Hypermetabolism in mice carrying a near-complete human chromosome 21

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1. 

   Version published to 10.7554/elife.86023 on eLife

   May 30, 2023
2. 

   eLife

   Mar 27, 2023

   Authorr Response

   Reviewer #2 (Public Review):

   This manuscript is clear in that it shows no/minimal weight gain in a mouse model of trisomy 21 compared to the control mouse, even under a high-calorie diet. The difference is the clear demonstration of the increased expression of sarcolipin. It is important that the expression of SERCA was also shown not different between the genotypes. Additionally, an important result is that manipulating the skeletal muscle was sufficient to promote weight loss without the need for hypermetabolism in other tissues such as adipose tissue.

   • A clear explanation of why the expression of sarcolipin/hypermetabolism is different between mouse and human under the same condition would be useful.

   Overexpression of sarcolipin is only seen in this particular mouse model carrying the near complete human …

   Authorr Response

   Reviewer #2 (Public Review):

   This manuscript is clear in that it shows no/minimal weight gain in a mouse model of trisomy 21 compared to the control mouse, even under a high-calorie diet. The difference is the clear demonstration of the increased expression of sarcolipin. It is important that the expression of SERCA was also shown not different between the genotypes. Additionally, an important result is that manipulating the skeletal muscle was sufficient to promote weight loss without the need for hypermetabolism in other tissues such as adipose tissue.

   • A clear explanation of why the expression of sarcolipin/hypermetabolism is different between mouse and human under the same condition would be useful.

   Overexpression of sarcolipin is only seen in this particular mouse model carrying the near complete human chromosome 21. In another widely used mouse model (Ts65Dn) of Down syndrome where all the triplicated genes (~40% of the human Chr21 orthologs) are of mouse origin, we did not observe the same overexpression of sarcolipin (PMID: 36587842). The reason for this is presently unknown. Human Chr21 contains a significant number of non-coding human genes (>400) with uncertain effects on the mouse transcriptome. Data in Figure 8 represents our efforts to understand what drives the overexpression of mouse sarcolipin (Sln) gene expression in the TcMAC21 mouse model. Although we narrowed it down and highlighted some potential candidate transcriptional drivers for Sln overexpression (Fig. 8), future work is clearly needed to confirm and establish if any of those candidates are the or one of the bona fide driver(s).

   • p.12-13 and15. The language around 'futile' cycling is not correct because Ca movement through the sarcoplasmic reticulum of the resting fiber is essential to the function of the muscle. Firstly, the cycle of Ca through the SR is through the ryanodine receptor (RyR) as well as due to slippage through the SERCA (PMID: 11306667, PMID: 35311921). This is not made clear anywhere in the manuscript. Ca leak out of the SR through RyR is an essential component to the control/setting of the resting cytoplasmic [Ca2+] via the activation of store-operated Ca2+ entry, which is in a balance with the activation of the PMCA on the t-system membrane (PMID: 35218018). The SERCA resequesters the leaked Ca2+ from the SR. It is not possible that the resting [Ca2+] is set by the reduced efficiency of the SERCA, as indicated in the ms (PMID: 20709761). It is expected that the mito [Ca2+] steady state is set by the raised resting cyto [Ca2+] (PMID: 20709761). Ca2+ transients during EC coupling will promote transient increases in mito Ca2+ (PMID: 21795684, PMID: 36121378), but not steady-state increases. Some of these problems are highlighted by the errors in the diagram Fig 5D: please change/correct (i) the invagination of the sarcolemma is called the t-system; (ii) the cycle of Ca leak through the SR starts with RyR Ca leak, where the Ca is resequestered by the SERCA, in addition to Ca slippage through the pump. Draw a RyR opposite the t-system on the SR terminal cisternae. The heat generated by SERCA is absorbed in the cytoplasm, metabolites enter the mito and the OxPhos generates heat (PMID: 31346851). (iii) Ca does not enter mito because it cannot get into the SR (the resting cyto Ca is controlled by the t-system/plasma membrane, PMID: 20709761, PMID: 35218018). Please redraw.

