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A prospective observational cross-sectional study on the association of non-alcoholic fatty liver disease and insulin resistance in acute ischemic stroke in tertiary care hospital in Central India

[version 1; peer review: awaiting peer review]
Dr. Jayanth Kumar
https://orcid.org/0009-0002-6085-3542
1, Dr Anuj Varma1, Sourya Acharya1, Sunil Kumar
https://orcid.org/0000-0001-9905-4831
1
Dr. Jayanth Kumar
https://orcid.org/0009-0002-6085-3542
1, Dr Anuj Varma1, Sourya Acharya1, Sunil Kumar
https://orcid.org/0000-0001-9905-4831
1
PUBLISHED 01 May 2024
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS AWAITING PEER REVIEW

This article is included in the Datta Meghe Institute of Higher Education and Research collection.

Abstract

Background

Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance have individually been associated with increased cardiovascular risk, and their potential interplay in the context of Acute Ischemic Stroke remains a subject of investigation. This study aims to elucidate the association between NAFLD, insulin resistance, and Acute Ischemic Stroke, exploring risk factors, etiology, and potential implications for patient management.

Methods

This prospective observational cross-sectional study will be conducted at Acharya Vinoba Bhave Rural Hospital, enrolling patients aged 40-79 diagnosed with acute ischemic stroke between 2022 and 2025. Diagnostic confirmation will involve brain imaging, and abdominal ultrasonography will assess NAFLD severity. Biochemical parameters will be measured for insulin resistance evaluation, including blood glucose, glycosylated hemoglobin (HbA1c), and fasting insulin levels. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) will be calculated. Statistical analyses, including descriptive statistics and bivariate and multivariate analyses, will be applied to explore associations and potential risk factors.

Expected Outcome

Anticipated findings include insights into the prevalence and severity of NAFLD in Acute Ischemic Stroke patients, the association between insulin resistance and stroke incidence, and potential risk factors contributing to this association. The study provides a foundation for understanding the complex relationship between NAFLD, insulin resistance, and Acute Ischemic Stroke, offering valuable implications for risk stratification and patient management in clinical practice. Identifying modifiable risk factors may guide targeted interventions, contributing to improved outcomes in this patient population.

Keywords

Non-alcoholic Fatty Liver Disease (NAFLD), Insulin Resistance, Acute Ischemic Stroke, Risk Factors, Cross-sectional Study, Cardiovascular Risk

Corresponding author: Dr. Jayanth Kumar
Competing interests: No competing interests were disclosed.
Grant information: The author(s) declared that no grants were involved in supporting this work.
Copyright:  © 2024 Kumar DJ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Kumar DJ, Varma DA, Acharya S and Kumar S. A prospective observational cross-sectional study on the association of non-alcoholic fatty liver disease and insulin resistance in acute ischemic stroke in tertiary care hospital in Central India [version 1; peer review: awaiting peer review]. F1000Research 2024, 13:434 (https://doi.org/10.12688/f1000research.148150.1) First published: 01 May 2024, 13:434 (https://doi.org/10.12688/f1000research.148150.1) Latest published: 01 May 2024, 13:434 (https://doi.org/10.12688/f1000research.148150.1)

Introduction

Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance have emerged as significant contributors to cardiovascular morbidity, with a growing body of evidence suggesting their potential association with Acute Ischemic Stroke (AIS). NAFLD, characterized by hepatic fat accumulation in the absence of excessive alcohol consumption, has been recognized as a precursor to metabolic syndrome and cardiovascular disease.1 Insulin resistance, a key feature of metabolic syndrome, is implicated in the pathogenesis of NAFLD and atherosclerosis.2

The link between NAFLD and cardiovascular events, including stroke, has been the subject of several studies.3,4 Likewise, insulin resistance has been identified as a risk factor for cerebrovascular events.5 However, the concurrent investigation of NAFLD and insulin resistance in the context of AIS remains a relatively understudied area.

This study aims to fill this gap by conducting a prospective observational cross-sectional study to explore the association between NAFLD, insulin resistance, and AIS. Understanding the interplay between these metabolic factors and their potential contribution to the pathogenesis of AIS is crucial for refining risk stratification and developing targeted interventions for this patient population.

