Misadjustment of diurnal expression of core temperature and locomotor activity in lactating rabbits associated with maternal over-nutrition before and during pregnancy

Metabolic parameters ranging from circulating nutrient levels and substrate utilization to energy expenditure and thermogenesis are temporally modulated by the circadian timing system. During critical embryonic developmental periods, maternal over-nutrition could alter key elements in different tissues associated with the generation of circadian rhythmicity, compromising normal rhythmicity development. To address this issue, we determine whether maternal over-nutrition leads to alterations in the development of circadian rhythmicity at physiological and behavioral levels in the offspring. For this, female rabbits were fed a standard diet (SD) or high-fat and carbohydrate diet (HFCD) before mating and during gestation. Core body temperature and gross locomotor activity were continuously recorded in newborn rabbits, daily measurements of body weight and the amount of milk ingested was carried out. At the end of lactation, tissue samples, including brown adipose tissue (BAT) and white adipose tissue (WAT), were obtained for determining the expression of uncoupling protein-1 (UCP1) and cell death-inducing DNA fragmentation factor-like effector A (CIDEA) genes. HFCD pups exhibited conspicuous differences in the development of the daily rhythm of temperature and locomotor activity compared to the SD pups, including a significant increase in the daily mean core temperature, changes in the time when temperature or activity remains above the average, shifts in the acrophase, decrease in the duration and intensity of the anticipatory rise previous to nursing, and changes in frequency of the rhythms. HFCD pups exhibited a significant increase in BAT thermogenesis markers, and a decrease of these markers in WAT, indicating more heat generation by brown adipocytes and alterations in the browning process. These results indicate that maternal over-nutrition alters offspring homeostatic and chronostatic regulation at the physiological and behavioral levels. Further studies are needed to determine whether these alterations are associated with the changes in the organization of the circadian system of the progeny.

120 The sentence "…woman in reproductive age" was changed to "…woman of reproductive age".

120-Be more specific about what conditions these women exhibit
123 The sentence "…exhibit some of these conditions" was changed to "…exhibit obesity and metabolic alterations".

123-Within the introduction there must be justification for why rabbits are used as a model species for maternal overnutrition, and specifically why they can be a good model for humans, since research in human subjects is directly described in the introduction
143-160 A justification about the rabbit model was included: "The pathogenesis of human obesity and development of metabolic syndrome (MS) is not fully understood, in order to elucidate the mechanisms and develop new therapeutic strategies, it is essential to have an appropriate animal model that shares the most important aspects of the disease process with humans. Rabbits are a widely used experimental model in biomedical research, and have been proposed as an experimental alternative for the study of MS and its complications, such as atherosclerosis and coronary heart disease, which is the major cause of death in MS patients. Unlike rodents, rabbits have close similarities to human cardiovascular and lipoprotein profiles, with higher levels of apoB-containing low density lipoproteins, and abundant cholesteryl ester transfer protein in plasma, an important regulator of reverse cholesterol transport [Fan et al. 1999;Fan et al. 2015;Fan and Watanabe, 2003;Kawai et al 2006;Furukawa et al 2014;Yin et al. 2002;Such et al 2008;Noujaim et al 2010;Zarzoso et al 2012]. In addition, rabbits fed a high fat and sugar diet develop many characteristics of MS observed in humans [Carroll et al. 1996;Zhang et al 2008].
Furthermore, rabbits are an ideal model for the study of transgenerational effects of maternal overnutrition in newborn metabolic regulation, since the placental structure and materno-fetal blood flow interrelationships are closer to the human, in comparison to other models, such as rodents. Humans have discoid and hemochorial type placentas, and the number of trophoblastic layers in the placental barrier, such as the border between fetal and maternal blood systems, differs between species. In humans it is hemomonochorial, with only one layer, while in rabbits it is hemodichorial and in rodents hemotrichorial [Leiser and Kauffman 1994;Review in Perry, 1981;Carter, 2007;Hafez and Tsutsumi, 1966;Furukawa et al.2011]".

