Changes in the brain vessels in patients with non-alcoholic fatty liver disease and carbohydrate metabolism disorder

Background. The studies of brain vessels in patients with non-alcoholic fatty liver disease (NAFLD) can reveal new aspects of early detection of cerebral hemodynamic disorders, and will also allow the identification of a high-risk patients group. The purpose was to investigate the peculiarities of changes in the brain vessels in patients with NAFLD in combination with insulin resistance (IR) or type 2 diabetes mellitus (T2DM). Materials and methods. The study involved 74 patients with NAFLD (34 patients with non-alcoholic fatty hepatosis (NAFH) and 40 patients with non-alcoholic steatohepatitis (NASH)). The comparison group included 38 patients (16 patients with  IR and 22 patients with T2DM). The condition of  the blood  flow  in  the brain was studied using ultrasound duplex scan of the extracranial portion of the common carotid artery (eCCA), the external carotid artery (eCA), the extracranial portion of the internal carotid artery, middle cerebral artery, the vertebral artery (VA), ophthalmic artery. The peak systolic velocity (PSV) and the time-average maximum (TAMX) of blood flow velocity in the studied vessels was determined. Results. The asymmetric damage to the vessels of the brain with more severe left ventricular dysfunction with NAFH is conspicuous; it is more pronounced in patients with the NASH stage, especially in combination with IR. Hemodynamically, the most significant changes were found in eCCA, namely, the decrease in PSV was found to be 38.1 ± 2.3 cm/s on the right, and 36.6 ± 3.4 cm/s on the left, TAMX reduction 20.4 ± 2.1 cm/s on the right, and 16.7 ± 1.8 cm/s on the left. Conclusions. The patients with NAFLD, as well as IR and T2DM had a decrease in the speed of blood flow in extracranial vessels of the brain, mainly in the eCCA, eCA and VA (segment 3). The most pronounced blood flow deficiency has been observed in patients with NASH in combination with IR, mainly on the left, especially in еCCA.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is a clinical diagnosis that includes the presence of 5 % or more hepatic cells with fat accumulation as determined by liver imaging or biopsy in the absence of secondary causes of hepatic steatosis. NAFLD spans the spectrum of simple steatosis or non-alcoholic fatty liver to non-alcoholic steatohepatitis (NASH) which is defined histologically as hepatic steatosis, hepatic inflammation, and hepatocellular ballooning with or without fibrosis [1].
NAFLD is prevalent worldwide and is the most common chronic liver disease in Western countries. Its increasing prevalence is associated with major risk factors such as obesity, dyslipidemia, type 2 diabetes mellitus (T2DM), and the metabolic syndrome [2].
NAFLD is a systemic disorder of energy, glucose, and lipid homeostasis with hepatic manifestations [3]. Metabolic disorders, such as lipid accumulation, insulin resistance (IR), and inflammation, have been implicated in the pathogenesis of NAFLD, but the underlying mechanisms, including those that drive disease progression, are not fully understood [4]. Therefore, the non-alcoholic fatty liver di sease covers a range of diseases closely related to metabolic risk factors. As of today, the commonality of pathogenic mechanisms for NAFLD and metabolic syndrome has been proven, and their association with type 2 diabetes www.gastro.org.ua, http://gastro.zaslavsky.com.ua Патологія печінки і жовчовивідної системи / Pathology of Liver and Biliary Excretion System mellitus, cardiovascular diseases and severe forms of liver di sease, including cirrhosis and hepatocellular carcinoma, is evident [5].
It is believed that IR plays a central role in the development of NAFLD [6]. Patients with insulin resistance, as seen in obesity, metabolic syndrome, hypertension, and/or type 2 diabetes mellitus frequently exhibit endothelial dysfunction and are prone to develop arterial atherosclerosis, hypertension and metabolic disorders such as dysglycemia and elevated plasma free fatty acid levels. As a result of IR in patients with metabolic syndrome, type 2 diabetes, NAFLD, etc., endothelial dysfunction is formed, as well as vascular insulin resistance, combined with metabolic insulin resistance, that contribute to the development of hypertension and cardiovascular complications [7]. The associated physio logical mechanisms are not fully appreciated yet, however, it seems that augmented oxidative stress, a physiological imbalance is a possible mechanism involved in various vascular beds with insulin resistance and hyperglycemia [8].
A growing body of literature now recognizes that deterioration in vascular function over time is a major contributor to the process of cognitive aging. Liver disease, particularly NAFLD, may be involved in the process of cognitive aging through several potential mechanisms. First, persons with NAFLD have a high prevalence of individual vascular risk factors (e.g., hypertension) that contribute to the progression of cognitive aging. Additional risk factors that may accelerate vascular aging include microvascular endothelial dysfunction in concert with high levels of liver-derived gamma-glutamyltransferase (GGT) and insulin-like growth factor-1. Second, the concurrent presence of chronic systemic inflammation and the metabolic syndrome, which are highly prevalent in NAFLD, have been shown to contribute to cognitive impairment. Finally, obesity, in particular visceral adiposity, is related to neurodegenerative, vascular, and metabolic processes that affect brain structures underlying cognitive function [9].
Therefore, the studies of brain vessels in patients with NAFLD can reveal new aspects of early detection of cerebral hemodynamic disorders, and will also allow the identification of a high-risk patients group who will need to undergo a correction of existing disorders in order to prevent cardiovascular catastrophe, especially when combined with NAFLD with IR, T2DM and obesity.
The purpose of the study was to investigate the peculiarities of changes in the brain vessels in patients with NAFLD in combination with IR or T2DM.
In order to achieve the aim of the research, the patients in the basic group (Group 1) were further divided into subgroups depending on the presence of type 2 diabetes (moderate to severe) or IR (Fig. 1).
All studies were performed with patients' consent (written consent for performing appropriate diagnostic and therapeutic measures was received from all of the patients), and the methodology of their implementation met the World Medical Association Declaration of Helsinki (1975) and its revised version of 1983, and European Convention on Human Rights, as well as biomedicine and legislation of Ukraine.
All the examined patients were subject to general clinical, anthropometric, instrumental, and laboratory tests. The diagnosis was verified by complaints and history taking. In anthropometric study, height, weight, and waist circumferences were determined, and body mass index (BMI) was calculated. According to WHO recommendations, patients were divided based on BMI, where BMI of 16.0 and less corresponded to an expressed deficiency of body mass; 16.0-17.9 -insufficient body mass; 18.0-24.9 -normal body mass; 25.0-29.9 -excess weight; 30.0-34.9 -class I obesity; 35.0-39.9 -class II obesity; 40.0 and above -class III obesity (morbid obesity) [10].
Ultrasound examination of the abdominal cavity was performed in all patients according to generally accepted method. Standard and biochemical blood serum tests have been performed to determine the functional state of the liver, lipid metabolism indexes and carbohydrate metabolism (glucose, insulin, glycosylated hemoglobin (HbA1c, %) indexes).  [11].