Aim

The primary aim of this study is to investigate the association between Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance in patients with Acute Ischemic Stroke.

Objectives

  • 1. Risk factors and precipitating factors of NAFLD in acute ischemic stroke: Identify and analyze the risk factors and precipitating factors associated with the development of NAFLD in patients diagnosed with Acute Ischemic Stroke.

  • 2. Association between insulin resistance and acute ischemic stroke: Examine and quantify the relationship between insulin resistance and the occurrence of Acute Ischemic Stroke in the study population.

  • 3. Etiology of NAFLD and insulin resistance in acute ischemic stroke: Investigate and understand the underlying causes and etiological factors contributing to NAFLD and insulin resistance development in patients experiencing Acute Ischemic Stroke.

Methods

Study setting

The study will be conducted at the Acharya Vinoba Bhave Rural Hospital (AVBRH) Department of Medicine. AVBRH is a tertiary care teaching hospital located in the rural area of Wardha district. The hospital provides access to a diverse patient population and appropriate medical facilities for diagnosing and managing Acute Ischemic Stroke, NAFLD, and insulin resistance.

Study duration

The study will span from 2022 to 2025. This timeline allows for the recruitment of a sufficient number of participants, comprehensive data collection, and thorough analysis to achieve the study objectives. The extended duration also accounts for potential variations in patient enrollment and ensures the inclusion of a representative sample.

Study design

The research will adopt a Prospective Observational Cross-sectional study design. This design enables data collection from a defined group of participants at a single point in time. The prospective nature ensures that data is gathered in real-time, allowing for the evaluation of cause-and-effect relationships between NAFLD, insulin resistance, and Acute Ischemic Stroke.

Inclusion criteria

  • 1. Age: Participants must be above 18 years of age.

  • 2. Impaired fasting glucose: Individuals with impaired fasting glucose, defined as a fasting plasma glucose level between 110 and 125 mg/dl.

  • 3. Normal fasting insulin: Normal range of fasting insulin levels, within the 2.6-24.9 μ U/ml range.

  • 4. Liver function test derangement: Patients who exhibit deranged Liver Function Test values without a history of alcohol intake.

  • 5. Diagnostic confirmation: Individuals diagnosed by brain computed tomography (CT) or magnetic resonance imaging (MRI) in the hospital.

  • 6. Neurological deficits: Patients with symptoms of acute ischemic stroke presenting with neurological deficits.

  • 7. Timely diagnosis: Inclusion is limited to patients with the first episode of ischemic stroke diagnosed within seven days from the onset of stroke symptoms.

  • 8. Consent: Individuals who provide informed consent for being included in the study.

Exclusion criteria

  • 1. Pregnancy: Pregnant women are excluded from the study due to potential confounding factors and unique physiological changes during pregnancy.

  • 2. Severe renal or hematological diseases: Individuals with severe renal or hematological diseases, as these conditions may independently affect study parameters.

  • 3. Previous stroke disability: Patients with a history of previous stroke disability are excluded to focus on the acute phase of ischemic stroke.

  • 4. Lipid lowering agents: Individuals who have been on lipid-lowering agents within six months before enrollment, as these medications may influence lipid metabolism.

  • 5. Trauma: Patients with traumatic injuries are excluded to maintain the homogeneity of the study population.

  • 6. Cancer diagnosis: Individuals diagnosed with cancer are excluded due to the potential impact of cancer and its treatment on study parameters.

Enrollment

The enrollment process for this prospective observational cross-sectional study will commence with identifying eligible patients admitted to the Department of Medicine at Acharya Vinoba Bhave Rural Hospital (AVBRH) with a confirmed diagnosis of Acute Ischemic Stroke. The research team will then approach these patients, providing detailed information about the study’s objectives and procedures. Patients will be given ample time to ask questions, and written informed consent will be obtained from those willing to participate. Once consent is obtained, a baseline assessment will be conducted, capturing demographic information and relevant medical history. Subsequently, individuals confirmed to have experienced Acute Ischemic Stroke through brain computed tomography (CT) or magnetic resonance imaging (MRI) will progress to the next enrollment stage.

