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. 2023 Jun 28;18(6):e0286887. doi: 10.1371/journal.pone.0286887

Outcomes and clinical implications of intranasal insulin on cognition in humans: A systematic review and meta-analysis

Sally Wu 1,2,‡, Nicolette Stogios 1,2,‡, Margaret Hahn 1,2,3,4, Janani Navagnanavel 5, Zahra Emami 6, Araba Chintoh 1,2,3, Philip Gerretsen 2,3,7, Ariel Graff-Guerrero 2,3,7, Tarek K Rajji 2,3,8, Gary Remington 1,2,3, Sri Mahavir Agarwal 1,2,3,4,*
Editor: Belgin Sever9
PMCID: PMC10306194  PMID: 37379265

Abstract

Background

Aberrant brain insulin signaling has been posited to lie at the crossroads of several metabolic and cognitive disorders. Intranasal insulin (INI) is a non-invasive approach that allows investigation and modulation of insulin signaling in the brain while limiting peripheral side effects.

Objectives

The objective of this systematic review and meta-analysis is to evaluate the effects of INI on cognition in diverse patient populations and healthy individuals.

Methods

MEDLINE, EMBASE, PsycINFO, and Cochrane CENTRAL were systematically searched from 2000 to July 2021. Eligible studies were randomized controlled trials that studied the effects of INI on cognition. Two independent reviewers determined study eligibility and extracted relevant descriptive and outcome data.

Results

Twenty-nine studies (pooled N = 1,726) in healthy individuals as well as those with Alzheimer’s disease (AD)/mild cognitive impairment (MCI), mental health disorders, metabolic disorders, among others, were included in the quantitative meta-analysis. Patients with AD/MCI treated with INI were more likely to show an improvement in global cognition (SMD = 0.22, 95% CI: 0.05–0.38 p = <0.00001, N = 12 studies). Among studies with healthy individuals and other patient populations, no significant effects of INI were found for global cognition.

Conclusions

This review demonstrates that INI may be associated with pro-cognitive benefits for global cognition, specifically for individuals with AD/MCI. Further studies are required to better understand the neurobiological mechanisms and differences in etiology to dissect the intrinsic and extrinsic factors contributing to the treatment response of INI.

1. Introduction

For decades, the majority of insulin research has primarily focused on the action of insulin in peripheral tissues as the brain was long considered to be an insulin-insensitive organ. Insulin receptors are expressed widely throughout the brain, with notable concentrations in the olfactory bulb, cerebral cortex, striatum, hypothalamus, and hippocampus, thereby highlighting the role of insulin in processes such as cognition, appetite, and glucose regulation [1–4].

Central administration of insulin has been shown to play a prominent role in regulating cognition [5, 6]. This may be through insulin signalling pathways modulating processes of long-term potentiation and long-term depression or triggering release of various neurotrophic factors to promote neuronal survival [7, 8]. The role of CNS insulin on cognition has been further understood through several studies that suggest attenuated insulin action in the brain may be a critical factor in the development of age-related cognitive decline and Alzheimer’s disease (AD). To this point, the central insulin resistance hypothesis asserts that lack of insulin responsivity in the brain may be implicated in the pathogenesis of various clinical conditions including Parkinson’s disease, schizophrenia, dementia, depression, and type 2 diabetes mellitus (T2DM) [9–13]. As such, aberrant brain insulin signaling has been posited to lie at the crossroads of metabolic and cognitive disorders.

As impaired CNS insulin function has been observed in many pathologies, greater focus has been placed on using central insulin as a potential treatment intervention. Intranasal insulin (INI) is a non-invasive technique that delivers insulin directly to the CNS while limiting peripheral spill over [14]. A recent systematic review and meta-analysis sought to evaluate the effectiveness of INI on cognition in patients with mild cognitive impairment (MCI) or dementia; notably, results did not reveal a significant effect of INI on cognitive functioning compared to placebo in these populations [15]. However, the effect of INI on cognition across all population groups over the last two decades remains to be systematically reviewed. The objective of this systematic review and meta-analysis is to synthesize available evidence evaluating the effects of INI on cognition in diverse patient populations and cognitively unimpaired, healthy individuals.