Figure 1 -Distribution of patients with NAFLD depending on the presence of IR or T2DM
The degree of liver damage has been calculated using surrogate markers of fibrosis with the help of online calculators: 1. NAFLD fibrosis score: taking into account the age of the patients (years), BMI (kg/m 2 ), glucose tolerance or diabetes mellitus, alanine aminotransferase (ALT, U/L), albumin (g/L), and the number of platelets (×10 9 /L). The indicator corresponds: < -1.455: predictor of absence of significant fibrosis (F0-F2 fibrosis); ≤ -1.455 to ≤ 0.675: undetermined score; > 0.675: predictor of presence of significant fibrosis (F3-F4 fibrosis).
2. Fibrosis 4 calculator: this formula takes into account the age of a patient (years), level of ALT (U/L), AST (U/L) and platelet count (×10 9 /L). Hence: -an index lower than 1.45 -the probability of fibrosis presence is low (around 90 %); -an index larger than 3.25 -the probability of fibrosis presence is high (around 90 %).
Diagnosis of type 2 diabetes mellitus has been established according to IDF recommendations (2005), as well as taking into account the criteria of unified clinical protocol (the Order of the Ministry of Health of Ukraine dated December 21, 2012, No. 1118) [12]. The severity of T2DM was evaluated by HbA1c index (norm being up to 6.0 %).
Ultrasound evaluation of the head vessels was performed on Aloka ProSound 3500 SX (SSD-3500) (Hitachi Aloka Medical, China) apparatus using Aloka UST-5524-7.5 sen-sor. The condition of the blood flow in the brain was studied using ultrasound duplex scan of the extracranial portion of the common carotid artery (eCCA), the external carotid artery (eCA), the extracranial portion of the internal carotid artery (eICA), middle cerebral artery (MCA), the vertebral artery (VA), ophthalmic artery (ОА). The peak systolic velocity (PSV) and the time-average maximum (TAMX) of blood flow velocity in the studied vessels was determined. For the differential diagnosis of the atherosclerotic changes in brain vessels in the examined patients, eCCA posterior wall intima-media complex thickness was measured, since this is a marker of carotid atherosclerosis. The normal thickness of the intima-media complex for all patients is less than 1.0 mm. If the thickness index of intima-media complex of CCA posterior wall in the examined patients exceeded 1.0 mm, the patients were excluded from the study, and the results were considered as manifestations of atherosclerotic lesions of the brain vessels.
The criteria for exclusion of patients from the study were also: type 1 diabetes, type 2 diabetes (moderate to severe degrees, with severe manifestations of diabetic angioneuropathy), chronic hepatitis, alcoholic, viral (hepatitis B, C, D) etiologies, autoimmune hepatitis.
The scientific research was carried out within the scientific research work framework No. 851 "Mechanisms of the formation of complications in liver diseases and pancreas, methods of their treatment and prevention" (state registration number: 0115U001103), as well as the general department topic of the Department of Propaedeutics of Internal Diseases.
The analysis and processing of the examination results were carried out using the computer program Statistics for Windows 10.0 (StatSoft Inc., USA) using parametric and non-parametric methods of evaluating the results obtained.

