Prevalence of non-alcoholic fatty liver disease in patients with prediabetes

Background. Diabetes mellitus (DM) is one of the largest global health emergencies. Prediabetes is an early stage in hyperglycemia continuum where individual is at an increased risk for development of DM. NAFLD represents a range of liver disorders characterized by hepatic steatosis or accumulation of fat in the liver cells in the absence of excessive alcohol consumption, viral or drug related etiologies. However, not many studies have been conducted to study the prevalence of non-alcoholic fatty liver disease (NAFLD) in persons with prediabetes. This study is an endeavor in that direction. Materials and methods. This was a cross-sectional observational study. 100 prediabetic patients, fulfilling the criteria as under, were included in the study over a period from November 2017 to March 2019, after informed consent. Investigations carried out on the patients included baseline biochemical parameters like complete hemogram, fasting plasma glucose, liver function tests, kidney function tests, serum electrolytes and specialized investigations like HbA1c, 2-hour-OGTT and serum insulin levels. Results. The study included 38 males and 62 females, with the median age for the study population being 46 years. The mean BMI was found to be 24.29 ± 3.98 kg/m2, and the mean waist circumference was found to be 81.26 ± 8.71 cm. A significant association was found between the level of fatty echotexture on ultrasound and BMI (p = 0.003), and gender (0.05). 30 % population was found to be insulin sensitive, 22 % was found to be depicting early insulin resistance and 48 % had significant insulin resistance. There was a statistically significant correlation between ultrasound and fibroscan findings. A significant statistical correlation was found between HOMA IR and level of fatty echotexture on ultrasound, as well as median  liver stiffness on  fibroscan. Conclusions. We  found a significant correlation between insulin resistance and presence of NAFLD. Also, significant associations were observed between various demographic characteristics and grade of steatosis. There is a need to undertake further studies on a larger scale, to substantiate the observations of this study. This understanding is expected to go a long way in generating awareness and optimizing public health strategies.


Introduction
Diabetes mellitus (DM) is one of the largest global health emergencies. Prediabetes is an early stage in hyperglycemia continuum where individual is at an increased risk for development of DM. The global DM prevalence in 2019 is estimated to be 9.3 % (463 million people), rising to 10.2 % (578 million) by 2030 and 10.9 % (700 million) by 2045. The global prevalence of impaired glucose tolerance is estimated to be 7.5 % (374 million) in 2019 and projected to reach 8.0 % (454 million) by 2030 and 8.6 % (548 million) by 2045 [1].
Insulin resistance has been defined as a metabolic state in which a greater than normal amount of insulin is required to get a physiologically normal response [3]. In insulin resistance muscle, fat and liver cells do not respond properly to insulin and cannot easily absorb glucose from the blood stream. Failure of insulin to inhibit triglycerides (TGs) lipolysis in insulin-resistant states leads to an oversupply of free fatty acids to the liver, excess hepatic TGs synthesis and intracellular accumulation of toxic lipid products play a key role in the development of hepatic steatosis in non-alcoholic fatty liver disease (NAFLD).
NAFLD represents a range of liver disorders characterized by hepatic steatosis or accumulation of fat in the liver cells in the absence of excessive alcohol consumption, viral Vol. 17, No. 1, 2021 Оригінальні дослідження /Original Researches/ or drug related etiologies [4]. It is the most common cause of liver disease in the absence of viral hepatitis or alcohol excess, in the industrialised world affecting approximately 15-50 % of adult population. It includes a wide spectrum of liver disorders, ranging from pure steatosis to cirrhosis, liver failure and even hepatocellular carcinoma. Two hallmark components of pathophysiology of hepatic steatosis include mitochondrial dysfunction and insulin resistance [5].
Review of literature reveals a clear link between NAFLD, insulin resistant states such as obesity, type 2 DM and prediabetes [6] and significant correlation between the presence of NAFLD in prediabetic patients [7][8][9]. Various studies also found significant correlation between demographic characteristics like age, BMI and the prevalence of NAFLD [7,8]. Studies conducted by S. Suresh et al.
[10] and A. Bugianesi et al. [11] found statistically significant correlation between the presence of NAFLD and level of insulin resistance.
An association between insulin resistance and NAFLD is now well recognized and accepted, but it remains a subject of active research whether increase in TGs, metabolic intermediates cause insulin resistance or insulin resistance causes excessive accumulation of fats in liver [12].
Some investigators hypothesize that liver fat accumulation is a result, of peripheral insulin resistance in obesity. In skeletal muscle, peripheral insulin resistance will mainly affect a large portion of the total glucose uptake (> 80-90 %), whereas in adipose tissue, insulin resistance induces an impaired anti-lipolytic action of insulin and therefore an increased release of non-esterified fatty acids [13]. Also, insulin clearance is affected in advanced liver disease, which is considered as one of the main causes of hyperinsulinemia in liver cirrhosis [14].
Various studies, all over the world, have concluded an increasing incidence of hepatic steatosis in DM patients. However, not many studies have been conducted to study the prevalence of NAFLD in persons with prediabetes. This study is an endeavor in that direction.

