Severe liver steatosis as potential risk factor of cardiovascular disease in patients with type 2 diabetes: a prospective cohort study

doi:10.21203/rs.3.rs-3131077/v1

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Severe liver steatosis as potential risk factor of cardiovascular disease in patients with type 2 diabetes: a prospective cohort study

https://doi.org/10.21203/rs.3.rs-3131077/v1

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Background and Aims:

Non-alcoholic fatty liver disease (NAFLD), accelerates the progression of atherosclerosis and development of cardiovascular diseases (CVD) which is the most common cause of mortality in NAFLD patients.

Methods and results

In this prospective cohort study, 1197 patients with type 2 diabetes (T2D) were divided in to two groups (360 patients with NAFLD and 847 without) and were followed for median of 5 years for incidence of CVD. Cox regression analysis was used to assess the association between NAFLD, level of liver enzymes, and aspartate aminotransferase to platelet ratio index (APRI), and incidence risk of CVD and its subgroups (i.e., Myocardial Infarction, chronic heart disease, coronary artery bypass grafting, and percutaneous coronary intervention. There was a significant positive association between CVD incidence and NAFLD (HR = 1.487, 95% CI = 1.041–2.124, p-value = 0.029). Although patients with NAFLD had higher ALT and AST levels (p-value = < 0.001), there was no significant association between liver enzymes and the incidence risk of CVD when adjusting for different variables. Furthermore, NAFLD was linked to APRI Q (2), APRI Q(3), and APRIQ(4) NAFLD (1.365 (1.046–1.781), 1.623 (1.234–2.135), and 3.373 (2.509–4.536), respectively.

Conclusion

NAFLD increased the incidence risk of CVD in T2D. However, there was no association between liver enzymes (ALT, AST, ALK-P, and GGT) and a higher incidence risk of CVD in T2D when adjusted for confounding variables.

Non-alcoholic fatty liver disease

NAFLD

Cardiovascular diseases

Type 2 diabetes

T2D

Cardiovascular risk factors

Chronic macro vascular complications

ALT

alanine aminotransferase

AST

aspartate aminotransferase

ALKP

alkaline phosphatase

GGT

gamma glutamyl transferase

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in western countries.(1) The worldwide prevalence of NAFLD is estimated at 20 percent, and it is expected to be more when it is in combination with obesity and diabetes.(2, 3) NAFLD spectrum ranges from simple steatosis to non-alcoholic steatohepatitis.(4)

Although NAFLD etiology is unknown, insulin resistance is a key mechanism of lipid deposition in hepatocytes leading to steatosis and potentially steatohepatitis in patients with diabetes.(4–6) Obesity and T2D that are associated with peripheral insulin resistance(7, 8), result in secretion of multiple secreted factors, i.e., free fatty acids, tumor necrosis factor-alpha, interleukin-6 and other proinflammatory cytokines leading to an acceleration of steatosis and increasing oxidative stress, inflammation, dyslipidemia, visceral fat, low adiponectin, ectopic adipose tissue distribution, endothelial dysfunction and postprandial dyslipidemia that are the main factors that lead to and further aggravate the course of NAFLD, as well as accelerating the progression of atherosclerosis and development of cardiovascular diseases(CVD) (9–12)which is the most common cause of mortality in NAFLD patients(13–15). For instance, Targher et al. in a cross-sectional study, demonstrated that NAFLD is associated with a higher prevalence of CVD, independent of classical risk factors, and 69 percent of 2839 patients with diabetes had NAFLD.(16)According to a wide range of current evidence, the role of NAFLD in the incidence risk of CVD is not clear yet. A group of studies showed that there was an association between NAFLD and the risk of CVD in patients with diabetes (16–19), while other studies could not approve this association(20, 21). A possible explanation for this heterogeneity in the previous studies could be due to small sample size, different methods used for detection of NAFLD (e.g. ultrasound, computer tomography, and liver biopsy), the difference in study participants, and the duration of study and follow-up periods. Therefore, it is necessary to assess the possible role of NAFLD as a new cardio-metabolic risk factor for long-term CVD outcomes, regardless of the association with traditional risk factors, in patients with diabetes.