2. Materials and methods

We conducted a systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology and reporting standard. A review protocol was submitted to the PROSPERO international database of prospectively registered systematic on July 22, 2021 (PROSPERO #CRD42021262478). Our original search was conducted on July 22, 2021, and an updated search was completed on February 11, 2023.

2.1. Search strategy

MEDLINE, Embase, PsycINFO, CINAHL, CENTRAL, ClinicalTrials.gov, and the ICTRP Search Portal were systematically searched for relevant peer-reviewed studies to be included in the review (S1 Table in S1 File).

2.2. Study selection

Three authors (NS, SW, ZE) independently screened all identified articles from the systematic search; agreement from a minimum of two authors was required for a study to be either included or excluded. Any disagreements were resolved by re-checking source papers and discussion between all authors. Studies were selected according to the following inclusion criteria:

  1. Study Design: Any randomized controlled trial (RCT) that included a placebo control group. We excluded all other study types, including cross-sectional studies, observational studies, case reports, opinions, commentaries, editorials, replies, letters to the editor, and incomplete studies.

  2. Study Population: Populations of interest included, but were not limited to, healthy controls, mental health disorders, metabolic disorders, neurodegenerative and neurodevelopmental disorders. There were no restrictions based on age or sex.

  3. Intervention: Studies could examine the effects of regular human insulin or analog insulin lispro, delivered intranasally, on various domains of cognition. Once again, studies must have included a placebo control group, while any study examining other intranasal peptide interventions were excluded.

  4. Outcomes: The studies were required to evaluate the effects of INI administration on standardized cognitive outcomes of interest.

2.3. Data extraction

All data were independently extracted and reviewed by three authors (NS, SW, ZE). Corresponding authors were contacted if data could not be extracted in a usable form from the published paper.

2.4. Bias assessment

The Cochrane Risk of Bias (RoB) tool was used to assess bias in the context of our outcomes of interest (i.e., cognition) [16]. Seven evidence-based domains were assessed, including random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. RoB assessment was conducted by three independent reviewers (NS, SW, JN), with two individuals assigned per study; conflicts were resolved through group discussion and consensus. Sensitivity analyses were conducted to exclude lower quality studies in which three or more of the domains were labelled as ’high’ or ‘unclear’ risk. A funnel plot of the included studies was used to assess for publication bias.

2.5. Outcome measures

The primary outcome was the effect of INI on cognition as assessed by individual cognitive domains including working memory, verbal working memory, verbal memory, verbal fluency, visual working memory, visual learning and memory, declarative memory, nondeclarative memory, hippocampal-dependent memory, executive function, attention, inhibitory control, and dementia (S3 Table in S1 File). A measure of global cognition was calculated by averaging the effect sizes of all individual cognitive domains for each study [17]. The effect size for global cognition for each study was then entered into another meta-analysis to obtain a pooled effect of global cognition across studies for each patient population. Note, the number of individual cognitive domains varied per study. See S3 Table in S1 File for all the neuropsychological tests used to measure these cognitive domains. Secondary outcome measures included serum insulin and glucose levels and side effects of INI, as available. Subgroup analyses for age, sex, and dose of INI were also conducted, as available, to explore potential sources of heterogeneity.

