in the experiments. oGTT analysis should also include calculation of area under the curve. No explicit statement if correction for multiple testing is required or other measures to reduce false positive results.
Response: We have used only male mice to avoid sex bias. We will edit the OGTT analysis graph to include calculation area under the curve. Regarding the sample size, a mistake occurred when the figure legends have been written. Actually, in materials and methods section, we clearly indicated the number of animals used (n=8 mice for WD and n=6 mice for LPHC diet and not n=3). Information regarding the statistical analyses was included in Bioinformatics and Statistical Analysis section. In this section, we described how the correction for multiple testing was carried out (i.e. one-way ANOVA followed by Dunnetts correction). In the revised version, we will dedicate a separate section for statistical analysis to avoid misreading.
__**Minor comments:** __
o __Are prior studies referenced appropriately?* Relevant reference: Desjardins, E.M., Steinberg, G.R. Emerging Role of AMPK in Brown and Beige Adipose Tissue (BAT): Implications for Obesity, Insulin Resistance, and Type 2 Diabetes. Curr Diab Rep 18, 80 (2018). ____https://doi.org/10.1007/s11892-018-1049-6____ __
Response: We thank the Reviewer for this suggestion and we will include and appropriately discuss this paper.
o *Are the text and figures clear and accurate?* YES
__*Response: *__Ok, thanks for this positive evaluation.
Reviewer #3
__My main critique, coming from the perspective of a dietitian that works in human trials in the US, is that the diet called a "Western" diet is not similar to the diet that humans with metabolic problems typically eat… __
Response: OK. The aim of this work was to study at molecular level the responses of white adipose tissue to changes in protein to carbohydrate ratio. We completely agree with the Reviewer that “Western” diet is not an appropriate term to describe the diet that we have used; hence, we will change “Western diet” in “Control diet” throughout the manuscript. Actually, according to the general guidelines for nutrition studies on mice, when experimental animals are fed a special diet (i.e. LPHC in our study), the control animals should be fed a diet matched in every way to the special diet, except of course for the dietary variable (i.e. P/C ratio in our study) that the researcher is studying (Pellizzon and Ricci, The common use of improper control diets in diet-induced metabolic disease research confounds data interpretation: the fiber factor (2018). Nutrition & Metabolism 15:3).
__**Major comments:** __
__-The authors provide strong support their key findings. __
Response: We thank this reviewer for this positive evaluation.
__-The mice were on the LPHC diet for a short period of time (2 weeks). Ongoing amino acid deficiency has potential to promote frailty and other deleterious outcomes. No long-term diet outcomes can be inferred from this study. __
Response: OK. We will discuss this issue, highlighting that this dietary regimen should be recommended on human only for a short period and that further study is needed for understanding the long-term effects of LPHC diet.
__-The authors have provided no evidence that a LPHC diet improves human health, so I think they need to scale back those assertions, particularly as it relates to people shifting to a LPHC from what they currently eat, since people don't typically eat what the authors refer to as a "Western" diet as it's defined in this paper. __
Response: OK. We will reference studies in which LPHC diet has been suggested to improve human health.
__-As far as I can tell, no additional experiments are needed to support their claims identifying how the LPHC affects AMPK activated pathways in mice. __
Response: We thank this reviewer for this positive evaluation.
__-The methods are rigorous and sufficiently described to be reproducible. __
Response: We thank this reviewer for this positive evaluation.
__**Minor comments:** __
__-____Minor grammatical issues through e.g. "It is worth to notice" in last paragraph on page 12; there are font differences in the methods section __
Response: OK. We will correct these minor grammatical/font issues.
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Referee #3
Evidence, reproducibility and clarity
Summary:
The authors use a mouse model to compare molecular responses to a 23% protein/57 carbohydrate/20 fat diet to a 7% protein/73% carbohydrate/20% fat diet. The authors show that the low protein diet enhanced activation of biological pathways related to fatty acid catabolism including FAO, TCA cycle and electron transport chain in sWAT but not BA, similar to cold exposure. The authors use redundant assays and experiments in cell models to validate the genes and molecular pathways involved in the sWAT response to a low protein diet in mice. The authors show that AMPK activation promotes the induction of typical brown fat and …
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Referee #3
Evidence, reproducibility and clarity
Summary:
The authors use a mouse model to compare molecular responses to a 23% protein/57 carbohydrate/20 fat diet to a 7% protein/73% carbohydrate/20% fat diet. The authors show that the low protein diet enhanced activation of biological pathways related to fatty acid catabolism including FAO, TCA cycle and electron transport chain in sWAT but not BA, similar to cold exposure. The authors use redundant assays and experiments in cell models to validate the genes and molecular pathways involved in the sWAT response to a low protein diet in mice. The authors show that AMPK activation promotes the induction of typical brown fat and muscular genes in sWAT. The authors identify novel non-canonical pathways (Serca1 and Serca2a,) that are upregulated in sWAT browning.