   We have redrawn Fig. 6D diagram as suggested by the reviewer. We have also clarified the information as presented in revised Fig. 6D in the text and figure legend. Heat is generated by mitochondrial oxidative activity. In addition, ATP hydrolysis by the Ca2+ ATPase (SERCA pump) also generates heat (PMID: 12512777; PMID: 34826239; PMID: 11342561; PMID: 17018526; PMID: 12887329). In resting muscle, for every ATP hydrolyzed by the SERCA pump, 2 Ca2+ molecules get transported into the sarcoplasmic reticulum (SR) (PMID: 15189143). In the presence of sarcolipin (SLN), a higher number of ATP needs to be hydrolyzed to move the same number of Ca2+ molecules into the SR, due to Ca2+ slippage (PMID: 34826239; PMID: 23341466). In essence, ATP hydrolysis and Ca2+ transport into the SR by SERCA becomes uncoupled in the presence of SLN. This uncoupling of the SERCA pump, in the context of Ca2+ cycling in and out of the SR (also involving Ryr1), represents the ATP-consuming futile cycle in the skeletal muscle (PMID: 34741717). Since SLN is persistently overexpressed, the ATP-consuming futile activity of the SERCA pump is presumably happening in resting muscle, as well as during EC coupling (since the TcMAC21 mice are also hyperactive).

   • The changing of the properties of the muscle towards oxidative properties is consistent with the expression of sarcolipin in mouse muscle (all of it is in type II fibers). It is important to show whether the muscles have fiber-type shifts. Please report the fiber types of the muscles that have been surveyed in this project.

   In the qPCR data as shown in Figure 6C, we have profiled many genes associated with slow- and fast-twitched muscle fibers in gastrocnemius, and little if any changes were noted. At least at the level of the transcript, there is no indication of fiber type switching in gastrocnemius muscle. However, we did not perform the same qPCR analyses for all the other muscle types isolated (i.e., EDL, quadriceps, plantaris, soleus, and tongue). The main reason for this is that we had used all of these muscle tissues in our respirometry analysis as shown in Figure 6O-Q and Figure 6-Figure Supplement 4-9. Unfortunately, we did not have any leftover muscle tissues to profile muscle fiber types.

   • Non-shivering thermogenesis (NST) is mentioned in this manuscript as the means of hypermetabolism, as has the lengthened duration of the cyto Ca transients during EC coupling. It is not clear at all what the contribution of NST compared to the increased work of the SERCA to clear released Ca from the cyto to the hypermetabolism. What are the relative proportions? If sarcolipin is largely for NST, then hypermetabolism is about the resting muscle.

   In our view, the hypermetabolism we observed in the TcMAC21 mice is primarily due to SLN-mediated uncoupling of the SERCA pump. Chronic effects of SLN overexpression elevates ATP consumption by the SERCA pump and drives the catabolic process (i.e., increased mitochondrial OXPHOS) to generate the ATP needed to meet the demand created by the persistent uncoupling of the SERCA pump. However, the TcMAC21 mice are also hyperactive, and this can also contribute to increased metabolic rate. Since the mice are both hyperactive and hypermetabolic, we do not know the relative contribution of each to the overall phenotype of the mice.

   • The link that SLN is causing more ATP use at the pump but the heat generated by OxPhos in mito is important and should be made, see Barclays' work (eg. PMID: 31346851). A direct link between the SERCA function and mito function is occurring but I currently don't see one being made in the ms. This could be made clear in Fig 5D diagram.

   We have modified and clarified Figure 6D as suggested.

   p.22. "The reprogramming of glycolytic...elevated Ca transients...". The language is wrong here. Oxidative fibers do not have elevated Ca transients compared to glycolytic. The amplitude of Ca release is greater in glycolytic and the duration of the transient is longer in the oxidative (eg. PMID: 12813151).

   We have corrected this in the text and added the citation.

   • p.22. "as less calcium is being transported into the SR due to uncoupling of the SERCA pumps". The same amount of Ca is being transported, just at the expense of more ATP than would be the case in the absence of SLN. Otherwise, the SR Ca2+ content would not be at a steady state while the SR continuously leaks Ca2+.

   We have corrected this in the revised text. The incorrect statement has been deleted.