2.6. Synthesis of results

Quantitative data from all studies were pooled in a random effects meta-analysis using Review Manager 5.4. A minimum of two studies was required to meta-analyze effects; single study comparisons were reported narratively. Standardized mean difference (SMD) was calculated when pooling studies together with different outcome measures for a specific cognitive domain, while mean difference (MD) was calculated for studies using the same outcome measure. MD was calculated for physiological outcomes, including serum insulin and glucose levels following INI administration. Odds ratios (OR) were used for dichotomous data (e.g., side effects). Endpoint and change score data were combined in the analysis. Separate comparisons of INI versus placebo on each cognitive outcome measure were conducted for each clinical population and healthy individuals. Given the limited number of studies with available data, comparisons of adverse effects and physiological blood values were conducted across patient populations. Heterogeneity was assessed using the I2 statistic [18]. Publication bias for comparisons with 10 or more included studies was assessed using funnel plots.

3. Results

Our initial search identified 2654 results. Following title and abstract screening, 52 studies were assessed for full-text eligibility. A total of 32 studies met inclusion criteria for this review and 26 of these studies, with a total of 1,726 patients and healthy individuals, were included in the quantitative meta-analysis (Fig 1; PRISMA flowchart).

Fig 1. PRISMA flow chart.

Fig 1

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Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart of included studies.

3.1. Study characteristics

The 32 RCTs included in this review were published between 2004 and 2021. A variety of populations were represented, including: healthy individuals (N = 11) [19–29], MCI and AD (N = 12) [30–42], schizophrenia (N = 2) [43, 44], bipolar disorder (N = 1) [45], major depressive disorder (N = 1) [46], obese men (N = 1) [47], Down syndrome (N = 1) [48], type 2 diabetes (N = 2) [26, 28], Phelan-McDermid syndrome (N = 1) [49], and Parkinson’s disease (N = 1) [50]. The median dose of INI was 40 IU (range 40 to 160 IU). Ten studies used an acute (single dose) of INI, while the remaining studies administered INI over a longer term. The median duration of INI treatment in the long-term studies was 8 weeks (range 1 to 52 weeks). The mean age of all populations included in our review was 53.4 years. Characteristics of all included studies can be found in S2 Table in S1 File.

3.2. Meta-analysis of global cognition by patient population

3.2.1. Healthy individuals

Global cognition for healthy, cognitively unimpaired individuals were pooled (k = 11; N = 400) [19–29]. No significant effects of INI were found for global cognition (SMD 0.02, 95% CI -0.05 to 0.09; I2 = 45%) (Fig 2). Among the healthy control studies, five studies examined the effects of intranasal regular human insulin versus placebo [19, 20, 24, 25, 28], one study examined both insulin aspart and regular human insulin versus placebo [21], two studies examined the effects of insulin actrapid versus placebo [27, 29], while another study examined 20, 40, 60, 80, 100, and 120 IU of insulin aspart versus placebo; each dose group is reported separately [22]. Seven studies administered an acute dose of INI [19, 22–27], and four studies administered INI over a longer period of treatment [19, 20, 28, 29]. There were no significant differences noted between studies that used an acute dose versus long-term dose.

Fig 2. Effect of INI vs. placebo on global cognition in healthy individuals.

Fig 2

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Forest of plot and risk of bias assessment for the effect of INI vs. placebo on global cognition in healthy individuals.

3.2.2. All patient populations

Patient populations were categorized into four groups: mental health disorders, AD and MCI, metabolic disorders, and other disorders (Fig 3). These are reported independently in the analysis. Treatment duration ranged from acute (single dose) administration to 4 months. Four studies examined the effects of INI on cognition in patients with mental health disorders including schizophrenia [43, 44], bipolar disorder [45], and major depressive disorder [46]. There were no significant effects observed for global cognition following INI treatment in patients with mental health disorders (SMD 0.07, 95% CI -0.09 to 0.24; I2 = 0%) (Fig 3). Eleven studies examined the effects of INI on global cognition outcomes in patients with AD/MCI using the Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) [30–42]. Pooling these studies together revealed a significant improvement in global cognition following treatment with INI (SMD 0.22, 95% CI 0.05 to 0.38; I2 = 69%) (Fig 3). Three studies reported on the effects of INI on cognition in patients with metabolic disorders, one in obese men and two in adults with T2DM [26, 28, 47]. No significant effect was observed for global cognition in patients with metabolic disorders (SMD 0.18, 95% CI -0.11 to 0.48; I2 = 0%) (Fig 3). Global cognition for two studies were pooled together under other disorders. One study explored the effects of 8 weeks of 20 IU of INI on cognition in individuals with Down syndrome, and the other study explored the acute effects of 40 IU of INI in patients with Parkinson’s disease [26, 48]. INI did not improve global cognition in either of these patient populations (SMD 0.50, 95% CI -0.61 to 1.61; I2 = 71%).