This prospective cohort study aimed to investigate the association of NAFLD and liver enzymes (AST, ALT, ALK-P, and GGT) with the incidence of CVD in patients with T2D in a median of 5-year follow-up. (22, 23)

1. Study Population:

In this prospective study, we enrolled 1197 patients with history of T2D who attended Vali-Asr Hospital, which was affiliated with Tehran University of Medical Sciences. Baseline characteristics of the patients (cholesterol levels and other lipid and glycemic indices) were measured and 637patients with diabetes who did not have fatty liver and 560 patients with diabetes who had fatty liver were followed up for median of 5 years. A single blinded radiologist performed an abdominal ultra sound examination of liver and biliary tree (Hitachi EUB 405 apparatus equipped with a conve×3.5 MHz probe, intra- and inter observer coefficients of variation < 5%) to diagnose NAFLD. The NAFLD was diagnosed by definite presence of grade 3 hepatic steatosis that is defined based on hyper-echogenicity relative to renal parenchyma, reductions of ultra-sound beam, and/or the diaphragm and intrahepatic vessels/structures’ visualization reduced or absent, consistence evidence of severe hepatic steatosis with or without focal fatty sparing (24, 25). We excluded patients having or susceptible for other type of hepatitis and fatty liver (with a history of autoimmune hepatitis, alcohol abuse, viral hepatitis, hemochromatosis, Wilson’s disease, primary biliary cirrhosis, hepatotoxicity-inducing drugs usage, and/or rapid weight loss), and patients without fatty liver who were diagnosed with fatty liver during our follow up, so they were gone under physical examination and laboratory investigations (i.e. iron studies, ceruloplasmin, antinuclear and anti-smooth muscle antibody, and urinary copper). We also recorded the status of alcohol consumption, and possible chronic causes of non-NAFLD in each participant regularly. Participants with history of kidney disease (Creatinine (Cr) > 2 mg/dl or estimated glomerular filtration rate (eGFR) < 30 cc/min), familial hypercholesterolemia, liver dysfunction, epilepsy hypothyroidism, and hemoglobinopathy were excluded. In addition, women who were pregnant or taking oral contraceptives or hormone replacement therapy were excluded. Moreover, we excluded any patient whom drinks any amount of alcohol. Patients with other type of diabetes and metabolic conditions (type 1 diabetes, insulin-requiring type two diabetes, gestational diabetes, diabetes secondary to malignancies, pancreatitis and other metabolic conditions) were also excluded. After obtaining written consent form, total of 637 diabetic patients without fatty liver and 560 diabetic patients with fatty liver were selected. All participants went under physical examination and laboratory investigations at the baseline of study.

2.Examination:

Weight and height of included patients wearing light clothing and no shoes were measured. By dividing weight in kilograms by square of height in meters, body mass index (BMI) was calculated. The middle point between the iliac crest and rib cage while the patient was standing relaxed, was chosen to measure waist circumference using a non-stretchable measuring tape. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured three times with 5 minutes interval, after 10 minutes of seated rests using a calibrated Omron M7 digital sphyg142 manometers (Hoofddorp, The Netherlands) with appropriately sized cuffs which covered at least 80% of the subjects’ right arm. The mean value of second and third time were documented for further analysis. Hypertension was determined with SBP higher than 140 mmHg, or DBP higher than 90 mmHg, or previous diagnosis of hypertension and current treatment with antihypertensive drugs. Demographic information, current smoking, and medication usage status were self-reported during the interview.

3.Laboratory Investigations:

For laboratory evaluations, 10 ml of venous blood was drawn from each patient after 12-14h of overnight fasting. The sample were kept at a temperature of 4 to 8 ◦C until they were sent to the appropriate calibrating laboratories, and centrifuged (1500 rmp, for 10 min, at standard room temperature of 21 ◦C). At stored temperature of -70◦C, Laboratory evaluations were done on the extracted serum stored. High-performance liquid chromatography (A1C, DS5 Pink kit; Drew, Marseille, France) was used to investigate glycated hemoglobin (HbA1c). Enzymatic calorimetric methods by the glucose oxidase test were used to measure fasting blood sugar (FBS) and two-hour postprandial (2hPP) glucose. Total cholesterol, high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and triglycerides were measured using enzymatic methods. The central reference laboratory (Tehran, Iran) approved the kits used in this study.(26) Serum creatinine levels were evaluated using the Jaffe method (Parsazmun, Karaj, Iran). To calculate estimated glomerular filtration rate (eGFR), Modification of Diet in Renal Disease (MDRD) formula was used (eGFR = 186× (serum creatinine) − 1.154× (age)-0.203× [0.742 if female])(27). The APRI score was calculated by using [(AST/upper limit of the normal AST range) X 100]/Platelet Count formula. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK-P) and ɣ-glutamyl transferase (GGT) serum levels were analysed using enzymophotometry. The IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) method (ALT intra-assay CV = 3.7%, AST intra-assayCV = 2.5% and GGT intra-assay CV = 2.2%) used to measure serum ALT, AST and GGT and the DGKC (Deutsche GesellschaftfürKlinischeChemie) method (Intra-assay CV = 1.5%) used to measure serum ALK-P(28, 29). According to standardized criteria, ALT level > 30 IU/L in women and > 40 IU/L in men, AST level > 30 IU/L in women and > 36 IU/L in men, ALK-P levels > 306 U/L in both genders and GGT levels > 40 IU/L in women and > 60 U/L for men, considered elevated. These tests were performed using commercial Parsazmunkits (Tehran, Iran) and a Hitachi 704 automatic analyzer (Tokyo, Japan). For the diagnosis of viral hepatitis (hepatitis B and C), commercially available enzyme linked immunosorbent assay kits (DRG Diagnostics GmbH, Germany) used to measure hepatitis B antibody, hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B core antibody, hepatitis B antigen, and hepatitis C antibodies. All laboratory tests were assayed and attested by investigators and technicians blinded to the status of patients.