Enrolled participants will undergo abdominal ultrasonography to assess the presence and severity of Non-alcoholic Fatty Liver Disease (NAFLD). The diagnosis of NAFLD will be based on predefined ultrasound parameters, including parenchymal brightness, liver-to-kidney contrast, deep beam attenuation, bright vessel walls, and gallbladder wall definition. Blood samples will be collected from participants to evaluate biochemical parameters, including blood glucose levels, glycosylated hemoglobin (HbA1c), and fasting insulin levels. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) will be calculated based on these measurements.

Throughout the enrollment process, careful verification will be conducted to ensure that participants do not meet any of the predefined exclusion criteria. Patients meeting all inclusion criteria, undergoing diagnostic confirmation of Acute Ischemic Stroke, exhibiting NAFLD on abdominal ultrasonography, and having valid baseline blood biomarker measurements will be considered enrolled in the study. Ethical considerations, such as privacy, confidentiality, and voluntary participation, will be rigorously maintained by protocols approved by the Institute’s ethical committee. Participants will be informed about the study’s followup plans, including subsequent assessments and data collection points, and the research team will address any concerns or questions. Through this comprehensive enrollment process, the study aims to ensure the inclusion of eligible participants while upholding ethical standards and maintaining the integrity of the research.

Data collection process

The data collection process for this prospective observational cross-sectional study involves a systematic and comprehensive approach to gather pertinent information from enrolled participants. During the initial assessment, baseline demographic information, such as age, gender, and relevant medical history, will be meticulously recorded. Additionally, clinical parameters, including the waist/hip ratio (WHR) calculation, will contribute to a holistic understanding of the participant’s health status. Following the initial assessment, patients diagnosed with Acute Ischemic Stroke through brain computed tomography (CT) or magnetic resonance imaging (MRI) will have their diagnosis confirmed, and specific characteristics of the stroke will be documented. This step ensures accurate identification and categorization of the acute cerebrovascular events in the study population.

Abdominal ultrasonography will be employed as a diagnostic tool to assess the presence and severity of Non-alcoholic Fatty Liver Disease (NAFLD). The diagnosis will be based on predefined ultrasound parameters, offering a qualitative grading system to categorize the severity of NAFLD. This comprehensive evaluation allows for a nuanced understanding of the hepatic condition in the context of the study. Blood samples will be collected from participants to evaluate key biochemical parameters. Standardized digital glucometers will measure blood glucose levels, and the immunoturbidometric inhibition method, using a High-pressure Liquid Chromatography Procedure processed in an automated chemical analyzer, will analyze Glycosylated Hemoglobin (HbA1c) levels. Fasting insulin levels will be measured to calculate the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) using a specific formula.

All collected data will be meticulously recorded in a structured and secure database, ensuring the confidentiality of participant information through unique identifiers. Quality control measures will be implemented to uphold the accuracy and reliability of the data, with double-checks conducted during the data entry process. Potential followup assessments may be conducted according to the study’s predetermined timeline, allowing for longitudinal data capture. Ethical considerations, including participant privacy and confidentiality, will be strictly followed throughout the data collection process, aligning with informed consent and ethical approval protocols. This meticulous and ethical data collection approach aims to generate robust evidence regarding the intricate association between Non-alcoholic Fatty Liver Disease, insulin resistance, and Acute Ischemic Stroke within the designated study cohort.

Sample size

N=Z1−α/22∗p∗(1−p)D2.
Z1−α/2=1.96,at 5% level of significance

Prevalence rate of NAFLD = 9%.6

p = 0.09%

D = estimated error (6%) = 6%

=(1.96)2∗(0.009)∗(1−0.09)/(0.6)2=90

Minimum sample size required = 90

Statistical methods

In analyzing the data collected during this prospective observational cross-sectional study, statistical methods will be employed to address the study’s research questions and objectives. The initial step involves the application of descriptive statistics to succinctly summarize the demographic characteristics of the study population, as well as the distribution of clinical and biochemical parameters. Measures such as mean, median, standard deviation, and percentages will be utilized to provide a comprehensive overview.

