Supplementation of Non-Dairy Creamer-Enriched High-Fat Diet with D-Allulose Ameliorated Blood Glucose and Body Fat Accumulation in C57BL/6J Mice

Round  1

Reviewer 1 Report

Dear authors,

thank you for your manuscript "Supplementation of Non-dairy Creamer-enriched High-fat diet with D-Allulose Ameliorated Blood Glucose and Body Fat Accumulation in C57BL/6J Mice"

The manuscript is well written however you should address the following questions before it can be published:

Major comments

You chose to study the effects of D-allulose supplementation in mice fed with HFD with non-diary creamer. However, I believe the lack of control with a standard diet (chow diet alone and with the addition of d-allulose) hinders the results. The data obtained from these two groups would be valuable to clarify the effect of d-allulose supplementation.

The introduction needs to go deeper into the background of D-allulose supplementation effects and thus in the 2nd paragraph, the authors need to elaborate further. 

D-allulose limits fat absorption and more fat is excreted. Less fat reaches the blood circulation and is metabolised by the mice. So the changes you observed in the various genes, as well as the other biochemical indices, are not due to a direct effect of d-allulose but rather an indirect mechanism of action? Please elaborate more in the discussion. This is not clear in your manuscript.

Minor comments

Why did you use only male mice?

Please comment on the supplementation-dose of d-allulose (5%). How much d-allulose is too much?

Figure 1a and 1b: since the energy density of each diet is ~4 kcal/gr, the energy intake for the 2 groups (daily food intake of ~12 g/day) should be ~48 kcal/day. In Figure 1b the mean energy intake is ~3.2 kcal/day. Please explain.

Figures could be re-organised (for example the figures constituting Figure 5 can be merged into ones since the y-axis units are the same or figures 3a, 3b, 3c). Moreover, the authors can omit figures such as figure 1a, 1b, 1c, 1d, 1e, 2b,  and describe their results in the text.

Author Response

Major comments

Q1. You chose to study the effects of D-allulose supplementation in mice fed with HFD with non-diary creamer. However, I believe the lack of control with a standard diet (chow diet alone and with the addition of d-allulose) hinders the results. The data obtained from these two groups would be valuable to clarify the effect of d-allulose supplementation.

R1. Thank you for a kind comment. Your suggestion is very reasonable and considerable and we agree with reviewer’s comment. This study is focused on anti-obesity effect of d-allulose in obese mice fed HFD with non-diary creamer and its commercial availability, and thus we did not investigate the effect of d-allulose in mice fed chow diet. Although, we, unfortunately, did not include chow fed group, we also believe that the study on the effect of d-allulose in mice fed chow diet would be valuable to clarify the mechanism of d-allulos. We will consider your suggestion in future animal study. 

Q2. The introduction needs to go deeper into the background of D-allulose supplementation effects and thus in the 2nd paragraph, the authors need to elaborate further. 

R2. As your suggestion, we have revised the introduction section. Thank you for a kind comment.

Q3. D-allulose limits fat absorption and more fat is excreted. Less fat reaches the blood circulation and is metabolised by the mice. So the changes you observed in the various genes, as well as the other biochemical indices, are not due to a direct effect of d-allulose but rather an indirect mechanism of action? Please elaborate more in the discussion. This is not clear in your manuscript.

R3. As your suggestion, we have revised the discussion section at line 383-385. Thank you for a kind comment.

Minor comments

Q4. Why did you use only male mice?

R4. Obesity is greatly affected by hormones. Especially, estrogens protect from obesity and its complications, and ovariectomy eliminates the protection of female mice from becoming obese when exposed to high-fat diets. Therefore, male mice are more susceptible to obesity than female mice. For these reasons, male mice are used in many obesity studies.

Q5. Please comment on the supplementation-dose of d-allulose (5%). How much d-allulose is too much?