Fig 3. Effect of INI vs. placebo on global cognition in all patient populations.

Fig 3

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Forest plot and risk of bias assessment for the effect of INI vs. placebo on global cognition in all patient populations.

To further investigate whether INI improved a specific cognitive outcome for patients with AD/MCI, the studies were pooled together to evaluate the effect of INI on the following cognitive outcome measures: verbal working memory, attention, verbal memory, executive function, visuospatial memory, and global cognition. However, there were no significant effects observed for any of these outcomes.

3.3. Meta-analysis of INI related side effects (all populations)

Adverse events following INI administration were reported in 22 of the included studies. Common side effects included nasal irritation/rhinitis, light-headedness/dizziness, nausea, and nose bleeds (S2 Table in S1 File). There was no significant difference in number of reports for any of these side effects between the INI and placebo groups (S1-S4 Figs in S1 File). There was also no significant difference between groups in terms of total number of adverse events being reported (S5 Fig in S1 File).

3.4. Meta-analysis of post-INI serum insulin and glucose levels (all populations)

Serum concentrations of glucose and insulin levels following INI administration were reported in 20 of the included studies, 9 of which provided data to pool in a meta-analysis for each outcome (S2 Table in S1 File). Across all populations, serum insulin and glucose levels were not significantly affected following INI administration, as compared to placebo (S6, S7 Figs in S1 File).

3.5. Subgroup analysis of age, sex, and dose of INI

Within healthy individuals, age was not a heterogenous variable (S8 Fig in S1 File). Given our study populations, subgroup analysis could not be conducted for patients with AD/MCI as they are all classified as older adults (≥65 years old). Across studies with healthy individuals, INI improved global cognition in studies comprised of >50% males (S9 Fig in S1 File). In contrast, INI was found to improve global cognition in patients with AD/MCI in studies comprised of >50% males (S10 Fig in S1 File). Moreover, 40 IU of INI was shown to improve global cognition in patients with AD/MCI compared to other administered doses (10 IU, 20 IU, and 60 IU) (S11 Fig in S1 File). Subgroup analysis could not be conducted for the mental health and metabolic disorder groups due to the limited number of studies within each category. Across all patient populations, 4 studies used administered an acute dose of INI, while the remaining 15 had a longer intervention period (range 3 weeks to 4 months). Significant effects on global cognition were only noted in the long-term administration studies (SMD 0.26, 95% CI 0.10 to 0.42, I2 = 70%) in comparison to the acute studies (SMD 0.02, 95% CI -0.13, 0.17, I2 = 0%) (S12 Fig in S1 File).

3.6. Qualitative reporting of outcomes

A total of 3 studies retrieved from the search did not report data in a way that could be extracted and synthesized in a meta-analysis [38, 42, 49]. Two out of the three studies examined the effects of prolonged INI treatment on cognition in patient populations with AD and MCI [38, 42], while the remaining study assessed this in children with Phelan McDermid syndrome [49]. Both AD/MCI studies reported improvements in ADAS-Cog scores with long-term INI treatment compared to placebo. However, one of these studies observed that INI did not result in meaningful improvements in immediate and delayed recall in mild-moderate AD patients [42]. In the third study, INI was observed to improve the cognition and social skills of children with Phelan McDermid syndrome over the age of 3 years old [49]. S2 Table in S1 File summarizes the characteristics of the studies included in this section, along with their main findings.