Materials and methods
This was a cross-sectional observational study conducted at the Departments of Medicine and Biochemistry at Postgraduate Institute of Medical Education and Research, Dr. RML Hospital, New Delhi after receiving clearance from the Department and Institutional Review Board. 100 prediabetic patients, fulfilling the criteria as under, were included in the study over a period from November 2017 to March 2019, after informed consent.
Inclusion criteria: prediabetic population, > 18 years, with full informed consent having fasting plasma glucose between 100 to 125 mg/dl or 2-hour postprandial plasma glucose between 140-199 mg/dl (was included in the study reconfirming with standard 2 hour OGTT (after 75 gm glucose solution ingestion) or HbA1c 5.7 to 6.4 %.
2. Alcohol consumption > 20 g/day for males and > 10 g/day for females.
The study participants were first asked to fill a predetermined proforma with baseline demographic characteristics and then they underwent a detailed clinical examination. Investigations carried out on the patients included baseline biochemical parameters like complete hemogram, fasting plasma glucose, liver function tests, kidney function tests, serum electrolytes and specialized investigations like HbA1c, 2-hour-OGTT and serum insulin levels. The basal state insulin resistance of the patients was calculated using the HOMA2 IR calculator. (Homeostatic Model Assessment of Insulin Resistance). Ultrasound and transient elastography methods were used to assess for the presence of NAFLD and further evaluation of extent of fibrosis.

Statistical analysis
Categorical variables were presented in number and percentage (%) and continuous variables were presented as mean ± SD and median. Normality of data was tested by the Kolmogorov-Smirnov test. The data was entered in MS EXCEL spreadsheet and analysis was done using Statistical Package for Social Sciences (SPSS) version 21.0. A p value of < 0.05 was considered statistically significant.

Results
The study included 38 males and 62 females, with the median age for the study population being 46 years. The mean BMI was found to be 24.29 ± 3.98 kg/m 2 , and the mean waist circumference was found to be 81.26 ± 8.71 cm (table 1).
A significant association was found between the level of fatty echotexture on ultrasound and BMI (p = 0.003), and gender (0.05). Оригінальні дослідження /Original Researches/ 68 percent study population was found to have NAFLD on the basis of ultrasound, out of which 34 % had Grade I, 31 % had Grade II and 3 percent study population were found to have Grade III fatty liver. The extent of liver fibrosis was further evaluated in the study subjects with the help of fibroscan. 24 % subjects were found to have F2 fibrosis, 4 % had F3 fibrosis and 1 % was found to have cirrhosis. Mean serum insulin levels was found to be 10.95 ± 5.73 μIU/ml. Insulin resistance was determined via HOMA IR method. 30 % population was found to be insulin sensitive, 22 % was found to be depicting early insulin resistance and 48 % had significant insulin resistance (table 2).
There was a statistically significant correlation between ultrasound and fibroscan findings (p = 0.0001, r = 0.754). A significant statistical correlation was found between HOMA IR and level of fatty echotexture on ultrasound (p < 0.0001, r = 0.463), as well as median liver stiffness on fibroscan (p < 0.0001, r = 0.686) (table 3; figure 1). Amongst the glucose profile of the study population, the fasting blood glucose levels was found to be significantly correlated to the level of fatty echotexture on Ultrasound (p = 0.018), the level of insulin resistance (p = 0.026) and median liver stiffness on fibroscan (p = 0.008) (table 4; figure 2).