R5. Regarding its safety matter, D-allulose is generally recognized as safe (GRAS) based on USDA regulations (GRN no. 647 and 693). Also, our previous study has reported that a maximum single dose and maximum total daily intake of D-Allulose in human are 0.4 g/kg·BW and 0.9 g/kg·BW, respectively [1]. When convert the 5% (w/w) dose of d-allulose used in a mouse to a dose based on surface area for humans [2], about 0.45g/kg·BW dose of d-allulose is calculated for a human equivalent dose, which is lower than the maximum total daily intake of D-Allulose in human (0.9 g/kg·BW). Moreover, 5% D-allulose in rats maintained blood glucose levels within a normal range [3] and reduced body fat accumulation by enhancing 24-h energy expenditure with enhanced fat oxidation and reduced carbohydrate oxidation [4, 5], and 5% dietary D-allulose in HFD-fed mice led to the normalization of the metabolic status of diet-induced obesity by altering lipid-regulating enzyme activities and their gene-expression level along with fecal lipids [6].

①    Han, Y.; Choi, B.R.; Kim, S.Y.; Kim, S.B.; Kim, Y.H.; Kwon, E.Y.; Choi, M.S. Gastrointestinal Tolerance of D-Allulose in Healthy and Young Adults. A Non-Randomized Controlled Trial. Nutrients. 2018, 10(12), E2010.

②    Reagan-Shaw, S.; Nihal, M.; Ahmad, N. Dose translation from animal to human studies revisited. FASEB J. 2008, 22(3), 659-61.

③    Hossain, M.A.; Kitagaki, S.; Nakano, D.; Nishiyama, A.; Funamoto, Y.; Matsunaga, T.; Tsukamoto, I.; Yamaguchi, F.; Kamitori, K.; Dong, Y.; Hirata, Y.; Murao, K.; Toyoda, Y.; Tokuda, M. Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Biochem Biophys Res Commun. 2011, 405(1), 7-12.

④    Matsuo, T.; Izumori, K. Effects of dietary D-psicose on diurnal variation in plasma glucose and insulin concentrations of rats. Biosci Biotechnol Biochem. 2006, 70(9), 2081-5.

⑤    Nagata, Y.; Kanasaki, A.; Tamaru, S.; Tanaka, K. D-psicose, an epimer of D-fructose, favorably alters lipid metabolism in Sprague-Dawley rats. J Agric Food Chem. 2015, 63(12), 3168-76.

⑥    Han, Y.; Han, H.J.; Kim, A.H.; Choi, J.Y.; Cho, S.J.; Park, Y.B.; Jung, U.J.; Choi, M.S. d-Allulose supplementation normalized the body weight and fat-pad mass in diet-induced obese mice via the regulation of lipid metabolism under isocaloric fed condition. Mol Nutr Food Res. 2016, 60(7), 1695-706.

Q6. Figure 1a and 1b: since the energy density of each diet is ~4 kcal/gr, the energy intake for the 2 groups (daily food intake of ~12 g/day) should be ~48 kcal/day. In Figure 1b the mean energy intake is ~3.2 kcal/day. Please explain.

R6. Figure 1a and Figure 1b indicate energy intake and food intake, respectively. We have revised Figure 1a and 1b. Thank you.

Q7. Figures could be re-organised (for example the figures constituting Figure 5 can be merged into ones since the y-axis units are the same or figures 3a, 3b, 3c). Moreover, the authors can omit figures such as figure 1a, 1b, 1c, 1d, 1e, 2b, and describe their results in the text. 

R7. The y-axis units in Figure 3 and Figure 5 are the same, but figures are very different. Therefore, if these are presented as a single graph, it is difficult to observe the variation caused by allulose because of the relatively low y-axis figures in Figures 3B and 5B, C. Also, as your suggestion, we have revised results for each figure. Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Please, see the attached document.

Comments for author File: Comments.pdf

Author Response

MAJOR POINTS

Materials and methods

Q1. Possibly, 5% less sucrose in the diet could explain some of the effects observed in NDCA group compared to the NDC animals. The authors should have used the same percentage of sucrose in both diets and another sugar instead allulose (for example, tagatose or glucose at 5%) to carry out the comparisons.

R1. Thank you for a kind comment. In this study, there is a difference in kcal/g diet between the NDC and NDCA groups because of the difference in the amount of sucrose. For this reason, we conducted experiments under isocaloric diet conditions, and as a result energy intake was approximately equal in the NDC and NDCA groups. Therefore, anti-obesity effects observed in the NDCA group are not due to 5% less sucrose in the diet.