   • p.23. Tavi & Westerblad (PMID: 21911615) show how Ca transient amplitude and frequency signal in slow and fast twitch fibres. Here, we are not concerned with what is happening in myotubes, where the SR is less developed than in adult fibres.

   We did not use any myotubes in the present study. The myotube was mentioned in the context of discussing a published work (PMID: 30208317).

   Reviewer #3 (Public Review):

   Sarver et al., propose that TcMAC21 mice are hypermetabolic and that this is the cause of their reduced weight. Unfortunately, the developmental defects of TcMAC21 mice make this a challenging question to definitively answer. The authors claim that TcMAC21 mice are hypermetabolic due to a futile calcium cycling in skeletal muscle, which is caused by up-regulation of SLN. However, all of the data that would go into the energy balance equation (food intake, energy absorption, and energy expenditure) have been improperly analyzed. TcMAC21 pups are 8.5 g lighter than euploid littermates. The body weight data and images in Fig. 3A indicate that TcMAC21 mice runted. This difference is primarily a result of lower lean mass (FIG. 2B). This is important as it sets up many concerns that need to be addressed. Specific comments are noted below.

   There is no overt developmental defect in the TcMAC21 mice as their birth weight are not different from the euploid controls (PMID: 32597754). A “runted” mouse is considered very small, poorly developed, and less competitive (PMID: 22822473). The lean phenotype of TcMAC21 mice is due to their hypermetabolism and not the result of developmental defects. The absolute lean mass of TcMAC21 mice is lower than the euploid controls. This is to be expected. A human being that weighs 150 pounds will have less lean mass compared to another person weighing 250 pounds. Lean mass scales with body weight. This does not mean that there is a muscle deficit in the person weighing 150 pounds. That is the reason why the lean mass is also generally presented as % lean mass (after normalizing to body weight). This normalization can tell us whether the amount of lean mass is appropriate (or normal) for a given weight. The % lean mass is either not different between TcMAC21 or euploid mice fed a control chow (Fig. 2B) or significantly higher in TcMAC21 mice fed a high-fat diet (Fig. 3B). This tell us that there is no developmental deficit in the skeletal muscle (biggest contributor to lean mass) of TcMAC21. The amount of lean mass seen in TcMAC21 mice scale appropriately with their lower body weight. Our food intake and energy absorption data were correctly done and analyzed (addressed below). In fact, TcMAC21 mice have the same or slighter higher food intake (absolute amount without normalization) despite weighing much less than the euploid controls (Fig. 2C and Fig. 3A, and Supplementary File 2 and Supplementary File 5). A sick or runted mouse generally consumes much less food and are physically much less active. The TcMAC21 mice are actually hyperactive (Fig. 2D-F and Fig. 4D-F). All our data argue against the notion of “runting” or “developmental defects” in TcMAC21 mice, and instead support our conclusion that TcMAC21 mice are lean due to elevated activity and hypermetabolism.

   Specific comments:

   1. It is incorrect to normalize EE to lean mass if this parameter is different between groups. Normalizing the EE data to lean mass makes it appear as though TcMAC21 mice exhibited increased EE when in fact this is a mathematical artefact. EE data should simply be plotted as ml/h (or kcal/h) per mouse. Alternatively, ANCOVA can be applied using lean mass as a covariate. Excellent reviews on this topic have been written (PMID: 20103710; PMID: 22205519).

   Energy expenditure (EE) data should not be plotted as kcal/h per mouse, as indicated in the review article that the reviewer alluded to (PMID: 22205519). It is a given that EE increases as a function of body weight, as larger body mass requires greater energy to maintain. Plotting EE data per mouse (i.e., kcal/h) would lead to the erroneous conclusion that a fat mouse would have a higher EE compared to a lean mouse. Because lean mass is metabolically much more active than fat mass, normalizing EE data to lean mass is an acceptable way to plot EE data, although not ideal, as indicated by the review article the reviewer alluded to (PMID: 20103710). Often times, normalizing EE to lean mass gives similar results as the ANCOVA, as pointed out by the authors (PMID: 22205519). However, both review articles recommend ANCOVA (using body mass as a covariant of EE) as the preferred method to plot and evaluate EE data. Alongside the EE data (normalized to lean mass), we have now also included the ANCOVA data (Fig. 2D-F and Fig. 4D-F) where we used body weight as a covariate as recommended (PMID: 22205519). The results clearly indicate that the TcMAC21 mice have significantly higher EE compared to the euploid controls.