My main critique, coming from the perspective of a dietitian that works in human trials in the US, is that the diet called a "Western" diet is not similar to the diet that humans with metabolic problems typically eat. The typical US diet is closer to approximately a 17% protein/50% carbohydrate/33% split (https://doi.org/10.1016/j.nut.2015.02.007, https://doi.org/10.1038/s41430-017-0031-8). This level of protein utilized for the experimental "Western" diet here is comparable to levels used for "high protein" diets in some human studies (https://doi.org/10.1111/nure.12111).
Since the experimental diet differs substantially from what metabolically sick people typically eat, the ability to speculate how the findings from this study may apply to humans with metabolic diseases is very limited. This paper is really well-done, but I think the authors should call the experimental diet a high-protein, moderate carbohydrate diet (HPMC), not a "Western" diet. There are many who argue that such a HPMC is metabolically advantageous and promotes weight loss/improved body composition, so this study lays the groundwork for refuting that guidance. It would be exciting to see a head to head comparison of the two diets in humans in the future!
Major comments:
-The authors provide strong support their key findings
-The mice were on the LPHC diet for a short period of time (2 weeks). Ongoing amino acid deficiency has potential to promote frailty and other deleterious outcomes. No long-term diet outcomes can be inferred from this study.
-The authors have provided no evidence that a LPHC diet improves human health, so I think they need to scale back those assertions, particularly as it relates to people shifting to a LPHC from what they currently eat, since people don't typically eat what the authors refer to as a "Western" diet as it's defined in this paper.
-As far as I can tell, no additional experiments are needed to support their claims identifying how the LPHC affects AMPK activated pathways in mice.
-The methods are rigorous and sufficiently described to be reproducible
Minor comments:
-Minor grammatical issues through e.g. "It is worth to notice" in last paragraph on page 12; there are font differences in the methods section
Significance
The work is significant as it describes the metabolic effects of a LPHC at the molecular level for the first time. This paper demonstrates how a low protein diet may promote longevity and improve glucose metabolism, which has been shown to some extent in humans, but hasn't had a mechanistic explanation until now.
If similar findings were supported in longer term animal and human trials, it could lay the groundwork for modifying dietary recommendations to promote metabolic health and longevity.
This paper is of interest to basic scientists studying diet and energy metabolism. The potential health implications are interesting to people in healthcare and scientists studying human metabolism.
I am a dietitian who has conducted weight loss trials in humans, emphasizing varying macronutrient ratios. I have also done whole body metabolism work in humans using metabolic chambers. I have experience in urinary proteomics, but I lack sufficient expertise to scrutinize much of the methodology of the basic work you present here.
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Referee #2
Evidence, reproducibility and clarity
Aquilano et al. submitted a manuscript investigating the effects of a low-protein/high-carbohydrate diet on AMPK-dependent thermogenic activity in subcutaneous adipose tissue in mice presumably resulting in stimulated energy dissipation. Based on the observation that LPHC diets may promote metabolic benefits the authors aimed to study the underlying molecular functions. They focused mainly on a comparison of molecular markers for thermogenesis and the related metabolic pathways in brown and subcutaneous white adipose tissue in response to feeding mice a LPHC diet for two weeks. Using a proteomics approach first, they identified 75 …
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Referee #2
Evidence, reproducibility and clarity
Aquilano et al. submitted a manuscript investigating the effects of a low-protein/high-carbohydrate diet on AMPK-dependent thermogenic activity in subcutaneous adipose tissue in mice presumably resulting in stimulated energy dissipation. Based on the observation that LPHC diets may promote metabolic benefits the authors aimed to study the underlying molecular functions. They focused mainly on a comparison of molecular markers for thermogenesis and the related metabolic pathways in brown and subcutaneous white adipose tissue in response to feeding mice a LPHC diet for two weeks. Using a proteomics approach first, they identified 75 proteins differentially present in sWAT compared to BAT. These could be linked both to canonical as well as non-canonical (muscular) thermogenic functions as the authors state. Overall, they conclude that feeding a LPHC diet induces a white-to-brown conversion in sWAT. Deep RNA-sequencing identified 416 up and 52 down-regulated gene transcripts in sWAT. GO terms analysis showed enrichment for biological processed related to mitochondrial fatty acid catabolism, response to cold, and muscle contraction genes. Following up this rational, they conducted several experimental approaches to identify regulators in this system. For example, they tried to rule out that changes in gut microbiome composition could mediate metabolic benefits in response to LPHC diet. Finally, they hypothesized that nutrient shortage in particular amino acid lowering is responsible for sWAT browning. Here, AMPK seems to play a central role in the browning of sWAT in response to LPHC diet.
