Daytime Lipid Metabolism Modulated by CLOCK Gene Is Linked to Retinal Ganglion Cells Damage in Glaucoma

Round 1

Reviewer 1 Report

I have found the manuscript very interesting. The methodology is adequate. While in some way the results could be considered as preliminary, they sound worthy by their novelty. 

Author Response

We thank the reviewer for the comments and are grateful for the appreciation of this work, that encourage us to proceed with the further studies.

Reviewer 2 Report

            In “Daytime lipid metabolism modulated by CLOCK gene is linked to retinal ganglion cells damage in glaucoma”, Gubin and colleagues explore the relationship between lipid metabolism, retinal damage, and clock gene polymorphisms in human subjects.  They find that those with retinal damage from glaucoma tend to have dysregulated lipid and cholesterol levels.  The Authors also observed changes in circadian regulation of lipid and cholesterol levels associated with retinal damage.  Finally, some of these changes are associated with molecular clock gene polymorphisms, suggesting that circadian disruption itself can deregulate circulating lipids and cholesterol, which may contribute to further retinal damage.

            The findings in this paper are interesting and potentially impactful, and the Authors are generally careful to assign causality to either retinal damage from dysregulated lipids, or dysregulated lipids arising from retinal damage.  I especially appreciated the phrase “vicious circle” in the Discussion to describe the phenomeon they are witnessing.  My comments for revision are generally technical in nature, in particular make the paper easier to read and understand.

Major points

·         The n for each experiment must be included in the Figure or Figure Legend, even if the subject cohort was previously published elsewhere.  This information is not included in any of the Figures or Legends.

·         The Figures and Tables are out of order for how they are presented in the text, which made the paper very difficult to read.  For example, Figure 4 comes in the text before Figure 3, and Table 1 is placed after Figures 2 and 4 in the paper.  This was very confusing.  Please re-arrange or renumber the Figures so that they are more closely in line with when they are mentioned in the paper.

·         For Table 2 and the Results section that discuss it, it would be helpful for the Authors to detail how clock gene polymorphisms are associated with circadian function.  Do those with CLOCK rs1801260 3111T/C have decreased circadian function somehow?  This is unclear from the text.

Minor points

·         For all graphs, add an * for comparisons that are significant.

·         On Page 9, line 211, the Authors name a study from the “Republic of Macedonia”.  The current correct name for this nation is the “Republic of North Macedonia”, please correct this.

Author Response

Reviewer 2.

In “Daytime lipid metabolism modulated by CLOCK gene is linked to retinal ganglion cells damage in glaucoma”, Gubin and colleagues explore the relationship between lipid metabolism, retinal damage, and clock gene polymorphisms in human subjects.  They find that those with retinal damage from glaucoma tend to have dysregulated lipid and cholesterol levels.  The Authors also observed changes in circadian regulation of lipid and cholesterol levels associated with retinal damage.  Finally, some of these changes are associated with molecular clock gene polymorphisms, suggesting that circadian disruption itself can deregulate circulating lipids and cholesterol, which may contribute to further retinal damage.

            The findings in this paper are interesting and potentially impactful, and the Authors are generally careful to assign causality to either retinal damage from dysregulated lipids, or dysregulated lipids arising from retinal damage.  I especially appreciated the phrase “vicious circle” in the Discussion to describe the phenomeon they are witnessing.  My comments for revision are generally technical in nature, in particular make the paper easier to read and understand.

Response: We thank the reviewer for the comments and are grateful for the appreciation of this work, that encourage us to proceed with further studies.

Major points

• The n for each experiment must be included in the Figure or Figure Legend, even if the subject cohort was previously published elsewhere. This information is not included in any of the Figures or Legends.

Response: Done.

• The Figures and Tables are out of order for how they are presented in the text, which made the paper very difficult to read. For example, Figure 4 comes in the text before Figure 3, and Table 1 is placed after Figures 2 and 4 in the paper.  This was very confusing.  Please re-arrange or renumber the Figures so that they are more closely in line with when they are mentioned in the paper.