   1. It makes no sense to normalize food intake to weight, as it makes no sense to divide metabolic rate by weight as well (see above). If food intake is not normalized, this will clearly show that TcMAC21 mice eat much less than controls, and if plotted as cumulative food intake will show that TcMAC21 are smaller and gain less weight on a high-fat diet because they simply eat less. This further indicates that the major tenet of this paper is not correct.

   It is expected that a smaller mouse will eat less food compared to a bigger mouse. Normalizing food intake to body weight can tell you whether the amount of food intake is appropriate (or normal) for a given weight. Amazingly, despite a much lower body weight, ad libitum fed TcMAC21 mice consumed the same or a slightly higher absolute amount of food, without normalizing the data to body weight (Fig. 2C and Fig. 4A and Supplementary File 2 for the chow-fed group and Supplementary File 5 for the HFD-fed group). In fact, the absolute food intake (without normalization) in the refeeding period, after a fast, was significantly higher in the TcMAC21 mice relative to euploid controls (17.7 ± 0.082 vs. 13 ±0.87 kcal, P = 0.002; Supplementary File 5). Thus, relative to their body weight, ad libitum fed TcMAC21 consumed a significantly higher amount of calories (Fig. 2C and Fig. 4A). For transparency, we chose to show side-by-side both the absolute and relative food intake data. These results, along with the rest of the data, provide compelling evidence that hypermetabolism, and not reduced food intake, underlies the lean phenotype of the TcMAC21 mice.

   1. The authors have tried to address the smaller weight of TcMAC21 mice by including weight-matched wild-type mice. However, they only focus on analyzing surface temperature, which is not an indicator of thermogenesis. Moreover, there is no information on whether these weight-matched wild-type mice are similar in age or body composition to the TcMAC21 mice. Nevertheless, the increased surface temperature can also indicate increased heat conservation, which is opposite to thermogenesis. It would make sense that TcMAC21 mice with massive reductions in lean mass would activate compensatory mechanisms of heat conservation to offset increased heat dissipation to the environment. This does seem to be the case, based on the data shown in Fig. 6D (see below).

   Skin temperature has been widely and extensively used a proxy for thermogenesis, often in association with thermogenesis of brown adipose tissue (BAT), which is located just deep to the skin over the shoulder blades of the mouse. Mice fed a high-fat diet lose the “brownness” of their brown adipose tissue as excessive circulating lipid is stored in this depot. This is a well-known phenomenon. One can see this clearly in Figure 4K where the euploid BAT has accumulated a significant amount of lipid while the TcMAC21 BAT has not. The addition of weight-matched mice was solely to help indicate whether or not the BAT was a major contributor to the TcMAC21 hypermetabolic phenotype.

   We did not conduct body composition analysis on the weight-matched mice. With a body weight of less than 30 grams, these wild-type mice represent a similarly lean and healthy adult mouse. They are not age-matched (the control mice are younger) because this is not possible. A wild-type mouse of the same age of TcMAC21 (already on high-fat diet for 12 weeks or longer) will weigh significantly more than the TcMAC21, just as the age-matched euploid littermates weighed significantly more than the TcMAC21 mice.

   The idea of heat conservation is possible, but our data clearly indicate the TcMAC21 mice have elevated thermogenesis. The supporting data include: 1) increased deep colonic temperature; 2) activation of oxidative and thermogenic gene program in skeletal muscle; 3) overexpression of sarcolipin in the skeletal muscle, leading to futile SERCA pump activity and heat generation; 4) Increased skeletal muscle mitochondrial respiration; 5) elevated T3 levels; 6) increased physical activity level; 7) increased energy expenditure (EE normalized to lean mass or ANCOVA using body weight as a covariate). Taken together, these data provide compelling evidence to support our conclusion that the TcMAC21 mice are indeed hypermetabolic and have elevated thermogenesis.