4. Assessment of CVD:

Cardiovascular disease (CVD) was stated if patient had non-fatal coronary artery disease diagnosed by a physician, positive findings in non-invasive tests (i.e., myocardial perfusion scan, exercise tolerance test, transthoracic echocardiography, and multi-detector computed tomography coronary angiography) that promptly needed initiation of anti-ischemic treatment. Angina pectoris, coronary insufficiency, acute coronary syndrome (ACS), myocardial infarction (MI), coronary artery bypass graft surgery (CABG), coronary angioplasty, percutaneous coronary intervention, and other revascularization procedures were considered as non-fatal coronary artery disease.

In the outcome, revascularization defined as needing for PCI or CABG procedure. MI/ ACS defined as myocardial infarction which treated with pharmaceutical agents, without any invasive procedure. Congestive heart failure (CHF) diagnosed according to The Framingham Diagnostic criteria.

5.Statistical Analysis:

Version 25 of SPSS software and R programming language (V 4.2.2) were used to perform statistical analysis on the recorded data. To test for the normality of the study population, Kolmogorov–Smirnov and Shapiro-Wilk normality tests, P-P plot, and histograms were used. All of the tested variables were found to be normal as the null hypothesis was rejected for all of them. To discover any significant differences between the variables in two studied groups, univariable analysis of potential continuous and categorical risk factors was performed using t-test and chi-square test, respectively. Data are reported as mean ± standard deviation (SD) for continuous variables and as proportions for categorical variables. Multivariate Cox regression analysis was performed to assess the association between NAFLD and liver enzymes (i.e. AST, ALT, ALKP and GGT) and incidence of CVD and its subtypes (i.e. MI, PCI, CABG and CHF) during the follow-up period. We created two different model. The first one CVD considered as the outcome and the second one, CVD subtypes consider as the outcome. As for CVD, test exposures were adjusted for age, sex, duration of diabetes, 2hpp, FBS, HDL, LDL, creatinine and BMI, separately. As for CVD subtypes, adjustment was done for age, sex, duration of diabetes and FBS. The adjusted variables were selected based on the significance in the univariate model. All variables with P-value less than 0.1 entered the model. The level of statistical significance was set to be a p-value < 0.05.

Baseline characteristics of the study population:

In this study, 1197 patients (43.5% male) who met our inclusion criteria were enrolled and followed up for median of 5 years [Min = 1 year, Max = 40 years]. Patients with NAFLD (360 patients with mean age of 53.19 ± 10.36) had significantly lower age, duration of diabetes, duration of using oral antidiabetic drugs, and creatinine compared to patients without NAFLD (837 patients with mean age of 57.38 ± 11.31) (Fig. 1). However, patients with NAFLD had higher DBP, serum level of total cholesterol, uric acid, AST and ALT and also, their BMI was higher in comparison to patients without NAFLD. There were no significant differences in sex, SBP, FBS, 2hpp, HbA1c, HDL-c, LDL-c, triglycerides, ALK-P, and GGT between the two groups (Table 1).