Response: Misleading reference to Figure 4 when it was first mentioned prior to Figure 3 was withdrawn (L.89).

• For Table 2 and the Results section that discuss it, it would be helpful for the Authors to detail how clock gene polymorphisms are associated with circadian function. Do those with CLOCK rs1801260 3111T/C have decreased circadian function somehow?  This is unclear from the text.

Response: We thank the reviewer for this question, which is very interesting. There are several points of view on how sleep, chronotype and metabolism can be affected in minor allele carriers. Exact mechanisms are not yet fully elucidated, results obtained on different cohorts and populations appear partly contradictory. As suggested by the reviewer, we added to The Results section while discussing Table 2, the following sentence, L.148: “This single nucleotide polymorphism, SNP, was previously linked to the sleep-wake cycle, body weight, and lipid metabolism. Some studies considered minor C allele as protective, linked to lower cholesterol in Russian women [38].”

Minor points

• For all graphs, add an * for comparisons that are significant.

Response:  Done

• On Page 9, line 211, the Authors name a study from the “Republic of Macedonia”. The current correct name for this nation is the “Republic of North Macedonia”, please correct this.

Response: Done, now L.217.

Reviewer 3 Report

Gubin and coworkers investigated the possible occurrence of circadian alterations in lipid metabolism correlated to the reduced perceived light amplitude and to some CLOCK gene polymorphism in patients affected by glaucoma with altered photic transduction due to loss of retinal ganglion cells (RGCs).                                                                                               They found a close association of RGCs loss with changes in lipid metabolism in a time-dependent manner. In particular, HDL-cholesterol and tryglicerides showed a significant increase in the morning, while total cholesterol and LDL-cholesterol increased mainly in the evening with a strong evening gradient especially in patients with CLOCK_3111TT genotype.

This paper may be of some interest, however, I have some major concerns:                                                                                                                              1. This is an observational study, thus the patients were not synchronized on the same daytime schedule, regarding in particular the meal time and the sleep/wake cycle. It is well known that many rate-limiting enzymes in cholesterol and bile acid metabolism in the liver have diurnal patterns and misalignment of these endogenous cycles with food consumption may contribute to dyslipidemia. Moreover, misalignment of feeding with circadian cycles modulating insulin  sensitivity, nutrient storage, inflammation, and thermogenesis  may contribute to metabolic complications of obesity (Allada R & Bass J. Circadian mechanisms in Medicine. NEJM,2021).      This should be discussed.                                                                                                                                  2. Light is the most important synchronizing factor of circadian rhythms and retinal photoreceptors play a pivotal role in transmitting the light signal to the CLOCK in the suprachiasmatic nucleous.    The authors studied patients with reduced light perception due to RGCs loss, which is less severe than the complete absence of light signal as occurring in totally blind patients. However, the Authors ignored in Introduction and Discussion sections previous findings on blind patients (see for example about this on PubMed) regarding alterations of basal and rhythmic secretion of hormones involved also in the regulation of metabolism, including the liipidic one, with anabolic or catabolic action (cortisol, thyroid hormones, GH, IGF1).                                                                

Even if the authors studied the variations of lipid metabolism but not of the hormonal secretions, a discussion of these studies and the possible link to their results could increase the significance of their conclusions.                                                                                                      

Author Response

Reviewer 3.

Gubin and coworkers investigated the possible occurrence of circadian alterations in lipid metabolism correlated to the reduced perceived light amplitude and to some CLOCK gene polymorphism in patients affected by glaucoma with altered photic transduction due to loss of retinal ganglion cells (RGCs). 

They found a close association of RGCs loss with changes in lipid metabolism in a time-dependent manner. In particular, HDL-cholesterol and tryglicerides showed a significant increase in the morning, while total cholesterol and LDL-cholesterol increased mainly in the evening with a strong evening gradient especially in patients with CLOCK_3111TT genotype.