   1. A more optimal method of testing whether increased heat dissipation plays a role in the EE of TcMAC21 mice, is to measure EE at thermoneutrality, where energy dissipation to the environment will be minimized. Here the authors have attempted this in Fig. 6D. Unfortunately, the authors normalized EE to lean mass, artefactually elevating TcMAC21 EE. Despite this mistake, it now looks as though the large differences in EE that were seen at room temp have been attenuated, and only significantly limited to the dark phase. This indicates that in addition to the normalization artefact, higher heat dissipation from smaller TcMAC21 mice may also contribute to the elevated EE at 22C.

   It is well known that at thermoneutrality mouse will markedly reduce their EE. Therefore, it is not surprising that the TcMAC21 mice, housed at thermoneutrality, will have lower EE compared to the TcMAC21 mice housed at room temperature. This also holds true for the euploid controls. This is to be expected. Yet, remarkably, the TcMAC21 mice still have significantly higher EE compared to the euploid controls when housed at thermoneutrality. The TcMAC21 mice never reduce their EE to the level of the euploid controls. We have now included the ANCOVA data for EE using body weight as a covariate as recommended (PMID: 22205519) (Fig. 7F). The results clearly indicate that the TcMAC21 mice have significantly higher EE compared to euploid controls even at thermoneutrality. The data obtained at thermoneutrality, as well as the body weight-matched control experiment as shown in Figure 4I, argue against heat dissipation as the driver of increased EE. Instead, our data support hyperactivity and hypermetabolism as the driver of increased EE.

   1. In Fig. 6D, why is the hourly plot not shown here (like 2D and 4C)? The data clearly are not as striking as the EE data at 22C?

   Because of space limitation in Figure 7, we did not include the hourly tracing data and instead showed the overall energy expenditure (EE) during the light and dark cycle as bar graphs. Per reviewer request, we have now included the hourly tracing data in Fig. 7F, along with the ANCOVA data. The data clearly indicates that TcMAC21 mice, housed at thermoneutrality, have higher EE, especially in the dark cycle when they are active. This is quite remarkable. We know from many published studies that mice significantly reduce their EE when house at thermoneutrality. And yet, the TcMAC21 mice never reduce their EE to the level of euploid controls when housed at thermoneutrality.

   1. GTT was similar between TcMAC21 and controls (Fig. 3I). However, the smaller insulin response could be due to the fact that glucose was normalized to body weight. It would be better to normalize to lean mass, since that is different as well, or simply give all mice the same amount of glucose that the control group receives since this is how it is done in humans.

   The dose of glucose injection in GTT based on mouse weight is widely and extensively practiced across the metabolic community. The TcMAC21 mice are markedly more insulin sensitive, supported by multiple independent lines of evidence: 1) Overnight fasting blood glucose and insulin levels are significantly lower in TcMAC21 mice relative to euploid controls (Figure 3G). 2) Insulin tolerance test clearly indicate a substantial improvement in insulin sensitivity in TcMAC21 mice even though the insulin dose injected was much smaller (i.e., insulin dose was based on body weight) (Figure 3K). 3) The insulin response during refeeding, after an overnight fast, is dramatically lower even though the refeeding blood glucose levels rise to the same levels as the euploid controls (Fig. 3L-M). This is similar to the GTT data where the rate of glucose clearance in TcMAC21 mice is the same as the euploid controls despite a dramatically lower insulin response (Fig. 3I-J). Taken together, these data clearly indicate a markedly heightened insulin sensitivity in TcMAC21 mice relative to euploid controls.

   1. The fecal energy in Fig. 4B only measures the concentration of energy per gram of feces. However, this analysis has failed to take into account total fecal excretion, which should be used to multiply the energy density of the feces. Thus, these data are incomplete and not sufficient to exclude absorption differences between the groups. And it is now curious why if all other metabolic measurements (even though wrong), such as food intake and EE are normalized to body weight, why have the authors not normalized to body weight for the feces data? Is this because if this was done this would show massive elevating in fecal energy in TcMAC21 mice and thus falsify their hypothesis?