Major comments:
o Are the key conclusions convincing? YES interesting paper but see comments below.
o Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? YES - The relevance of the described effects for whole-body energy balance regulation is not shown. Indirect calorimetry could be interesting. The only whole-body effect (slightly improved glucose clearance in oGTT) was very small.
o Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary to evaluate the paper as it is, and do not ask authors to open new lines of experimentation. YES - 1) Indirect calorimetry could be very helpful to show effects on energy metabolism. 2) Can the authors discuss why they didn't conduct the experiment also under thermoneutral conditions?
o Are the suggested experiments realistic for the authors? It would help if you could add an estimated cost and time investment for substantial experiments. Maybe an additional collaborator is necessary.
o Are the data and the methods presented in such a way that they can be reproduced? YES mostly - but article numbers of all diets must be added and information if the all diets were purified diets. This could have effects on the gut microbiome.
o Are the experiments adequately replicated and statistical analysis adequate? Sample sizes are very low. The authors should explain why only males were used in the experiments. oGTT analysis should also include calculation of area under the curve. No explicit statement if correction for multiple testing is required or other measures to reduce false positive results.
Minor comments:
o Are prior studies referenced appropriately? Relevant reference: Desjardins, E.M., Steinberg, G.R. Emerging Role of AMPK in Brown and Beige Adipose Tissue (BAT): Implications for Obesity, Insulin Resistance, and Type 2 Diabetes. Curr Diab Rep 18, 80 (2018). https://doi.org/10.1007/s11892-018-1049-6
o Are the text and figures clear and accurate? YES
Significance
My expertise: Energy metabolism in rodent models for metabolic disease, body temperature regulation, body mass regulation
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Referee #1
Evidence, reproducibility and clarity
This work found that a LPHC meal activates browning of sWAT by ROS/AMPK pathway, and tried to clarify the detailed mechanism of the beneficial effect of LPHC diet. Although the paper contains scientific novelty and is well-written, most of the results are descriptive and deeper mechanistic study seems lacking. Here listed some comments and questions.
1.The stimulatory effect of LPHC diet on browning of some WATs has been previously reported (Nutrition, 42, 37-45 Oct 2017). Also, the activation of AMPK was observed in this study. However, the detailed mechanism responsible for AMPK activation by LPHC diet remains elusive in the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This work found that a LPHC meal activates browning of sWAT by ROS/AMPK pathway, and tried to clarify the detailed mechanism of the beneficial effect of LPHC diet. Although the paper contains scientific novelty and is well-written, most of the results are descriptive and deeper mechanistic study seems lacking. Here listed some comments and questions.
1.The stimulatory effect of LPHC diet on browning of some WATs has been previously reported (Nutrition, 42, 37-45 Oct 2017). Also, the activation of AMPK was observed in this study. However, the detailed mechanism responsible for AMPK activation by LPHC diet remains elusive in the present study, which lowers its scientific importance.
2.Different with WAT, LPHC diet increases glucose uptake and FA synthesis in BAT (Nutrition. 30 (4), 473-80 Apr 2014). Is it possible that AMPK activation in WAT due to the lowered glucose uptake, which might increase AMP/ATP ratio? It is recommended to determine the uptake of glucose in WAT.
- The present study indicates that the promotional effect of LPHC diet on WAT browning is dependent on mitochondrial ROS generation. However, it is still unknown why the production of ROS increased and why ROS could activate AMPK. The authors should clarify these critical steps.
4.The relationship between cytosolic calcium and AMPK was not clear. In addition to the fact that AMPK regulates SERCA to increase cytosolic Ca depicted in the present study, AMPK could also be activated by increased Ca via CaMKK. A recent study indicates that the activation of AMPK requires TRPV4-mediated Ca release from ER (Cell Metabolism Volume 30, Issue 3, 3 September 2019, Pages 508-524.e12). This issue should also be clarified.
Significance
This work indicates that LPHC diet promotes browing of WAT through activation of AMPK by elevating mitochondrial ROS production. Compared to previous studies, this work firstly found the critical importance of mitochondrial ROS in activation of AMPK through a series of works on omics data. However, they failed to clearly explain the detailed mechanism responsable for either enhanced mitochondrial ROS production by LPHC diet or activation of AMPK by mitochondrial ROS. Therefore, due to most of the conclusions have been presented in some previous published papers, the main novelty of the present work should be greatly improved by further mechanistic stidies.
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