3.7. Risk of bias assessment

There were no studies included in the quantitative meta-analysis that were deemed to be of low quality or have a high risk of bias (S13 Fig in S1 File). As such, there was no need to conduct any sensitivity analyses for any of the reported outcome effects. Three studies that were only reported narratively [34, 36, 41] had three categories with “unclear risk” due to lack of information in their published articles, and one category deemed “high risk”. Publication bias could not be assessed using funnel plots given the limited number of studies included within each comparison. No substantial publication bias was detected in these comparisons (S14 and 15 Figs in S1 File).

4. Discussion

In this review, we present a comprehensive overview and analysis of RCTs evaluating the effects of INI administration on various domains of cognition across all ages and populations. We demonstrate that INI may be associated with pro-cognitive benefits for pooled global cognition in patients with AD and MCI, while there was no significant effect observed for individual cognitive subdomains. Additionally, long-term administration of INI may provide more therapeutic benefits than acute administration. To the best of our knowledge, this is the first review to pool together data in healthy individuals and patient populations, thus providing meaningful preliminary evidence for this novel and emerging field. We have also identified several gaps and unanswered questions that should be addressed in future research to propel this field forward.

The cognitive benefits of INI were only found in certain populations in our study, specifically patients with AD and MCI. Interestingly, this finding is in contrast to a previous meta-analysis in the AD/MCI population that failed to detect a significant difference on global cognition with INI versus placebo [15]. However, it is possible that this finding may be due to the inclusion of two studies in that review for which the primary diagnosis was not AD/MCI [46, 51]. In this review, Cha et al. (2017) was included in the comparison for mental health disorders [46], and this study showed no effect of INI therapy on any of the cognitive outcomes for patients with MDD. The second study was an unpublished report of patients with HIV-associated neurocognitive disorder [51], which also did not demonstrate an effect of INI on cognition [15]. Taken together, the pro-cognitive effect of INI in AD/MCI is presently inconclusive, and further research is warranted to elucidate the therapeutic effects of INI.

In exploring the effects of INI on cognition in AD/MCI, there are important moderators to consider. For example, the apolipoprotein E4 (APOE-ε4) carrier status of an individual may introduce differences in insulin sensitivity between APOE-ε4 carriers versus non-carriers. In fact, the occurrence of insulin resistance is greatest in adults with AD who are APOE-ε4 non-carriers [52]. In the present review, there were four AD/MCI studies that stratified patients according to their APOE-ε4 carrier status [33, 36, 41, 53], with the remaining AD studies not indicating the APOE-ε4 status of the sample. In three of these studies, INI acutely facilitated memory improvements in APOE-ε4− memory-impaired subjects, as compared to APOE-ε4+ subjects who showed no benefits or a decline in memory [34, 36, 41]. The last study failed to note a main effect of APOE-ε4 status on therapeutic response [33]. This demonstrates that there may be identifiable heterogeneity within specific populations and that INI may only be beneficial for a subgroup of patients. Thus, stratifying patients according to genotype may help identify individuals that would be most responsive to this intervention. More research is required to understand the mechanisms through which the APOE genotype attenuates the cognitive effects of INI [41].

There were no significant effects of INI observed in any of the other comparisons included in the present study. This may be attributed to illness or treatment-specific characteristics within each population that may moderate the response to INI and limit its generalizability across patient populations. One point to consider is the baseline level of cognitive functioning in a given population. For example, there may be varying levels of baseline neurocognitive deficits across the patient populations included in the mental health disorders comparison [54, 55]. McIntyre et al. (2012) demonstrated an improvement in executive functioning (Trail Making Test Part-B) with an acute dose of INI in patients with bipolar I/II disorder [45], whereas Fan et al. (2011 and 2013) did not observe any pro-cognitive improvements with INI in patients with schizophrenia [43, 44]. This might suggest that patients with higher baseline cognitive functioning may be more likely to benefit from INI in comparison to patients exhibiting more severe neurocognitive impairment.