Results
The anthropometric study found that the prevai ling majority of the examined patients of both groups had overweight or obesity of varying classification (class I-II) ( Table 1).
It is worth noting that among the patients with NAFH (Subgroup 1.1) and T2DM (Subgroup 2.2) people with class I obesity prevailed (p < 0.05), while among the patients with NASH (Subgroup 1.2) and IR (Subgroup 2.1) people with overweight were determined significantly more often. At the Патологія печінки і жовчовивідної системи / Pathology of Liver and Biliary Excretion System same time, among the patients from Group I, higher BMI indexes within the limits of corresponding malnutrition degree were determined in those with NAFH (Subgroup 1.1), and among patients with carbohydrate metabolism disorder -among those examined for IR (Subgroup 2.1).
The study of the carbohydrate metabolism indexes confirmed the presence of IR in patients in Subgroup 2.1 and T2DM in patients in Subgroup 2.2. At the same time, an increase in insulin level in patients with T2DM (moderate to severe) is a compensatory mechanism of the body for insulin resistance, as indicated by the high index of HOMA-IR in patients in Subgroup 2.1.
While characterizing carbohydrate metabolism, namely IR, at NAFLD, differences were found in patients with NAFH and NASH, namely, more severe hyperinsulinemia in patients with NAFH ((44.7 ± 6.5) U/L), which is associated with a more severe IR. Fasting glycemic indices were also higher in NAFH patients: glucose level in patients in the Subgroup 1.1A was (7.95 ± 0.24) mmol/l, and HbA1c -(7.98 ± 0.36) %.
It should be noted that more severe disorders of carbohydrate metabolism were found when combined with type 2 diabetes or IR with NAFLD than without it. In this case, the maximum deviations from the parameters of the control group were established in patients in Subgroup 1.1A (NAFH in combination with IR) (Tables 2, 3).
The Tables 4, 5 demonstrate that all examined subgroups of both groups revealed changes in the velocity of blood flow in the vessels of the brain by the results of duplex scan.
The average blood flow velocity in both external and internal carotid arteries in patients with both T2DM (Subgroup 2.2) and IR (Subgroup 2.1) was determined. The maximum deviations from the norm were found in the blood flow rates according to eCCA, namely, the decrease of PSV to (50.2 ± 2.2) cm/s on the right and to (51.0 ± 3.6) cm/s to the left in patients in the Subgroup 2.1, and to (51.0 ± 1.8) cm/s on the right and up to (50.6 ± 2.4) cm/s to the left in the patients in the Subgroup 2.2. TAMX is also believed to be significantly lower in patients with type 2 diabetes. In the study of segments 3 and 4 of VA, a blood flow deficit on both sides in patients in Group 2 was also diagnosed. It should be noted that a significant difference in the studied parameters in patients with type 2 diabetes was not detected in Group 2 ( Table 4).
Patients with NAFLD revealed significant deviations from the norm in blood flow rates in the study of major vessels of the brain (Table 5). At the same time, in patients with NAFH in both 1.1A and 1.1B Subgroups, the indicators did not significantly differ from those in patients with IR ((Subgroup 2.1) or T2DM (Subgroup 2.2) without liver damage. Maximum changes in blood flow to extracranial vessels of the brain are found in patients with NASH (Subgroups 1.2). At the same time, the difference in these indicators, depending on the presence of IR (Subgroup 1.2A) or type 2 DM (Subgroup 1.2B) was not established.
The asymmetric damage to the vessels of the brain with more severe left ventricular dysfunction with NAFH is conspicuous; that is more pronounced in patients with the

Index
Control group Group 2 (n = 38)   A detailed statistical analysis enabled to establish the relationship between the violation of the blood flow in the brain vessels and IR severity.