   The fecal data the reviewer requested was originally in the supplemental figure section. We have now moved these data to the main figure to ensure that this will not be missed by any reader. As indicated in the text and in Fig. 4B, TcMAC21 mice fed a HFD show no difference in fecal frequency (movements/day), fecal weight (g/movement), fecal energy composition (cal/g) and total fecal energy (kcal/day). These data clearly indicate that the fecal energy content is not different between TcMAC21 and euploid mice. These results, along with the rest of the data in the paper, provide compelling evidence that hypermetabolism, and not reduced nutrient absorption in the gut, underlies the lean phenotype and resistance of TcMAC21 mice to weight gain when fed a high-fat diet.

   1. I cannot find any indication of sample size in any of the EE experiments, aside from the bar graph in Fig. 6D. In any case, this experiment only an n=4 to 5 per group. This is an extremely small number for these types of experiments, so how can the authors be sure of reproducibility with such a low sample size? Are all of the other EE experiments also of similarly small sample sizes?

   Sample size for all EE experiments were clearly indicated in the original text, figure legends, and figures themselves, as well as in all supplemental figures and Supplementary files. In addition, for transparency, we always include individual data points, whenever possible, for all our data figures. They were sufficiently powered (n = 8-9 per genotype) and the effect size was large. Sample size for all thermoneutral experiments were lower than both the chow-fed and HFD-fed experiments because these mice are hard to breed and in limited supply.

   Read the original source
3. 

   eLife

   Mar 24, 2023

   eLife assessment

   This important paper provides new insight into the effect of extra-copies of a chromosome, thus aneuploidy, on body metabolisms in mammals. The authors used various solid analyses on the metabolisms and physiology of the transgenic mouse with most of human chromosome 21 and presented convincing results to support the authors' claims. The work would be of interest to researchers who work on the physiology and biochemistry of body metabolisms in mammals.

   Read the original source
4. 

   eLife

   Mar 24, 2023

   Reviewer #1 (Public Review):

   Chromosomal aneuploidy in humans causes diseases such as Down syndrome associated with changes in cognitive and metabolic activities, but how extra copies of chromosomes cause the changes remains largely unknown. In this important paper, the authors characterized the metabolisms and physiology of the transgenic mouse with most of human chromosome 21 thoroughly and nicely showed the overexpression of sarcolipin which uncouples Ca2+ import with ATP hydrolysis of sarcoplasmic reticulum Ca2+ ATPase (SERCA), which results in heat production and hyperactive mitochondria activity.

   Read the original source
5. 

   eLife

   Mar 24, 2023

   Reviewer #2 (Public Review):

   This manuscript is clear in that it shows no/minimal weight gain in a mouse model of trisomy 21 compared to the control mouse, even under a high-calorie diet. The difference is the clear demonstration of the increased expression of sarcolipin. It is important that the expression of SERCA was also shown not different between the genotypes. Additionally, an important result is that manipulating the skeletal muscle was sufficient to promote weight loss without the need for hypermetabolism in other tissues such as adipose tissue.

   - A clear explanation of why the expression of sarcolipin/hypermetabolism is different between mouse and human under the same condition would be useful.

   - p.12-13 and15. The language around 'futile' cycling is not correct because Ca movement through the sarcoplasmic reticulum of the …

   Reviewer #2 (Public Review):

   This manuscript is clear in that it shows no/minimal weight gain in a mouse model of trisomy 21 compared to the control mouse, even under a high-calorie diet. The difference is the clear demonstration of the increased expression of sarcolipin. It is important that the expression of SERCA was also shown not different between the genotypes. Additionally, an important result is that manipulating the skeletal muscle was sufficient to promote weight loss without the need for hypermetabolism in other tissues such as adipose tissue.

   - A clear explanation of why the expression of sarcolipin/hypermetabolism is different between mouse and human under the same condition would be useful.