Table 1

Baseline characteristics of the study population based on the incidence of NAFLD
		With incident NAFLD	Without incident NAFLD	P-value
Sex	female	205	475	0.651
Sex	male	155	362	0.651
Age (year)		53.2 ± 10.4	57.4 ± 11.3	< 0.001
Duration of diabetes (year)		5.9 ± 5.6	8.0 ± 7.8	< 0.001
SBP (mmHg)		128.5 ± 15.0	135.6 ± 75.9	0.082
DBP (mmHg)		80.5 ± 7.2	78.8 ± 7.8	< 0.001
Drug duration (year)		2.5 ± 1.0	2.8 ± 2.5	< 0.002
FBS (mg/dL)		153.8 ± 55.0	155.6 ± 51.3	0.590
2hpp (mg/dL)		214.9 ± 92.7	216.1 ± 82.6	0.824
Hb AIC (%)		7.4 ± 1.5	7.9 ± 6.0	0.187
Total cholesterol (mg/dL)		184.0 ± 42.0	177.4 ± 43.1	0.015
HDL-c (mg/dL)		44.7 ± 12.9	45.1 ± 12.3	0.612
LDL-c (mg/dL)		103.4 ± 33.6	99.8 ± 34.3	0.092
TG (mg/dL)		184.2 ± 97.0	173.3 ± 102.5	0.089
Cr (mg/dL)		0.97 ± 0.19	1.01 ± 0.26	0.009
eGFR		76.5 ± 16.7	73.5 ± 18.5	0.008
Uric acid level (mg/dL)		5.4 ± 1.4	5.1 ± 1.3	0.003
BMI (Kg/m²)		30.9 ± 6.0	29.3 ± 5.3	< 0.001
AST(IU/L)		29.0 ± 18.4	23.8 ± 17.4	< 0.001
ALT(IU/L)		39.9 ± 27.4	29.0 ± 22.1	< 0.001
ALK-P(IU/L)		177.1 ± 83.8	172.2 ± 92.2	0.444
GGT(IU/L)		43.1 ± 56.1	42.2 ± 101.2	0.907
CVD (%)		67 (18.82%)	175 (20.80%)	0.434
MI		6 (1.67%)	17 (2.0%)	0.312
Revascularization		57 (15.83%)	157 (18.53%)	0.089
CHF		2 (0.56%)	2 (0.24%)	0.812
Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; FBS, fasting blood sugar; 2hpp, Two-Hour Postprandial Glucose; Hb AIC, hemoglobin A1C; HDL, High density lipoproteins; LDL, low density lipoproteins; TG, triglycerides, Cr, creatinine; UA, uric acid; BMI, body mass index; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALKP, alkaline phosphatase; GGT, glutamyl transferase; eGFR, estimated glomerular filtration rate; NAFLD, Non-alcoholic fatty liver disease; MI, Myocardial Infarction; CHF, Congestive Heart Failure

NAFLD and liver enzymes and incidence of CVD:

NAFLD was not associated with CVD incidence in univariate model (Fig. 2) (P = 0.064); hence, there was a significant positive association between NAFLD and the incidence of CVD after adjustment for confounding variables (aHR = 1.487 [1.041–2.124], P = 0.029).

There was no statistically significant association between the liver enzymes and incidence of CVD (Table 2).

Table 2

Association between incidence of CVD subgroups with NAFLD and liver enzymes
		AST	ALT	ALK-P	GGT	NAFLD
MI	Yes	28.41 ± 27.43	42.77 ± 55.95	172.63 ± 85.62	29.00 ± 19.72	6
	No	24.44 ± 18.38	30.01 ± 21.02	173.04 ± 85.62	42.33 ± 92.92	217
	P-value	0.327	0.298	0.985	0.593	0.607
	HR	1.00 [0.99, 1.02]	1.01 [1.01, 1.02]	1.00 [0.99, 1.01]	0.99 [0.97, 1.02]	1.46 [0.59, 3.65]
	P-value	0.732	0.005	0.961	0.451	0.416
Revascularization	Yes	24.12 ± 18.21	28.31 ± 12.12	164.23 ± 61.23	33.31 ± 20.65	57
	No	24.51 ± 18.23	30.28 ± 18.92	173.21 ± 78.64	42.58 ± 15.51	305
	P-value	0.338	0.341	0.531	0.772	0.144
	HR	0.99 [0.98, 1.02]	1.00 [0.98, 1.01]	1.00 [0.99, 1.01]	1.00 [0.99, 1.01]	1.20 [0.65, 2.24]
	P-value	0.881	0.722	0.659	0.774	0.561
CHF	Yes	18.21 ± 7.21	36.00 ± 5.62	237.21 ± 24.12	40.12 ± 12.15	2
	No	28.41 ± 8.21	30.59 ± 23.45	172.60 ± 89.42	42.6 ± 83.64	312
	p-value	0.291	0.712	0.036	0.977	0.357
	HR	1.01 [1.00, 1.01]	1.01 [1.00, 1.01]	1.02 [1.01, 1.04]	0.99 [0.98, 1.00]	2.12 [0.94, 4.12]
	P-value	0.912	0.212	0.012	0.843	0.081
ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALK-P, alkaline phosphatase; GGT, glutamyl transferase; NAFLD, Non-alcoholic fatty liver disease; MI, Myocardial Infarction; CHF, chronic heart disease; CABG, Coronary artery bypass grafting; PCI, percutaneous coronary intervention.

NAFLD and liver enzymes and incidence of CVD subgroups:

Among 1207 patients, 12, 62, 23, and 128 patients experienced congestive heart failure (CHF), coronary artery bypass grafting (CABG), myocardial infarction (MI), and percutaneous coronary intervention (PCI), respectively. There was no significant associations between NAFLD and liver enzymes with the incidence risk of CVD subgroups except for ALT levels and MI incidence (HR: 1.01 [1.01, 1.02], P = 0.005) and ALK-P levels with CHF (HR: 1.02 [1.01, 1.04], P = 0.012) (Table 2).

A. