d-Allulose Ameliorates Skeletal Muscle Insulin Resistance in High-Fat Diet-Fed Rats

Round 1

Reviewer 1 Report

Gou and colleagues reported on the effect of D-allulose on insulin resistance in a high-fat diet (HFD)-induced obese model. Six-week-old male Wistar rats were divided into three groups: CD (control), HFD (HFD+ cellulose) and HFA (HFD + D-allulose). The HE-clamp method was used for assessing the impact of D-allulose on systemic and skeletal muscle insulin resistance. More detailed, effects of D-allulose were investigated on body weight, white adipose tissue, glucose metabolism, insulin sensitivity, TNFa, adiponectin level, insulin signalling and glucose transporter 4. The authors demonstrated that D-allulose possesses protective effects against HFD-induced systemic and skeletal muscle insulin resistance.

The manuscript is well written, results are well documented and displayed in figures. However, there a number of issues that need to be addressed before the work is acceptable.

Results

The authors mentioned in the introduction that studies on the skeletal muscle, a critical tissue responsive to insulin, are lacking. Still, in this context, only under point 2.5 (line 147) the skeletal muscle was under the focus of investigations. Also the title implies main focus on skeletal muscle insulin resistance, although most finding related to systemic insulin resistance and characterization of adipose tissue!?

Only Irs1, p-Akt and Glut4 were included to investigate muscle insulin resistance. However, there are any changes analysed in the insulin receptor expression or distribution? Furthermore, GLUT4 is known to be change its intracellular distribution under insulin. Have the authors analysed these alterations in the intracellular distribution of GLUT4 since under increased insulin more GLUT4 is integrated into the membrane? In addition, PI-3-Kinase plays an important role in metabolic insulin signaling pathways! MAP-kinase cascade mediates insulin-dependent gene regulation.

Figures

Figures 1-4 are too small, they should be enlarged to better distinguish data groups and data points.

In Western blot images (Figs. 4 and 5) or the respective legends the molecular weight (kDa) should be indicated.

Western blot images are not convincing in many cases, in general n=3-4 bands per group (CD, HFC, HFA) should be presented in images.

Material and Methods

At which daytime points the measurements were done, because blood glucose and insulin underlying diurnal/circadian changes as displayed in daily fluctuations!

Author Response

Manuscript. Number: molecules-1385374

Title: d-Allulose Ameliorates Skeletal Muscle Insulin Resistance in High-Fat

 

We are grateful to reviewers for the critical comments and valuable suggestions that have helped us improve our paper considerably. As indicated in the following responses, we have taken all these comments and suggestions into account in the revised version of our manuscript (The corresponding revisions are highlighted in yellow.)

 

Reviewer #1 major comment 1: The authors mentioned in the introduction that studies on the skeletal muscle, a critical tissue responsive to insulin, are lacking. Still, in this context, only under point 2.5 (line 147) the skeletal muscle was under the focus of investigations. Also the title implies main focus on skeletal muscle insulin resistance, although most finding related to systemic insulin resistance and characterization of adipose tissue!?

 

Author response to Reviewer #1 Major comment 1: As the reviewer pointed out, we focused on skeletal muscles because there is still a lack of direct evidence regarding the effect of d-allulose on insulin resistance in skeletal muscles. Our results from the high-dose clamp (the second step of the HE clamp test) (Fig. 3) indicate that d-allulose can improve muscle-specific insulin resistance induced by HFD. Fig.4 and Fig.5 illustrate inflammatory cytokines released from adipose tissue changed the insulin signaling pathway activation in skeletal muscles. IRS-1 is the crucial regulator of the level of insulin signal activation. We believe this sequential picture of the signaling is the most novel findings in our paper.

 

We changed the first paragraph of the discussion (Line 170-181 in the revised manuscript) to explain further why we focus on the skeletal muscle and the significance of our study as below.