-There is a lot of information in the introduction describing the effects of maternal overnutrition on the circadian clock. However, there is no measurement of the circadian clock gene expression or circadian rhythms of energy metabolism within this paper. There should be a better connection between how the research presented in this paper relates to the circadian clock, or this information should be removed and the introduction should focus more on behavioral and body temperature outputs
161 Information associated to the molecular clockwork was reduced in the introduction.

143-A hypothesis must be provided
161-167 The hypothesis was modified.

-Objective does not include anything about UCP1 or CIDEA expression levels or blood metabolites, so why were these measured? Please add objectives related to these outcomes. Also, circadian rhythms in behavioral and physiologic markers weren't measured. In order to characterize a true circadian rhythm, responses must be measured in consistent lighting (i.e. 24 dim light). Please refer to the rhythms measured (really only locomoter activity) as a diurnal or daily rhythm.
167-171 The objective was modified.

183-185
Additional information about the experimental design was included.

-Please provide the total kcal/g of each diet
191-193 The total amount Kcal/kg of each diet was included in the text.

-Replace elaborated with a more appropriate word. Could just say HFCD contained SD supplemented with…
194 The word "was elaborated" was changed to "contained".

-Why where # of does unevenly distributed between treatments. Please provide justification for sample size with power calculation.
199 Due to the difficulties in obtaining the offspring of the HFCD does (important alterations during conception and spontaneous miscarriages) and greater number of dead offspring during the first weeks of life, we decided to increase the number of HFCD does in order to ensure enough animals for the protocol. In this manuscript we did not report any data about female rabbits, such information, in which the power calculation was provided, was submitted elsewhere.

-Please justify that human chorionic gonadotropin would not affect fetal metabolic programming
202 In our study we administered a single dose (30 UI) of human chorionic gonadotropin (hGC). It is well known that the half-life of this hormone is approximately 37 h [Feiman et al. 1968], therefore the transitory action of this hormone is short-lived to improve the conception rate. There are no reports in the literature about the effects of a single administration of hGC in fetal metabolic programming.

-mRNA levels were not determined by extraction. RNA was first extracted and then mRNA expression of UCP1 and CIDEA were determined by RT-qPCR. Correct wording
289 The sentence was corrected.

-Duration and intensity of the anticipatory component of are never defined. Please discuss how this was determined because it is completely unclear
339-346 Information about the duration and intensity of the anticipatory component was included.

-You report acrophase, but there is no description of how this is determined. Determination of acrophase must have been performed using cosinor rhythmometry but there is no description of this is the methods. Cosinor rhythmometry should be performed on data where a diurnal response is measured -so for body temp. and for locomotor activity.
Originally, in the manuscript we indicate that "To evaluate the rhythmicity in the rabbits' core body temperature and locomotor activity, we used a previously reported procedure [Montúfar-Chaveznava R, et al. 2013;Trejo-Muñoz L, et al. 2012]". This was replaced by a full description of the rhythmicity procedure (332-339).
It is well known that single Cosinor was developed to analyze short and sparse data series [Halberg et al., 1967]. In this work, as we have a long time series for body temperature and locomotor activity, we employed two different methods. The first method used was the Fourier theory, to determine the amplitude and phase (or acrophase) of specific sinusoids.
In particular, we employed the Fast Fourier Transform for frequency decomposition considering we have discrete data; next we selected the frequencies (or periods) of interest (i.e. 24h) and recovered the information from the polar representation of Fourier coefficients. In the second method used, since the 24 h phase obtained with Fast Fourier Transform does not always fit the data, we constructed a pulse sequence of a 24h period which was shifted along the data to find time pulse and data fits using a similarity metric.

-Time course data should be analyzed as repeated measures analysis with day as a repeated factor. A two
The data was analyzed as repeated measures, as mentioned.

-Replace " close similarities" with "no difference"
399 The term "close similarities" was replaced with "no difference".

-Replace "plasmatic level" with "plasma concentrations" here and elsewhere
400 The term "plasmatic levels" was replaced with "plasma concentrations".

Figures:
All the figures were modified according to comments.