   - p.12-13 and15. The language around 'futile' cycling is not correct because Ca movement through the sarcoplasmic reticulum of the resting fiber is essential to the function of the muscle. Firstly, the cycle of Ca through the SR is through the ryanodine receptor (RyR) as well as due to slippage through the SERCA (PMID: 11306667, PMID: 35311921). This is not made clear anywhere in the manuscript. Ca leak out of the SR through RyR is an essential component to the control/setting of the resting cytoplasmic [Ca2+] via the activation of store-operated Ca2+ entry, which is in a balance with the activation of the PMCA on the t-system membrane (PMID: 35218018). The SERCA resequesters the leaked Ca2+ from the SR. It is not possible that the resting [Ca2+] is set by the reduced efficiency of the SERCA, as indicated in the ms (PMID: 20709761). It is expected that the mito [Ca2+] steady state is set by the raised resting cyto [Ca2+] (PMID: 20709761). Ca2+ transients during EC coupling will promote transient increases in mito Ca2+ (PMID: 21795684, PMID: 36121378), but not steady-state increases. Some of these problems are highlighted by the errors in the diagram Fig 5D: please change/correct (i) the invagination of the sarcolemma is called the t-system; (ii) the cycle of Ca leak through the SR starts with RyR Ca leak, where the Ca is resequestered by the SERCA, in addition to Ca slippage through the pump. Draw a RyR opposite the t-system on the SR terminal cisternae. The heat generated by SERCA is absorbed in the cytoplasm, metabolites enter the mito and the OxPhos generates heat (PMID: 31346851). (iii) Ca does not enter mito because it cannot get into the SR (the resting cyto Ca is controlled by the t-system/plasma membrane, PMID: 20709761, PMID: 35218018). Please redraw.

   - The changing of the properties of the muscle towards oxidative properties is consistent with the expression of sarcolipin in mouse muscle (all of it is in type II fibers). It is important to show whether the muscles have fiber-type shifts. Please report the fiber types of the muscles that have been surveyed in this project.

   - Non-shivering thermogenesis (NST) is mentioned in this manuscript as the means of hypermetabolism, as has the lengthened duration of the cyto Ca transients during EC coupling. It is not clear at all what the contribution of NST compared to the increased work of the SERCA to clear released Ca from the cyto to the hypermetabolism. What are the relative proportions? If sarcolipin is largely for NST, then hypermetabolism is about the resting muscle.

   - The link that SLN is causing more ATP use at the pump but the heat generated by OxPhos in mito is important and should be made, see Barclays' work (eg. PMID: 31346851). A direct link between the SERCA function and mito function is occurring but I currently don't see one being made in the ms. This could be made clear in Fig 5D diagram.

   - p.22. "The reprogramming of glycolytic...elevated Ca transients...". The language is wrong here. Oxidative fibers do not have elevated Ca transients compared to glycolytic. The amplitude of Ca release is greater in glycolytic and the duration of the transient is longer in the oxidative (eg. PMID: 12813151).

   - p.22. "as less calcium is being transported into the SR due to uncoupling of the SERCA pumps". The same amount of Ca is being transported, just at the expense of more ATP than would be the case in the absence of SLN. Otherwise, the SR Ca2+ content would not be at a steady state while the SR continuously leaks Ca2+.

   - p.23. Tavi & Westerblad (PMID: 21911615) show how Ca transient amplitude and frequency signal in slow and fast twitch fibres. Here, we are not concerned with what is happening in myotubes, where the SR is less developed than in adult fibres.

   Read the original source
6. 

   eLife

   Mar 24, 2023

   Reviewer #3 (Public Review):

   Sarver et al., propose that TcMAC21 mice are hypermetabolic and that this is the cause of their reduced weight. Unfortunately, the developmental defects of TcMAC21 mice make this a challenging question to definitively answer. The authors claim that TcMAC21 mice are hypermetabolic due to a futile calcium cycling in skeletal muscle, which is caused by up-regulation of SLN. However, all of the data that would go into the energy balance equation (food intake, energy absorption, and energy expenditure) have been improperly analyzed. TcMAC21 pups are 8.5 g lighter than euploid littermates. The body weight data and images in Fig. 3A indicate that TcMAC21 mice runted. This difference is primarily a result of lower lean mass (FIG. 2B). This is important as it sets up many concerns that need to be addressed. Specific …

   Reviewer #3 (Public Review):