Discussion
The occurrence of NAFLD and the statistical correlation of its presence with insulin resistance were the primary outcomes of the study. In addition, the study also secondarily analyzed the demographic characteristics of the study population and grade of steatosis, fibrosis and level of insulin resistance. A comprehensive review of our various results is as under.
Our study found the occurrence of NAFLD to be 68 %. Out of these 68 subjects, 34 % had grade I, 31 % had grade II and 3 % study population were found to have grade III fatty liver.
In contrast, F. Mansour-Ghanaei et al. [7] found the prevalence of prediabetes around 48.69 %. A similar study conducted by V. Mohan et al. [9] in Chennai in 2008, found the prevalance of NAFLD in prediabetics in South India to be 33 percent. The higher occurrence of NAFLD in this study may be attributable to the differences in demographic characteristics and biochemical parameters of the study population among the studies. The mean age (46.49 ± 8.18 years) of the study population was higher in the current study in comparision to the study by F. Mansour-Ghanaei et al. (40 ± 10 years) [7]. Similarly, in comparision to the study conducted by V. Mohan et al. [9], the mean BMI (22.8 ± 3.8 kg/m 2 vs 24.29 ± 3.98 kg/m 2 ), fasting plasma glucose (99 ± 11 mg/dl vs 107.88 ± 10.41 mg/dl) and total cholesterol levels (175 ± 35 mg/dl vs 196.04 ± 45.54 mg/dl) of the subjects in this study were significantly higher.
In the current study mean liver stiffness measure was similarly observed to be 5, 8.43, 11.3 and 13.2 kPa, for study subjects with F0-F1, F2, F3 and F4 fibrosis respectively. A significant increase in mean fibroscan reading from F2 to F3 (p value = 0.002), and a marginal association between increase in liver stiffness measure readings from F2 to F4 (p value = 0.09) was noted, which is comparable to the findings of G. Zhang et al. [16]. 58 percent male study subjects (36 out of 62) and 84 percent females (32 out of 38) were found to have NAFLD, thus female gender was found to be associated with NAFLD (p = 0.05), a finding that is in concordance with the observations of G. Bedogni  A 5 year cohort based study was conducted by M. Liu et al. [19] which also found out the mean BMI of prediabetic study subjects with NAFLD to be 24.86 ± 2.67 kg/m 2 , which is comparable to the mean BMI calculated in the current study 24.29 ± 3.98 kg/m 2 . The positive statistical association between the BMI of study subjects and the presence of NAFLD (p = 0.003) is in concordance with the findings of F. Mansour-Ghanaei et al. [7].
The level of insulin resistance was calculated by the HOMA IR computational method, requiring serum fasting blood glucose, and serum fasting insulin levels. A significant correlation was found between the level of insulin resistance and grade of fatty liver on ultrasound with p value < 0.0001, r = 0.463. Similar observations have been made in various studies in the past -G. Privitera et al. [20] and M. Li et al. [21]. Similarly, C. Ortiz-Lopez et al. [6], had studied 118 apparantly healthy individuals with a diagnosis of NAFLD. They also found a significantly raised HOMA IR value of 3.5 ± 0.2 μU/mmol in subjects with NAFLD, than the subjects without NAFLD (1.2 ± 0.4 μU/mmol), with a p value < 0.001.
The level of insulin resistance was also found to be significantly related to fasting blood glucose values (p = 0.026) of study subjects. Y. Singh et al. [22] also concluded a correlation of fasting plasma glucose was found with HOMA IR values in this study as well (r = 0.350, p < 0.0001) in their study carried out in Delhi.

Conclusions
In this small study conducted at ABVIMS and Dr RML Hospital, New Delhi, we found a significant correlation between insulin resistance and presence of NAFLD. Also, significant associations were observed between various demographic characteristics and grade of steatosis. There is a need to undertake further studies on a larger scale, to substantiate the observations of this study. This understanding is expected to go a long way in generating awareness and optimizing public health strategies.

Limitations of the current study
The current study was a hospital based cross-sectional study comprising of 100 prediabetic study subjects. Certain limitations that were observed are as follows.
The study had a small sample size (n = 100), which cannot be extrapolated to conclusions at a large scale. Regional variations were not taken into account in this study.
Cause-effect conclusions cannot be made definitively owing to cross-sectional nature of the study.

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