Indicator
More often, the correlation between the violation of the blood flow in the vessels of the brain (in eCCA, eCA

Note: the difference between the indices in patients of the Group 2 and the control group is significant: * -p < 0.05.
and VA (segment 3)) and IR severity with the HOMA index was found only in patients with NASH in combination with IR (Subgroup 1.2A). In patients in Subgroup 1.1A, the dependence is established only on the parameters of blood flow on eCCA and VA, and in patients 2.1 -only on eCCA (Table 6). Consequently, IR plays a leading role in the formation of a blood flow deficit in the main vessels of the brain in patients with NAFLD.

Discussion
The obtained results indicate that NAFLD, IR, T2DM, and their combination are more likely to occur in persons with excessive body weight and obesity. The evaluation of carbohydrate metabolism indexes showed that IR is more pronounced in patients with NAFH stage than with www.gastro.org.ua, http://gastro.zaslavsky.com.ua Патологія печінки і жовчовивідної системи / Pathology of Liver and Biliary Excretion System

Index NAFLD patients in the basic Group 1
Arterial segment  Various studies paid attention to psychological status, cognitive impairment, as well as their association with changes in the structure of the brain (with a decrease in the volume of white matter) and the severity of fibrosis at NAFH (B. Filipović et al., 2018) [13]. It is believed that cerebral hemodynamics due to disruptions in the microcirculation can lead to structural changes in the brain that underlie cognitive impairment (L.B. VanWagner et al., 2017) [9].

Table 6 -Correlation between parameters of blood flow in great brain blood vessels and IR based on HOMA-IR index
According to A. Rasool et al. (2017), all patients with NAFLD should be subjected to a study of the intima-media complex of the posterior wall of the common carotid artery, since NAFLD is associated with carotid atherosclerosis and, consequently, coronary atherosclerosis [14]. In the study of Y.J. Lee et al. (2018), NAFLD was associated with a subclinical form of cerebrovascular atherosclerosis in an asymptomatic general population of Korean men. Higher prevalence of subclinical form of cerebroid-cardiac atherosclerosis was observed in patients with NAFLD compared with controls without NAFLD. In addition, NAFLD was associated with a higher risk of atherosclerosis in large vessels [15]. Another study (L. Airaghi et al., 2018) also examined subclinical cerebrovascular lesions in patients with NAFLD, but without risk factors for atherosclerosis [16].
The results obtained by us testify to the impairment of the vascular pool of the brachiocephalic trunk in patients with NAFLD, type 2 diabetes and IR. There was no significant difference between the indicators of cerebral hemodynamics in patients with type 2 diabetes and IR (without liver damage). In fact, it does not differ from the data and results of patients with NAFLD (regardless of the presence of IR or DM). Our data indicate the maximum slowing of blood flow to the vessels of the brain in patients with NAFLD at the stage of steatohepatitis (regardless of the presence of IR or DM). In this case, asymmetry with more pronounced deceleration of velocity indicators to the left is revealed.
Consequently, insulin resistance plays a leading role in the formation of vascular tract defects in patients with metabolic disorders (liver steatosis, obesity, T2DM, etc.). Vascular changes are more pronounced in patients with NASH, which is probably due to a decrease in functional reserve of the liver, which in turn contributes to the reduction of the production/deactivation of vascular active biological substances, which together with the IR is the basis of the vascular system damage, including cerebral hemodynamics.
Further research is needed to better understand the progression of vascular lesions in NAFLD and in the carbohydrate metabolism, taking into account risk factors, severity of liver damage, IR and type 2 diabetes as well as cognitive impairment in these patients.

Conclusions
1. The patients with NAFLD (NAFH and NASH), as well as IR and T2DM had a decrease in the velocity of blood flow in extracranial vessels of the brain, mainly in the com-mon carotid artery, external carotid artery and vertebral artery (segment 3).
2. There was no statistically significant difference between the indicators of cerebral hemodynamics in patients with IR, type 2 diabetes mellitus (mild course) and NAFH (regardless of type of IR or DM).
3. The most pronounced blood flow deficiency has been observed in patients with NASH in combination with IR, mainly on the left, especially in еCCA (PSV reduction to (38.1 ± 2.3) cm/s on the right and up to (36.6 ± 3.4) cm/s on the left, and also TAMX decrease to (20.4 ± 2.1) cm/s on the right and to (16.7 ± 1.8) cm/s on the left).

Conflicts of interests.
Authors declare the absence of any conflicts of interests that might be construed to influence the results or interpretation of their manuscript.