Bivariate analysis will be conducted to explore unadjusted associations between Non-alcoholic Fatty Liver Disease (NAFLD), insulin resistance, and Acute Ischemic Stroke. Chi-square tests, Fisher’s exact tests for categorical variables, t-tests, or Mann-Whitney U tests for continuous variables will be instrumental in this exploration. Following this, multivariate analysis, particularly logistic regression, will be employed to assess the independent association between NAFLD, insulin resistance, and the occurrence of Acute Ischemic Stroke. This approach allows for the control of potential confounding variables, enhancing the depth of understanding regarding these interrelationships.

To explore the relationships between continuous variables, correlation analysis, utilizing coefficients such as Pearson or Spearman, will be employed to ascertain the strength and direction of associations. Subgroup analysis will be undertaken to assess whether the identified associations vary within specific subgroups based on demographic or clinical characteristics, providing a nuanced perspective on potential heterogeneity within the study population.

Sensitivity analysis, an essential component of the statistical approach, will be conducted to test the robustness of the findings. This involves assessing the impact of excluding certain subgroups or outliers on the overall results. The previously calculated sample size, ensuring adequate power to detect statistically significant associations, will guide the reliability of the study outcomes.

Statistical analyses will be performed using appropriate software, such as SPSS version 23, depending on the required tests and analyses. The level of significance will be set at 0.05, with a 95% confidence interval, to determine statistical significance in hypothesis testing. Throughout these analyses, ethical considerations will remain paramount, ensuring the confidentiality of participant data and adherence to ethical guidelines. In employing these comprehensive statistical methods, the study aims to derive meaningful insights into the complex associations between NAFLD, insulin resistance, and Acute Ischemic Stroke, contributing to the broader understanding of these health conditions.

Ethical considerations

The Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (DU) has granted its approval to the study protocol (Reference number: DMIHER (DU)/IEC/2023/119. Date:11-04-2023). Prior to commencing the study, we will obtain written informed consent from all participants, providing them with a comprehensive explanation of the study’s objectives.

Limitation

This study, while designed to investigate the intricate relationship between Non-alcoholic Fatty Liver Disease (NAFLD), insulin resistance, and Acute Ischemic Stroke, has its limitations. Conducted in a single tertiary care teaching hospital in a rural setting, the findings may only be universally applicable to some populations, warranting caution in generalizing the results. The cross-sectional design, chosen for its practicality, inherently restricts the establishment of causality and the exploration of temporal relationships among the studied variables. Additionally, reliance on self-reported information or medical records for patient history may introduce data incompleteness. The calculated sample size, though optimized for statistical power, might need to be revised in detecting subtle associations, especially in subgroup analyses. Diagnostic techniques for NAFLD and insulin resistance, though standardized, have inherent limitations, and the potential influence of unmeasured confounding factors could impact the observed associations. The study’s duration may not fully capture the long-term consequences of NAFLD and insulin resistance, and the findings may be influenced by specific ethnic and cultural factors prevalent in the study region. Despite these constraints, this study endeavors to contribute valuable insights into the complex interplay between NAFLD, insulin resistance, and Acute Ischemic Stroke, recognizing the need for cautious interpretation and offering a foundation for future research and clinical considerations.

Dissemination

After the completion of the study, we will publish in an indexed journal or conferences.

Study status

The study has yet to start after publication of protocol we will start recruitment in the study.

Discussion

The investigation into the association between Non-alcoholic Fatty Liver Disease (NAFLD), insulin resistance, and Acute Ischemic Stroke (AIS) yields crucial insights into the complex interplay of metabolic factors contributing to cardiovascular morbidity.7 The existing literature has established both NAFLD and insulin resistance as independent risk factors for cardiovascular diseases, including stroke.8,9 However, our study takes a step further by concurrently exploring these factors in the context of AIS. The observed prevalence and severity of NAFLD in AIS patients will be a key focal point of discussion. Existing evidence suggests that NAFLD is associated with an increased risk of cardiovascular events, potentially mediated through mechanisms such as inflammation, oxidative stress, and endothelial dysfunction.10,11 Our findings may contribute to the growing body of knowledge on the role of NAFLD in AIS pathogenesis.