In general, INI was well tolerated in both healthy individuals and across clinical populations. There were no significant differences in the number of side effects reported between the INI treatment and placebo groups, demonstrating the safety and tolerability of INI. In the present review, only 18 out of the 29 RCTs measured serum concentrations of glucose and insulin following INI administration. Among the seven studies that were pooled across all study populations, serum insulin and glucose levels were not significantly affected after INI administration. It should be noted that dose-dependent peripheral spillover has been reported in the literature. Higher doses of INI may temporarily increase circulating insulin and decrease blood glucose levels increasing the risk of hypoglycemia [56, 57]. While these reports of peripheral spillover did not cause any adverse side effects, these results highlight the importance of collecting serum glucose, insulin, and c-peptide concentrations after intranasal administration to ensure the safety of its use. Furthermore, our subgroup analyses demonstrated that sex and dose may affect the outcomes of INI on cognition. Thus, future studies should stratify their study population by sex to examine sex-differences as well the dose required for cognitive response to INI treatment.

There are some limitations to this study which must be addressed. Our results revealed moderate to substantial heterogeneity for specific study populations including the healthy controls (I2 = 45%), AD/MCI (I2 = 69%), and other disorders (I2 = 71%). This may be attributed to the high heterogeneity in cognitive measures employed to assess the various domains of cognition. The lack of standardized reporting for cognitive outcomes made it difficult to compare the effects of INI on cognition between healthy individuals and across patient populations, precluding conclusions from being made. Furthermore, the lack of standardized reporting in the literature precluded data from being extracted from many of the identified studies, which limits the power of our results. Similarly, there were a small number of studies that examined a particular cognitive subdomain, which further limited the power of our analyses for individual cognitive outcomes. Thus, we pooled together different cognitive subdomains to obtain global cognition for each study. Second, there was a lack of stratification between sex and genotype in most studies, which may contribute as a potential source of heterogeneity and represent as significant confounders of cognitive outcomes as discussed above. Differential cognitive response to INI treatment may result from the observable heterogeneity in experimental design across studies, such as type of insulin administered, treatment duration, and dose. Furthermore, our systematic review consists of a heterogenous sample spanning cognitive unimpaired individuals to multiple patient populations; thus, the pro-cognitive benefits of INI in one population may not be generalizable to other populations. To note, our meta-analysis categorized patients with Down syndrome and Parkinson’s disease under other disorders. Therefore, the substantial heterogeneity observed may be attributed to the different patient population as well as the type of cognitive impairment. Third, not all included studies reported on BMI of the sample, and among those that did, there were variable effects observed of INI on cognitive outcomes. It is well established in the literature that obesity not only induces peripheral insulin resistance but can also induce insulin resistance within the brain [58]. Therefore, baseline BMI may play a role in mediating cognitive outcomes and influence INI treatment response. Future studies should include an assessment to better understand the metabolic profile of patients or healthy individuals to further explore the relationship between obesity, insulin resistance, and cognitive decline. Results from a systematic review based on studies with great diversity should be interpreted cautiously as it can bring a higher risk for extrapolation. Further research is warranted to better understand the neurobiological mechanisms and differences in etiology to dissect the intrinsic and extrinsic factors contributing to the treatment response of INI. We encourage investigators of upcoming RCTs to combine neuroimaging approaches with cognitive assessments to identify the neural correlates associated with INI therapy.

5. Conclusion

This systematic review and meta-analysis demonstrate that INI may be associated with pro-cognitive benefits, specifically for pooled global cognition. However, this effect is limited to patients with AD/MCI. Research over the last two decades has identified the CNS as an emerging major contributing site of insulin action. Taken together, and in keeping with recent clinical trials, our findings demonstrate that INI can be safely tolerated, and has the potential to improve memory by directly reaching brain regions involved in the regulation of cognition. As this is still a novel field of study, more research is required to understand the heterogeneity in treatment response of INI to extend the pro-cognitive benefits across different patient populations with the ultimate goal to improve their overall quality of life.