Results

Q2. The list of parameters that these authors have determined is quite extensive. However, it would be very useful to include the insulinemia and the glucose/insulin ratio, since this hormone plays a key role in lipid and glucose metabolism.

R2. Thank you for a kind comment. Your suggestion is very reasonable and considerable and we agree with reviewer’s comment. We will consider doing an additional experiment including plasma insulin and glucagon levels to more clearly address the mechanism involved in glucose metabolism for an additional paper.

Conclusions

Q3. As these authors comment in Conclusions, this study confirms the beneficial effects of allulose in an HFD, however, in this case, in the presence of non-dairy creamer. However, it is not clear for this Reviewer (or the authors do not explain it sufficiently) what is original in the present study. More saturated fat in HFD plus NDC than in HFD (since corn oil has been replaced by palm oil)? The presence of cis fatty acids instead of trans fatty acids, since hidrogenated palm oil was used as a non-dairy creamer? These authors should provide reports that show the differences between HFD vs HFD with NDC to reinforce the importance of the present study.

R3. Thank you for a kind comment. This study is focused on 1) anti-obesity effect of d-allulose in obese mice fed HFD with non-diary creamer and 2) its commercial availability. Non-dairy creamers, generally used as milk substitutes for coffee, often contain hydrogenated fat, which is artificially produced by converting unsaturated fatty acids to saturated fatty acids. In general, a high saturated fatty acid intake induces dyslipidemia, which is associated with the risk of cardiovascular disease via abnormal regulation of blood lipid levels, by elevating total cholesterol, TG, and low-density lipoprotein (LDL) cholesterol, and decreasing high-density lipoprotein (HDL) cholesterol. In Korea, there is a strong preference for instant coffee mix containing non-dairy creamers and sugar. Therefore, if allulose can ameliorate the deleterious effects of non-dairy creamers, it is desirable that the commercial value of allulose as a substitute for sugar is high in the process of making instant coffee. However, as your suggestion, we also consider that a comparative study with HFD and NDC is necessary, and we plan to carry out another study to analyze mRNA-seq for allulose supplemented mice. We may consider a comparative study with HFD and NDC based on your valuable comments. Thank you for kind suggestions.

MINOR POINTS

Materials and methods

Q4. The composition of non-dairy creamer is displayed in the text, however the percentages of fat and carbohydrate are not indicated for NDC and NDCA diets.

R4. We have newly inserted the percentages of fat and carbohydrate for NDC and NDCA diets at line 86. Thank you.

Q5. Why did they use 5% allulose?

R5. Regarding its safety matter, D-allulose is generally recognized as safe (GRAS) based on USDA regulations (GRN no. 647 and 693). Also, our previous study has reported that a maximum single dose and maximum total daily intake of D-Allulose in human are 0.4 g/kg·BW and 0.9 g/kg·BW, respectively [1]. When convert the 5% (w/w) dose of d-allulose used in a mouse to a dose based on surface area for humans [2], about 0.45g/kg·BW dose of d-allulose is calculated for a human equivalent dose, which is lower than the maximum total daily intake of D-Allulose in human (0.9 g/kg·BW). Moreover, 5% D-allulose in rats maintained blood glucose levels within a normal range [3] and reduced body fat accumulation by enhancing 24-h energy expenditure with enhanced fat oxidation and reduced carbohydrate oxidation [4, 5], and 5% dietary D-allulose in HFD-fed mice led to the normalization of the metabolic status of diet-induced obesity by altering lipid-regulating enzyme activities and their gene-expression level along with fecal lipids [6]. For these reasons, we used 5% allulose in this study. Thank you.

①    Han, Y.; Choi, B.R.; Kim, S.Y.; Kim, S.B.; Kim, Y.H.; Kwon, E.Y.; Choi, M.S. Gastrointestinal Tolerance of D-Allulose in Healthy and Young Adults. A Non-Randomized Controlled Trial. Nutrients. 2018, 10(12), E2010.