This paper may be of some interest; however, I have some major concerns: 1. This is an observational study, thus the patients were not synchronized on the same daytime schedule, regarding in particular the meal time and the sleep/wake cycle. It is well known that many rate-limiting enzymes in cholesterol and bile acid metabolism in the liver have diurnal patterns and misalignment of these endogenous cycles with food consumption may contribute to dyslipidemia. Moreover, misalignment of feeding with circadian cycles modulating insulin sensitivity, nutrient storage, inflammation, and thermogenesis may contribute to metabolic complications of obesity (Allada R & Bass J. Circadian mechanisms in Medicine. NEJM,2021).      This should be discussed.

Response:

We are thankful to the reviewer for this important comment. We agree fully that results obtained in patients’ real-life are observational, and patients may differ in their individual circadian phases depending on intrinsic tau, ambient light exposure, and habitual food consumption. Therefore, misalignment between these interrelated factors could differ between the patients, and further studies are needed to elucidate this agenda for a better understanding of fundamental basics. On the other hand, there is a need to obtain clinically valuable results to facilitate translation of fundamentals of in-lab chronobiologic results into clinical practice for applied purposes (doi: 10.1016/j.cmet.2019.06.019; doi: 10.1371/journal.pbio.3001567). From this point of view, introducing “same daytime schedule” synchronization to patients’ daytime routines will end up with inevitable "in-lab bias" and complicate direct clinical application of the results. We believe that studies from “both ends” – controlled, synchronized, in-lab; and observational, in-real-life will help one another to comprehend the whole picture.  

To further discuss putative consequences of misalignment caused by light and feeding, we added the following text in the Discussion section, at L.302: “Implementing circadian research for applied clinical purposes is important for translational circadian medicine [73, 74]. his study is just one of this kind. Since light and feeding contribute to circadian alignment, further in-depth studies are needed to assess their roles. Light, being a principal synchronizer, can also modulate mealtime. Circadian misalignment between feeding and clock affects anabolic-catabolic balance, including insulin sensitivity, and inflammation [75]. Further studies should also include feeding- and light-controlled protocols to obtain a more complete picture of interrelations within the vicious circle of compromised light perception and altered metabolism in glaucoma and other diseases in which light perception is altered.”  

2. Light is the most important synchronizing factor of circadian rhythms and retinal photoreceptors play a pivotal role in transmitting the light signal to the CLOCK in the suprachiasmatic nucleous. The authors studied patients with reduced light perception due to RGCs loss, which is less severe than the complete absence of light signal as occurring in totally blind patients. However, the Authors ignored in Introduction and Discussion sections previous findings on blind patients (see for example about this on PubMed) regarding alterations of basal and rhythmic secretion of hormones involved also in the regulation of metabolism, including the liipidic one, with anabolic or catabolic action (cortisol, thyroid hormones, GH, IGF1).

Even if the authors studied the variations of lipid metabolism but not of the hormonal secretions, a discussion of these studies and the possible link to their results could increase the significance of their conclusions.

              Response:

We agree with the reviewer that complete loss of light signal in totally blind patients with ipRGCs loss constitute an important example of what may occur to metabolic coordination when daylight perception is abolished. We added the following paragraph in the Discussion, at L.278: “Alterations to the photic signalling and transduction cause disruption of circadian rhythms, the severity of which depends on impairment of ipRGCs, and melatonin production can also be affected by its receptor gene polymorphisms [19, 21]. The fact that when visual function is lost, photic signalling is still transferred to the central clock prompted the discovery of ipRGCs [66-68]. Studies in totally blind people revealed that circadian rhythms of melatonin and cortisol were not equally compromised when light perception is not possible. Lockley and al. [69] described that 23% of individuals with no light perception remained entrained, and even more (37%) in another study [70], while the majority was non-entrained or had abnormal phase positions [69, 70]. Individual differences may be either due to genetic polymorphisms in clock, clock-controlled, or melatonin receptors’ genes, or to differences in non-photic cues that likely include food consumption. Since loss of light perception affects pineal and pituitary functions, down-streaming hormones are also compromised [71, 72], leading to the occurrence of complex metabolic consequences.”

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Round 2

Reviewer 3 Report

The authors have correctly addressed my criticisms.