To Reviewer #3

In this manuscript, Passarelli and Fabres-Machado aimed at summarizing the current

knowledge about AGEs-induced and ER/inflammation-mediated modulation of the expression of

GLUT4, as a marker of glycemic homeostasis and of cardiovascular disease (CVD)

development/progression. Although the topic is of interest, and well written overall, the

manuscript is unnecessarily long and does not go to the point announced in the title:

“Endoplasmic Reticulum Stress and Inflammation: Participation in Advanced Glycation End

Products-Mediated Pathophysiology of Diabetes Mellitus”. Furthermore, the main conclusions

get buried in a lengthy “conclusions” section. Specific issues are listed below.

Thank you for the comments that will discussed below.

According to Reviewer #2’s comment, the title was revised to point out the AGE effects

and the participation of ER-stress in GLUT4 regulation and atherogenesis development in DM.

Besides, as suggested, the “Concluding Remarks” section was shortened.

Main issues:

  1. Although most sections in this manuscript are well documented, many of them deviate

attention from the declared goal of the review making it unnecessarily long. The authors should

focus on discussing the relationship between AGEs in the pathophysiology of diabetes.

Ok. Some items and subitems of the manuscript were merged; from items 4 to 7, only 3

remained (specifying their subjects) as follows:

4. AGEs, ER-stress and inflammation related regulation of GLUT4 expression

5. AGEs, ER-stress and inflammation related atherogenesis and CVD development

 6. AGEs, ER-stress and inflammation in other DM-related diseases

Importantly, the manuscript focuses only on the AGEs effects (upon GLUT4 regulation

and CVD) mediated by UPR and/or inflammation (which is the scope of this special issue).

2. /In line xx, the authors state, somewhat simplistically, that “...whereas T2D subjects develop

impaired insulin secretion, T1D subjects develop IR.” To back this assertion they cite only 3

references. The issue is much more complex than this and should not be treated lightly.

The main idea is to point out that IR is not only a feature of T2D, but it is also present in

hyperinsulinized T1D subjects. However, the manuscript is not the place to discuss that in detail.

By searching “diabetes type 1 and insulin resistance”, PubMed displays 3,029 results, dating

back to the fifties. It should be reminded that the first descriptions of insulin resistance were in

uncontrolled T1D patients who, despite the very high doses of insulin, did not achieve a

reasonable glycemic control (Please see: “Extreme insulin resistance in diabetic coma: report of

a fatal case of juvenile diabetes treated with 7,555 units of insulin in 45 hours.” ROSE WR,

KAPLAN R, PICARD HN. Diabetes. 1953, 2:462-4). Nowadays, it is well known that insulin-

treated T1D subjects present a plasma insulin concentration at least 3- to 4-fold higher than the

normal values, because of the route of insulin injection (sc), which does not provide hormone to

the liver through the portal vein. Only later, in the 1980s, the knowledge that insulin resistance in

apparently healthy patients should be a cause of the development of T2D became consolidated,

including the characterization of the involved molecular mechanisms.

The references cited in the manuscript include:

1) A systematic review and meta-analysis (including 38 publications) evaluating IR assessed by

glucose clamp in TD1 subjects (Donga et al., 2015);

2) An experimental study in rats demonstrating the molecular mechanisms involved in the

development of IR in T1D rats treated with insulin (as the best glycemic control is achieved, the

most hyperinsulinemic and insulin resistant the rats become (Okamoto et al., 2011).

Because of that, the increased use of insulin sensitizers has been considered as an

adjunctive therapy in T1D subjects (for a review: Priya and Kalra. A review of insulin resistance

in type 1 diabetes: Is there a place for adjunctive metformin? Diabetes Ther. 2018, 9:349-361).

However, once again we point out that this manuscript is not the suitable place to discuss

insulin resistance in T1D in detail.

3. Why the authors need a section on the “state of the art in DM and its complications”, which

again, is oversimplified and does not contribute to the stated goal of the manuscript.

Furthermore, why they even go into discussion the origin and causes of the metabolic

syndrome? All this, once again, deviates attention from AGEs and the complications of diabetes

mellitus. Excellent reviews are available on these topics.

We need this section to provide a conceptual support to the topics that will be discussed

in the light of ER- and inflammatory-stress mediated regulation of 1) GLUT4 and IR and 2)

atherogenesis and CVD; as well as the AGEs participation. No other topic than that related to

diabetic pathophysiology was included in this section. Regarding the origin of the metabolic

syndrome, it has been cited because of the Banting Lecture by Reaven in 1988 is a hallmark in

the knowledge of the association of IR and CVD (both fundamental topics of this manuscript).

 
4. The section on GLUT4 has the same problems mentioned in item 3 above even though it is

part of the topic. Perhaps the authors should focus on the relationships between AGEs and

GLUT4.

  Ok. Description of the sections was revised.

5. Figure 1, on the other hands, suggests that IR and even DM2 relates mainly or only to GLUT4

decreased translocation to the cell membranes with the consequent decrease in glucose uptake.

This is another conceptual error in that it ignores the role of insulin in the suppression of

glucose production by the liver, a key contributor to the hyperglycemia of DM2.

Although the legend of Figure 1 does not state the target tissues, the Figure clearly shows

that the described GLUT4-related regulation was restricted to adipose and muscle tissues, targets

in which AGEs-induced and ER-stress/inflammation-mediated effects were extensively

investigated.

Regarding the contested relevance of the role of GLUT4 in IR, we regret to say that the

Reviewer is making statements that, in reality, we have not done. At the beginning of section

2.1., it read: “Insulin resistance involves complex and variable disorders in insulin signaling

pathway, which eventually culminates in impaired glucose utilization by muscle and adipose

tissues, contributing to glycemic control impairment [11,13].” In this comment, we do not ignore

the involvement of other mechanisms in IR, including hyperinsulinemia, increased hepatic

glucose outflow, increased renal glucose resorption, among others…

Besides, we end this paragraph stating: “Because of that, in the present manuscript,

muscle and adipose tissues regulation of SLC2A4/GLUT4 is reviewed as a marker of glycemic

control in DM.” Thus, reduced GLUT4-mediated glucose uptake in adipose tissue is one (among
others) marker of insulin resistance and impaired glycemic control, the same way that several

diseases present dozens of markers.

Additionally, I cannot help complaining about the lack of politeness of this comment,

stating that the importance of GLUT4 in IR “is another conceptual error in that it ignores the

role of insulin in the suppression of glucose production by the liver”. I am sure the Reviewer

would never react like this in relation to Dr. Amira Klip, concerning her iconic review about

GLUT4 published in JBC (2019).

Please, observe pieces of the abstract of the Review.

………………………
 Finally, concerning the incontestable role of hepatic glucose production in diabetic

insulin resistance (but not addressed in the present manuscript), we would like to point out that

the isolated effect of AGEs in hepatic insulin resistance has not been demonstrated yet.

Conversely, the “hormetic” effect was clearly reported in detail (see below).

6. Why is section 7.2 “ER-stress and inflammation in cancer” relevant to this manuscript stated

goal(s)?

Firstly, because cancer is one of the foci of this special issue entitled: “Unfolded Protein

Response in Inflammation and Cancer”. Secondly, because conditions related to DM/AGEs such

as breast cancer and the antitumorigenic effect of metformin, both involving

ER-stress/inflammation mechanisms, would significantly contribute to this topic. Please observe

that, at the beginning of Section 7 (now 5) we stated: “Here, we have selected two conditions for

a brief review, based on their association with DM and AGE: the non-alcoholic fatty liver

disease (NAFLD) and cancer.