②    Reagan-Shaw, S.; Nihal, M.; Ahmad, N. Dose translation from animal to human studies revisited. FASEB J. 2008, 22(3), 659-61.

③    Hossain, M.A.; Kitagaki, S.; Nakano, D.; Nishiyama, A.; Funamoto, Y.; Matsunaga, T.; Tsukamoto, I.; Yamaguchi, F.; Kamitori, K.; Dong, Y.; Hirata, Y.; Murao, K.; Toyoda, Y.; Tokuda, M. Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Biochem Biophys Res Commun. 2011, 405(1), 7-12.

④    Matsuo, T.; Izumori, K. Effects of dietary D-psicose on diurnal variation in plasma glucose and insulin concentrations of rats. Biosci Biotechnol Biochem. 2006, 70(9), 2081-5.

⑤    Nagata, Y.; Kanasaki, A.; Tamaru, S.; Tanaka, K. D-psicose, an epimer of D-fructose, favorably alters lipid metabolism in Sprague-Dawley rats. J Agric Food Chem. 2015, 63(12), 3168-76.

⑥    Han, Y.; Han, H.J.; Kim, A.H.; Choi, J.Y.; Cho, S.J.; Park, Y.B.; Jung, U.J.; Choi, M.S. d-Allulose supplementation normalized the body weight and fat-pad mass in diet-induced obese mice via the regulation of lipid metabolism under isocaloric fed condition. Mol Nutr Food Res. 2016, 60(7), 1695-706.

Results

      Q6. How was FER ratio calculated? Please, indicate it.

R6. FER ratio was calculated as follows: body weight gain/energy intakes per day. We have newly inserted it at line 184-185. Thank you.

Q7. Are there any explanation for the increases observed in kidney and muscle weights after allulose treatment? What muscle are they using? Please, indicate it.

R7. In our previous study [1], HFD significantly decreased kidney and muscle weights compared to the normal diet. In this study, we presented kidney and muscle weights in g/100g body weight. Therefore, increased kidney and muscle weights by allulose are associated with the reduced weight by allulose, which is related to the normalization of kidney and muscle weights reduced by a high-fat diet. Also, the muscle weight includes soleus and gastronemius muscle. Thank you.

①    Choi, B.R.; Kwon, E.Y.; Kim, H.J.; Choi, M.S. Role of Synbiotics Containing d-Allulose in the Alteration of Body Fat and Hepatic Lipids in Diet-Induced Obese Mice. Nutrients. 2018, 10(11), E1797.

Q8. Please, indicate in the text at which figure are being referred when the results of UCP1 and SIRT in BAT are mentioned.

R8. We have newly inserted the results of UCP1 and SIRT in BAT at line 193-195. Thank you.

Q9. Fecal lipids are presented as mmol/day. Therefore, the authors should indicate in Material and methods how the faeces were obtained.

R9. We have revised y-axis units to “mmol/g feces”. Also, we have newly inserted methods about fecal collection at line 114-115.

      Q10. GOT and GPT are expressed as karmen/mL. What do these units mean?

R10. Karmen/mL is a common unit used to indicate levels of GOP and GPT [1-3], and GOP and GPT levels are expressed in International units or karmen unit. Karmen units/ml can be converted into International units per litre by multiplying by 3/6∙22 or 0∙48. Thank you.

①    Choi, B.R.; Kwon, E.Y.; Kim, H.J.; Choi, M.S. Role of Synbiotics Containing d-Allulose in the Alteration of Body Fat and Hepatic Lipids in Diet-Induced Obese Mice. Nutrients. 2018, 10(11), E1797.

②    Hollands, M.; Logan, J.E. An Examination of Commercial Kits for the Determination of Glutamic Oxaloacetic Transaminase (GOT) and Glutamic Pyruvic Transaminase (GPT) in Serum. Can Med Assoc J. 1966, 95(7), 303-6.

③    Hollands, M.A.; Logan, J.E. An Examination of Commercial Kits for the Determination of Glutamic Oxaloacetic Transaminase (GOT) and Glutamic Pyruvic Transaminase (GPT) in Serum. Canad. Med. Ass. J. 1966, 95, 303-306.