Insulin resistance, as a central element of metabolic syndrome, is known to play a role in atherosclerosis and may contribute to the risk of cerebrovascular events.5 Investigating the association between insulin resistance and AIS in our study population will provide valuable insights into the metabolic factors influencing stroke incidence. The identification of potential risk factors contributing to the observed associations, including demographic and clinical variables, will be discussed in the context of their implications for risk stratification. Understanding these risk factors may guide targeted interventions to mitigate the impact of NAFLD and insulin resistance on AIS outcomes.

It is important to acknowledge the limitations of the study, including the single-center design and the cross-sectional nature, which hinder the establishment of causality and the exploration of temporal relationships. Additionally, the study’s generalizability may be limited to the specific demographic characteristics of the study population.

Ethical considerations

The Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (DU) has granted its approval to the study protocol (Reference number: DMIHER (DU)/IEC/2023/119. Date: 11-04-2023). Prior to commencing the study, we will obtain written informed consent from all participants, providing them with a comprehensive explanation of the study’s objectives.

Data availability

No data are associated with this article.

References

  • 1.  Younossi ZM, Koenig AB, Abdelatif D, et al.: Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016; 64: 73–84. PubMed Abstract | Publisher Full Text
  • 2.  Reaven GM: Banting lecture 1988. Role of insulin resistance in human disease. Diabetes. 1988; 37: 1595–1607. Publisher Full Text
  • 3.  Targher G, Day CP, Bonora E: Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease. N. Engl. J. Med. 2010; 363: 1341–1350. Publisher Full Text
  • 4.  Adams LA, Lymp JF, St Sauver J, et al.: The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology. 2005; 129: 113–121. PubMed Abstract | Publisher Full Text
  • 5.  Howard G, O’Leary DH, Zaccaro D, et al.: Insulin sensitivity and atherosclerosis. The Insulin Resistance Atherosclerosis Study (IRAS) Investigators. Circulation. 1996; 93: 1809–1817. Publisher Full Text
  • 6.  Teng ML, Ng CH, Huang DQ, et al.: Global incidence and prevalence of nonalcoholic fatty liver disease. Clin. Mol. Hepatol. 2023; 29: S32–S42. PubMed Abstract | Publisher Full Text | Free Full Text
  • 7.  Thiruvoipati T, Kielhorn CE, Armstrong EJ: Peripheral artery disease in patients with diabetes: Epidemiology, mechanisms, and outcomes. World J. Diabetes. 2015; 6: 961–969. PubMed Abstract | Publisher Full Text | Free Full Text
  • 8.  Yoneda M, Yamamoto T, Honda Y, et al.: Risk of cardiovascular disease in patients with fatty liver disease as defined from the metabolic dysfunction associated fatty liver disease or nonalcoholic fatty liver disease point of view: a retrospective nationwide claims database study in Japan. J. Gastroenterol. 2021; 56: 1022–1032. PubMed Abstract | Publisher Full Text | Free Full Text
  • 9.  Reaven GM: Role of insulin resistance in human disease (syndrome X): an expanded definition. Annu. Rev. Med. 1993; 44: 121–131. PubMed Abstract | Publisher Full Text
  • 10.  Younossi ZM, Golabi P, Paik JM, et al.: The global epidemiology of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH): a systematic review. Hepatology. 2023; 77: 1335–1347. PubMed Abstract | Publisher Full Text | Free Full Text
  • 11.  Lindenmeyer CC, McCullough AJ: The Natural History of Nonalcoholic Fatty Liver Disease - An Evolving View. Clin. Liver Dis. 2018; 22: 11–21. PubMed Abstract | Publisher Full Text | Free Full Text

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Kumar DJ, Varma DA, Acharya S and Kumar S. A prospective observational cross-sectional study on the association of non-alcoholic fatty liver disease and insulin resistance in acute ischemic stroke in tertiary care hospital in Central India [version 1; peer review: awaiting peer review]. F1000Research 2024, 13:434 (https://doi.org/10.12688/f1000research.148150.1)
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