Euglycemic Hyperinsulinemia Lowers Blood Pressure and Impedes Microvascular Perfusion More Effectively in Persons with Cardio-Metabolic Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled "Euglycemic hyperinsulinemia lowers blood pressure and impedes microvascular perfusion more effectively in persons with cardio-metabolic disease" quantifies, for the first time, the paradoxical microvascular vasoconstriction in response to insulin among individuals with cardio-metabolic disease (CMD). It also reports a new observation that insulin lowers central aortic pressure in humans, particularly in those with CMD. The authors propose a novel mechanism linking impaired baroreflex responses to reduced microvascular perfusion. Additionally, the use of contrast-enhanced ultrasound (CEU) across diverse CMD groups under tightly controlled conditions strengthens the study's impact. Together, these findings expand the understanding of vascular insulin resistance and its role in CMD pathophysiology.

Despite its strengths, the manuscript has notable limitations. 
First, the healthy control group was significantly younger than the cardio-metabolic disease (CMD) groups, introducing potential age-related confounding in vascular responses. 
Second, although the sex distribution across groups was balanced, the study did not stratify analyses by sex, despite known sex differences in vascular insulin sensitivity.
Third,while functional vascular outcomes were rigorously assessed, the absence of biochemical markers of endothelial function (e.g., nitric oxide, endothelin-1) limits mechanistic insight. 
Finally, by excluding individuals with overt cardiovascular disease, the study may underestimate the true prevalence and extent of paradoxical microvascular vasoconstriction in the broader CMD population.

The study is scientifically sound and offers valuable insights, but it requires additional analysis and, more comprehensive discussion.

Author Response

Response to reviewer #1 general comments

We thank the reviewer for his/her thoughtful critique. We were pleased that they found these studies “expand the understanding of vascular insulin resistance and its role in cardio metabolic disease pathophysiology”.

They also cited limitations of our findings. They expressed concern with regard to the control group being significantly younger than the CMD groups which introduces the potential for age-related differences confounding in vascular responses. We agree, but we felt that it was important to get a truly vascularly healthy group to define the “normal” vascular responses to insulin and in a practical way this is most readily done by studying young adults. The reviewer also would like to have data on the effects of sex. We agree this is an important issue. However, at the outset we recognized this would require a significant expansion in the number of subjects required in each group of CMD. As we continue to do these types of studies our expectation is that we will accrue substantially more subjects among several groups (type 2 diabetes, and metabolic syndrome) and perhaps be able to address the issue of gender more appropriately. We have added several sentences to the discussion to simply highlight the importance of gender in vascular function and that it is deserving of further study using the tools we have implemented here.

We note that both men and women were included, though not in equal numbers, in each of the five study groups. We did not see any sex-based differences in either the microvascular or blood pressure responses. However, we recognize that the study was not powered for such a subgroup analysis. To truly resolve the question sex-based differences in insulin’s action on blood pressure or microvascular perfusion will require larger prospectively designed studies.

Comment #3-referee would like to see more data on biochemical markers of endothelial function.

Response #3-to meaningfully measure biochemical responses by skeletal muscle microvasculature requires invasive procedures (e.g. arterial venous sampling, or endothelial cell isolation), or use of inhibitors (e.g. L-NMMA). These techniques warrant focused study now that the phenomena of microvascular insulin resistance has been more clarified by the work presented in this manuscript.

Comment #4 - Finally, by excluding individuals with overt cardiovascular disease, the study may underestimate the true prevalence and extent of paradoxical microvascular vasoconstriction in the broader CMD population.

Response #4-We did not include subjects with overt cardiovascular disease. We agree this may lead us to underestimate the prevalence of paradoxical microvascular constriction in response to insulin in CMD. This deliberate exclusion was predicated on the knowledge that individuals with overt cardiovascular disease are typically on multiple medications that impact vascular function and insulin responses and it is much more difficult to get a “clean” biologic response uncontaminated by drug effects.

Finally, we thank the referee for concluding that the studies were scientifically sound and offer valuable insights, and we appreciate his/her enthusiasm for more comprehensive data on this topic.

 

Reviewer 2 Report

Comments and Suggestions for Authors

This is a professionally written and scientifically sound manuscript that compares the vasodilatory response to euglycemic insulin in healthy controls vs patients with cardiometabolic diseases. The data are robust, the statistical analysis is sound, and the conclusions are well supported. Nevertheless, there are areas where the manuscript could be strengthened:

1. The control subjects were younger and had lower BMI than the patient groups. As both age and adiposity affect vascular insulin sensitivity and endothelial function, this might introduce potential confounders that ultimately complicate interpretation. The authors should discuss this limitation or adjust statistics through a sensitivity analysis that accounts for such discrepancy.
2. The manuscript highlights a duality in insulin responsiveness, addressing the occurrence of responder vs. non-responder phenotypes. This intriguing finding might deserve further exploration. The authors are encouraged to launch hypotheses on the potential underlying mechanisms, that might include genetic polymorphisms, variability in endothelial NO synthase activity, or divergences in sympathetic nervous system responsiveness.
3. The rationale for selecting a 2-h insulin infusion and observation period is not provided. Clarify whether this duration was based on prior literature, pilot data, or physiological reasoning.
4. Given the known sex-specific differences in endothelial function and cardiometabolic risk, the manuscript would benefit from an analysis of sex-based differences.
5. Consider replacing tables with graphs that visually depict the observed changes, which is predicted to enhance data clarity and reader engagement.
6. The observation that diastolic pressures remained unchanged in both groups is noteworthy. Consider providing a physiological explanation for this outcome.
7. Could differential natriuresis between controls and patients have contributed to the observed vascular responses? If urinary sodium data were collected, the authors should consider presenting it; if not, this limitation should be acknowledged.