 

In this study, the HE-clamp test was used to show that d-allulose supplementation ameliorated insulin resistance in the HFD group to the level in the CD group. The 2^nd step of the HE-clamp test specifically evaluates skeletal muscle insulin resistance because the high-dose insulin infusion sufficiently suppresses hepatic gluconeogenesis. There is still a lack of direct evidence regarding the effects of d-allulose on insulin resistance in skeletal muscles. Furthermore, the study on the interplay between major insulin target tissues, muscles, liver, and fats, is required to understand the underlying mechanisms of insulin resistance. We showed that d-allulose administration restored the balance between proinflammatory and anti-inflammatory adipokines in HFD-fed rats. The subsequent changes in insulin signaling, especially IRS-1, indicate that the link between the adipose tissue and skeletal muscle plays a critical role in the improvement of HFD-induced muscular insulin resistance by d-allulose in Wistar rats. 

 

Reviewer #1 major comment 2: Only Irs1, p-Akt and Glut4 were included to investigate muscle insulin resistance. However, there are any changes analysed in the insulin receptor expression or distribution? Furthermore, GLUT4 is known to be change its intracellular distribution under insulin. Have the authors analysed these alterations in the intracellular distribution of GLUT4 since under increased insulin more GLUT4 is integrated into the membrane? In addition, PI-3-Kinase plays an important role in metabolic insulin signaling pathways! MAP-kinase cascade mediates insulin-dependent gene regulation.

 

Author response to Reviewer #1 Major comment 2: To confirm the HE clamp's finding that d-allulose restored insulin signal sensitivity in skeletal muscle of HFD-fed rats and to clarify the mechanism, we examined the insulin signaling induced by insulin injection. As factors controlling insulin signal activation, we think IRS-1 phosphorylation and Glut-4 expression play a major role in determining insulin signaling. Accordingly, we investigated these molecules. We also addressed the possibility that other insulin signal-related molecules the reviewer indicated can be regulated differentially by d-allulose.

 

We added the second paragraph of the discussion (Line 182-195 in the revised manuscript) to explain why we examined Akt, IRS-1, and Glut-4 in the present study as below. We added two new references 11 and 15.

 

11. da Silva Rosa, S.C.; Nayak, N.; Caymo, A.M.; Gordon, J.W. Mechanisms of muscle insulin resistance and the cross-talk with liver and adipose tissue. Physiol Rep 2020, 8, e14607.
12. Garvey, W.T.; Maianu, L.; Zhu, J.H.; Brechtel-Hook, G.; Wallace, P.; Baron, A.D. Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance. J. Clin. Invest. 1998, 101, 2377-2386.

 

The impaired insulin signaling cascade activation and impaired Glut-4 function is the primary defect in skeletal insulin resistance [11]. In the present study, we focused on IRS-1 phosphorylation and Glut-4 expression. d-allulose increased IRS-1 tyrosine phosphorylation and decreased serine 307 phosphorylation. These results are consistent with suppressed TNF-α expression and phosphorylation of JNK and the increase in adiponectin secretion. Adiponectin reduces serine 307 phosphorylation of IRS-1 to sensitize insulin signaling in the muscle [12]. The Glut-4 expression was also increased by d-allulose administration. A study by Lee, D et al. demonstrated the mRNA upregulation of Glut-4, IRS-1, phosphatidylinositol-4,5-bisphosphate 3 (PI-3) kinase catalytic subunit alpha, and AKT2 in c57BL/KsJ-db/db mice [13]. In our analysis of protein expression levels, Glut-4 expression was increased by d-allulose treatment, but not IRS-1 or Akt. Glut-4 translocation defects were proposed as the mechanism of insulin resistance [14]. The possibility that d-allulose may change a specific step of the insulin signaling cascade should be investigated in a future study.

 

 

We deleted the part of the third paragraph to avoid the duplicate explanation.