Discussion

      Q11. A study of 8 weeks should not be considered as a short-term dietary treatment.

R11. In the previous study [1], obese mice using HFD with 40 kcal% Fat showed immune system-related pathological changes such as insulin resistance, glucose intolerance and an elevated atherogenic index after week 16. Also, in another previous study [2], 5% dietary d-allulose led to the normalization of the metabolic status of diet-induced obesity by altering lipid-regulating enzyme activities and their gene-expression level along with fecal lipids in C57BL/6J mice fed HFD for 16 weeks. Therefore, in this study, the experimental period of 8 weeks is considered to be a short-term to observe the metabolic syndrome-related pathological changes such as insulin resistance. However, we agree to your opinion to a certain extent, and thus we have deleted the word “short-term” in this manuscript. Thank you.

①    Do, G.M.; Oh, H.Y.; Kwon, E.Y.; Cho, Y.Y.; Shin, S.K.; Park, H.J. et al. Long-term adaptation of global transcription and metabolism in the liver of high-fat diet-fed C57BL/6J mice. Mol. Nutr. Food. Res. 2011, 55, S173–S185.

②    Han, Y.; Han, H.J.; Kim, A.H.; Choi, J.Y.; Cho, S.J.; Park, Y.B.; Jung, U.J.; Choi, M.S. d-Allulose supplementation normalized the body weight and fat-pad mass in diet-induced obese mice via the regulation of lipid metabolism under isocaloric fed condition. Mol Nutr Food Res. 2016, 60(7), 1695-706.

Q12. An increase in UCP1 in BAT (and thermogenesis) also could help to explain the decrease in triglyceridemia. Please, indicate it.

R12. We have newly inserted the information about the relationship between an increase in UCP1 in BAT and triglyceride at line 355-357. Thank you.

Q13. Both a decrease in NPC1L1 and an increase in SRB1 can affect cholesterol absorption and, however, the level of cholesterol in faeces does not change between the two groups. How can it be explained?

R13. In the present study, the mRNA expressions of SRB1 and NPC1L1 in the small intestine were significantly decreased by D-allulose supplementation in the NDCA group. However, mRNA expression levels of CD36, ABCA1, and FAS in the small intestine were not different. Also, fecal and plasma cholesterol levels were not changed by D-allulose supplementation in this experiment. These results may be related to a relatively short duration (8 weeks) of this study when compared to a previous 16-week trial in C57BL/6J mice. In a previous 16-week trial in C57BL/6J mice, D-allulose supplemented mice with HFD revealed that small intestinal mRNA expression of factors related to lipid-regulatory absorption, such as CD36, ATP-binding cassette sub-family G member 5, ATP-binding cassette sub-family G member 8, and fatty acid transporter 4, was significantly decreased and the fecal cholesterol level was increased [1]. Therefore, long-term supplementation of D-allulose for at least 16 weeks or longer period in mice fed HFD would be effective in increasing fecal cholesterol level and reducing plasma cholesterol level.

①    Han, Y.; Han, H.J.; Kim, A.H.; Choi, J.Y.; Cho, S.J.; Park, Y.B.; Jung, U.J.; Choi, M.S. d-Allulose supplementation normalized the body weight and fat-pad mass in diet-induced obese mice via the regulation of lipid metabolism under isocaloric fed condition. Mol Nutr Food Res. 2016, 60(7), 1695-706.

Author Response File: Author Response.pdf

Round  2

Reviewer 1 Report

Thank you for your response. I find the paper interesting and the scope matches the topics covered by the journal.

Author Response

Thank you for your kind comment.

Reviewer 2 Report

Please, see enclosed document.

Comments for author File: Comments.pdf

Author Response

Question. As indicated in the Instructions for Authors:
“SI Units (International System of Units) should be used. Imperial, US customary and other units should be converted to SI units whenever possible.”
Therefore, in Table 2, GOT and GPT should be expressed in International Units, not as karmen/mL.

Answer: We ahve revised Unit of GOT and GPT to "Inthernational Units (IU/L)". Thank you.