Author Response

Reviewer #2-General comments-This is a professionally written and scientifically sound manuscript that compares the vasodilatory response to euglycemic insulin in healthy controls vs patients with cardiometabolic diseases. The data are robust, the statistical analysis is sound, and the conclusions are well supported. Nevertheless, there are areas where the manuscript could be strengthened:

 

Response to reviewer #2 general comments-we appreciate the reviewers comments that the manuscript is professionally written and scientifically sound that the data are robust, the statistical analysis is sound conclusions are well supported. Below we respond to some specific shortcomings noted by reviewer #2.

 

Comment #1- The control subjects were younger and had lower BMI than the patient groups. As both age and adiposity affect vascular insulin sensitivity and endothelial function, this might introduce potential confounders that ultimately complicate interpretation. The authors should discuss this limitation or adjust statistics through a sensitivity analysis that accounts for such discrepancy.

Response to comment #1-we agree with the reviewer that the age differences between the control and CMD subjects could be contributing to the observed differences in vascular function. As we indicated in responses to reviewer #1, we felt it was most important to have control data in entirely healthy individuals. We agree that age differences could be contributing to differences in vascular function noted between the control and CMD groups. We have expanded the discussion of this limitation. We anticipate that over the coming years we will expand the age range of the controls and the CMD subjects to be able to strengthen the comparison. We would prefer that empirical approach over adjustments statistical sensitivity.

 

Comment #2-The manuscript highlights a duality in insulin responsiveness, addressing the occurrence of responder vs. non-responder phenotypes. This intriguing finding might deserve further exploration. The authors are encouraged to launch hypotheses on the potential underlying mechanisms, that might include genetic polymorphisms, variability in endothelial NO synthase activity, or divergences in sympathetic nervous system responsiveness.

 

Response to comment #2-we agree that the duality of insulin responsiveness is an intriguing finding. We have elected not to include discussion of the many possible factors potentially contributing to this as it would be too speculative absent more mechanistic data.

Comment #3 - The rationale for selecting a 2-h insulin infusion and observation period is not provided. Clarify whether this duration was based on prior literature, pilot data, or physiological reasoning.

 

Response to comment #3-We have previously studied the time course for insulin's effects on the microvascular perfusion. We found both in healthy rodents and humans that microvascular recruitment is detectable within 15 minutes and endures for at least several hours. The two hour time frame allows for stable detection of metabolic insulin sensitivity and has been used for such purpose in multiple studies. We have indicated in the revised methods the reason for selecting the time course. We have added the following sentence to the methods. We chose two hours of insulin infusion for vascular and metabolic measurements as prior studies have shown that insulin’s actions reach a near study-state within this interval.

 

Comment #4-Given the known sex-specific differences in endothelial function and cardiometabolic risk, the manuscript would benefit from an analysis of sex-based differences.

 

Response to comment #4-we agree completely with this comment but the study was not nearly powered to detect differences in either vascular or metabolic insulin resistance based on sex. As we indicated in response to reviewer #1 we have modestly expanded the discussion of this topic to recognize its importance but believe it will require considerably more study.

 

Comment #5-Consider replacing tables with graphs that visually depict the observed changes, which is predicted to enhance data clarity and reader engagement.

 

 

Response to comment #5-we appreciate the suggestion and do that routinely for in person presentation of data. However, we preferred the precision of the tabular format for printed material. All a matter of taste.

 

Comment #6-The observation that diastolic pressures remained unchanged in both groups is noteworthy. Consider providing a physiological explanation for this outcome.

Response #6 We have added the following sentences to the discussion: It is noteworthy that while declines in systolic pressure in the CMD subjects were significant, there were also non-significant downward trends of both brachial and central aortic diastolic pressures. The better preservation of diastolic pressure may be related to the fluid volume replacement which was occurring with the glucose and insulin infusions.

 

Comment #7 Could differential natriuresis between controls and patients have contributed to the observed vascular responses? If urinary sodium data were collected, the authors should consider presenting it; if not, this limitation should be acknowledged.

Response #7-this is an interesting thought. Unfortunately, neither urine volume nor urinary sodium were measured during the insulin infusion. Subjects did not receive any intravenous saline solution. However, their sodium balance of the day or two prior to study is not controlled. Lacking relevant data, we have not altered the manuscript to discuss this issue.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors.

Thank you for adequately addressing all concerns:

1. Age difference in controls: Justified as necessary to define normal vascular responses; limitation acknowledged in the discussion.

2. Sex differences: Authors clarify inclusion of both sexes and note the study wasn’t powered for sex-based analysis, which is now highlighted in the revised discussion.

3. Endothelial biomarkers: Explained as requiring invasive methods beyond this study’s scope, but noted for future work.

4. Exclusion of overt CVD: Reasonable to avoid confounding by medications; limitation acknowledged.

The manuscript is scientifically sound, the responses are appropriate, and the revisions are clear.

Recommendation: Accept without further revision.

Reviewer 2 Report

Comments and Suggestions for Authors

All my concerns have been adequately considered by the Authors, with no exception. While some of them were followed, others were not, but this is part of the freedom of Science.