   Sarver et al., propose that TcMAC21 mice are hypermetabolic and that this is the cause of their reduced weight. Unfortunately, the developmental defects of TcMAC21 mice make this a challenging question to definitively answer. The authors claim that TcMAC21 mice are hypermetabolic due to a futile calcium cycling in skeletal muscle, which is caused by up-regulation of SLN. However, all of the data that would go into the energy balance equation (food intake, energy absorption, and energy expenditure) have been improperly analyzed. TcMAC21 pups are 8.5 g lighter than euploid littermates. The body weight data and images in Fig. 3A indicate that TcMAC21 mice runted. This difference is primarily a result of lower lean mass (FIG. 2B). This is important as it sets up many concerns that need to be addressed. Specific comments are noted below.

   Specific comments:

   1. It is incorrect to normalize EE to lean mass if this parameter is different between groups. Normalizing the EE data to lean mass makes it appear as though TcMAC21 mice exhibited increased EE when in fact this is a mathematical artefact. EE data should simply be plotted as ml/h (or kcal/h) per mouse. Alternatively, ANCOVA can be applied using lean mass as a covariate. Excellent reviews on this topic have been written (PMID: 20103710; PMID: 22205519).

   2. It makes no sense to normalize food intake to weight, as it makes no sense to divide metabolic rate by weight as well (see above). If food intake is not normalized, this will clearly show that TcMAC21 mice eat much less than controls, and if plotted as cumulative food intake will show that TcMAC21 are smaller and gain less weight on a high-fat diet because they simply eat less. This further indicates that the major tenet of this paper is not correct.

   3. The authors have tried to address the smaller weight of TcMAC21 mice by including weight-matched wild-type mice. However, they only focus on analyzing surface temperature, which is not an indicator of thermogenesis. Moreover, there is no information on whether these weight-matched wild-type mice are similar in age or body composition to the TcMAC21 mice. Nevertheless, the increased surface temperature can also indicate increased heat conservation, which is opposite to thermogenesis. It would make sense that TcMAC21 mice with massive reductions in lean mass would activate compensatory mechanisms of heat conservation to offset increased heat dissipation to the environment. This does seem to be the case, based on the data shown in Fig. 6D (see below).

   4. A more optimal method of testing whether increased heat dissipation plays a role in the EE of TcMAC21 mice, is to measure EE at thermoneutrality, where energy dissipation to the environment will be minimized. Here the authors have attempted this in Fig. 6D. Unfortunately, the authors normalized EE to lean mass, artefactually elevating TcMAC21 EE. Despite this mistake, it now looks as though the large differences in EE that were seen at room temp have been attenuated, and only significantly limited to the dark phase. This indicates that in addition to the normalization artefact, higher heat dissipation from smaller TcMAC21 mice may also contribute to the elevated EE at 22C.

   5. In Fig. 6D, why is the hourly plot not shown here (like 2D and 4C)? The data clearly are not as striking as the EE data at 22C?

   6. GTT was similar between TcMAC21 and controls (Fig. 3I). However, the smaller insulin response could be due to the fact that glucose was normalized to body weight. It would be better to normalize to lean mass, since that is different as well, or simply give all mice the same amount of glucose that the control group receives since this is how it is done in humans.

   7. The fecal energy in Fig. 4B only measures the concentration of energy per gram of feces. However, this analysis has failed to take into account total fecal excretion, which should be used to multiply the energy density of the feces. Thus, these data are incomplete and not sufficient to exclude absorption differences between the groups. And it is now curious why if all other metabolic measurements (even though wrong), such as food intake and EE are normalized to body weight, why have the authors not normalized to body weight for the feces data? Is this because if this was done this would show massive elevating in fecal energy in TcMAC21 mice and thus falsify their hypothesis?

   8. I cannot find any indication of sample size in any of the EE experiments, aside from the bar graph in Fig. 6D. In any case, this experiment only an n=4 to 5 per group. This is an extremely small number for these types of experiments, so how can the authors be sure of reproducibility with such a low sample size? Are all of the other EE experiments also of similarly small sample sizes?

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7. 

   Version published to 10.1101/2023.01.30.526183v1 on bioRxiv

   Jan 31, 2023