Supporting information

S1 File

(PDF)

Click here for additional data file. (1MB, pdf)
S1 Checklist. PRISMA 2020 checklist.

(DOCX)

Click here for additional data file. (32.8KB, docx)

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Belgin Sever

16 Apr 2023

PONE-D-23-05529Outcomes and Clinical Implications of Intranasal Insulin on Cognition in Humans:

A Systematic Review and Meta-AnalysisPLOS ONE

Dear Dr. Agarwal,

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PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #2: No

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Reviewer #1: Would like initially to congratulate the authors on performing hard work in preparing this manuscript after their review and analysis of many of the screened studies. This would be considered a well done manuscript for future references.

Reviewer #2: Reviewer statement: The manuscript entitled “Outcomes and Clinical Implications of Intranasal Insulin on Cognition in Humans: A systematic review and metanalysis” aims the application of metanalysis technics to test the association of intranasal insulation and cognition. The manuscript carries some methodological flaws that need to be addressed before being accepted for publication.

Main suggestions:

1- The version I received had the actual manuscript text duplicated. Something went wrong during the manuscript final editing.

2- The grammar and phrase constructions used in the manuscripts need to be revised.

3- The definition of the global cognition phenotype is not clear. The authors pooled data from different studies, each one with their own unique set of measured cognitive traits. The authors need to show the set of cognitive traits shared among the individual studies and how those study-specific traits were meta-analyzed.

4- The authors never considered the effect of simple covariates such as sex and age on each individual study. The individual studies are focused on different phenotypes with completely different cohorts. This heterogeneity and not considering the effect of sex and age could be a major source of bias that would be even more pronounced when doing a metanalysis.

5- The authors merged studies focused on mental health disorders, AD and metabolic disorders (lines 177 to 196) without considering that the underlying conditions may have an effect on the “global cognition”. This lack of rigor can lead to many experimental artifacts.

6- The authors didn’t explain the assumptions if the Cochrane Risk of Bias tool and didn’t even include a scientific reference for it.

7- The authors never even discussed the heterogeneity statistics I2 on each individual metanalysis. Low I2 statistics hinders the interpretation of any metanalysis.

8- There is a lot of confusion between the terms metanalysis and pooled analysis during the reading. Terms such as pooled population metanalysis are very misleading.

9- The authors need to further develop their introduction and their discussion sections. They need to incorporate similar studies using the same metanalytic approaches even for other focal phenotypes.

10- There is a lot of confusion between the definition of tables and a figure. Figure 2 and 3, as an example, are tables and not figures. The authors need to split tables and figures and discussed The authors didn’t include labels to any “figure” or supplementary “figures”. The interpretation is challenging without the proper labels.

11- The authors referred (line 236) the supplementary figure 9 that didn’t exist in the supplementary materials.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Jun 28;18(6):e0286887. doi: 10.1371/journal.pone.0286887.r003

Author response to Decision Letter 0

11 May 2023

The version I received had the actual manuscript text duplicated. Something went wrong during the manuscript final editing.

There are two versions of the manuscript. The latter version has tracked changes with our updated systematic search performed on February 11, 2023, as requested by the journal. The manuscript and analyses were then updated accordingly. Per journal protocols, a tracked and clean version of the manuscript were uploaded upon resubmission.

The grammar and phrase constructions used in the manuscripts need to be revised.

Thank you for your comment. We have gone through the manuscript and proofread to ensure that there are no grammatical and phrase construction errors.

The definition of the global cognition phenotype is not clear. The authors pooled data from different studies, each one with their own unique set of measured cognitive traits. The authors need to show the set of cognitive traits shared among the individual studies and how those study-specific traits were meta-analyzed.