 

We used the HE-clamp method to demonstrate for the first time that d-allulose administration ameliorates HFD-induced insulin resistance. Notably, the HE-clamp showed similar insulin sensitivity between the HFA and standard CD groups, despite the presence of higher visceral fat in the HFA group than in the CD group. Our results are consistent with those of others demonstrating that d-allulose in drinking water improves insulin sensitivity without affecting body weight or fat mass [16,17], indicating its mechanism is independent of weight loss or fat reduction. In the two-step HE-clamp test, low-dose insulin infusion GIR refers to insulin sensitivity of the liver and peripheral tissues [18], whereas high-dose insulin infusion GIR refers to the insulin sensitivity index of the skeletal muscle [19]. Although d-allulose supplementation was shown to increase the mRNA expression of IRS-1, Akt, and Glut-4 in the muscle [15], ours is the first study to focus on the protein levels of these signaling molecules. We found that the phosphorylation expression of IRS-1 and Akt decreased after HFD, so did the expression of Glut4 protein. Interestingly, after d-allulose supplementation, the above changes were restored to the level of the chow diet group.

 

 

Reviewer #1 comment on figures 1: Figures 1-4 are too small, they should be enlarged to better distinguish data groups and data points.

 

Author response to Reviewer #1 on figures 1: we enlarged the figure 1-4, and replaced them in the revised manuscript.

 

Reviewer #1 comment on figures 2: In Western blot images (Figs. 4 and 5) or the respective legends the molecular weight (kDa) should be indicated.

 

Author response to Reviewer #1 on figures 2: We added the molecular weight next to the western blot images.

 

Reviewer #1 comment on figures 3: Western blot images are not convincing in many cases, in general n=3-4 bands per group (CD, HFC, HFA) should be presented in images.

 

Author response to Reviewer #1 on figures 3: We presented the representative image. We ran samples only in duplicate. We will show all the images we used for the quantitative analysis as supplementary figures.

 

Reviewer #1 comment on Material and Methods 1: At which daytime points the measurements were done, because blood glucose and insulin underlying diurnal/circadian changes as displayed in daily fluctuations!

 

Author response to Reviewer #1 comment on Material and Methods 1: We added the description about the lighting condition (Line 262 in the revised manuscript) and the time points of the measurement of each test (Line 277-278, 283, 289-290, 303-304).

 

 

Thank you very much for your valuable comments and help.

Author Response File: Author Response.docx

Reviewer 2 Report

The work by Gou and colleagues explores the effects of D-Allulose in a model of HFD in rats.

The authors provided evidences that D-Allulose improved metabolic performances as shown by better responses after ipGTT, ipITT and HE clamp.

 

Importantly the authors showed that D-Allulose improved insulin signaling in skeletal muscle, that was impaired after the HFD.

 

The authors provided solid evidences, but the work has some issues that should be addressed:

• English must be reviewed carefully by a professional or native English proof-reader;
• Line 210: “miniaturization of fat” is not appropriate
• Line 299: “muscle tissue was harvested from the inferior vena cava”. What did the authors mean with this statement? Please explain.
• In Figure 5 the authors should also show the basal level of phosphor proteins to appreciate the effect of insulin.
• To improve a little bit the mechanistic part of the work the authors could perform some staining for lipid and glycogen (ORO and PAS, respectively) on muscle sections.
• The authors performed insulin signaling studies on soleus, which is a slow twitch, oxidative muscle. While there are no meaningful reason that other muscle, such as Tibialis, should perform differently, a comment on this matter would be relevant in the discussion.
• Do the authors have any data or any comment on muscle mass on the three different experimental groups?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have addressed the most issues and suggestions and improved the manuscript.

I have only found minor mistakes, which should be addressed. Please check the manuscript carefully.

Line 172: Delete space in the word “target”.

Line 183: Delete space between words TNF-alpha and expression.

Line 185: Correct “Lee D et al.” into “Lee et al.”

e.g. lines 390 and 447: Correctly use the citation style (Journal in italics).

Include reference 10 (Natsume et al. 2021) as published!