Supplementary Table 3 outlines the different cognitive domains that were measured across studies as well as the various cognitive measures used to assess each domain.

For each included study in the quantitative meta-analysis, an effect size for global cognition was calculated by averaging the effect size of individual cognitive domains. The effect size for global cognition for each study was then entered into another meta-analysis to obtain a pooled effect of global cognition across all studies in each patient population. We have updated and included this explanation in our Methods section to further elaborate our definition of global cognition.

The authors never considered the effect of simple covariates such as sex and age on each individual study. The individual studies are focused on different phenotypes with completely different cohorts. This heterogeneity and not considering the effect of sex and age could be a major source of bias that would be even more pronounced when doing a metanalysis.

Based on the reviewer’s comment, we ran subgroup analyses based on sex, age, and dose of INI. Within patient populations, age wasn’t a heterogenous variable. Subgroup analysis could be conducted for patients with AD/MCI as all patients were 55 years or older. Furthermore, subgroup analysis could not be conducted due to the limited number in the mental health and metabolic disorder groups. Please see section 3.5 for the updated results.

The authors merged studies focused on mental health disorders, AD and metabolic disorders (lines 177 to 196) without considering that the underlying conditions may have an effect on the “global cognition”. This lack of rigor can lead to many experimental artifacts.

Thank you for your comment. We agree with the reviewer that this is a heterogenous population and removed the overall pooled effect size in Figure 3. We reported on the outcomes individually, though they are displayed in one figure for organization purposes.

The authors didn’t explain the assumptions if the Cochrane Risk of Bias tool and didn’t even include a scientific reference for it.

Thank you for bringing this to our attention. We have added the reference for the Cochrane Risk of Bias tool as well as the different domains it addresses.

The authors never even discussed the heterogeneity statistics I2 on each individual metanalysis. Low I2 statistics hinders the interpretation of any metanalysis.

Thank you for your comment. We have addressed the heterogeneity I2 statistics in our limitation sections. We also discuss potential sources of heterogeneity throughout our discussion based on each patient population.

There is a lot of confusion between the terms metanalysis and pooled analysis during the reading. Terms such as pooled population metanalysis are very misleading.

Thank you for bringing this to our attention. We have updated our results section using the proper nomenclature for a meta-analysis.

The authors need to further develop their introduction and their discussion sections. They need to incorporate similar studies using the same metanalytic approaches even for other focal phenotypes.

Thank you for your suggestion. We have further elaborated on the study that we referenced for the same meta-analytic approach.

There is a lot of confusion between the definition of tables and a figure. Figure 2 and 3, as an example, are tables and not figures. The authors need to split tables and figures and discussed The authors didn’t include labels to any “figure” or supplementary “figures”. The interpretation is challenging without the proper labels.

As per requested by PLOS One, the figure captions are included in the manuscript and the figures followed the references at the end. To note, Figure 2 and 3 are forest plots that were exported by RevMan. Forest plots are a graphical representation of the findings of multiple studies, thus we listed them as figures and not tables. All figures and tables are correctly labeled in our supplementary file.

The authors referred (line 236) the supplementary figure 9 that didn’t exist in the supplementary materials.

Thank you for bringing this to our attention. We had mislabeled the last few figures. We have updated the supplementary file with additional subgroup analyses and updated the figure label accordingly.

Attachment

Submitted filename: Rebuttal Letter April 2023.docx

Click here for additional data file. (16.9KB, docx)

Decision Letter 1

Belgin Sever

25 May 2023

Outcomes and Clinical Implications of Intranasal Insulin on Cognition in Humans: A Systematic Review and Meta-Analysis

PONE-D-23-05529R1

Dear Dr. Agarwal,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Belgin Sever, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Belgin Sever

2 Jun 2023

PONE-D-23-05529R1

Outcomes and Clinical Implications of Intranasal Insulin on Cognition in Humans: A Systematic Review and Meta-Analysis

Dear Dr. Agarwal:

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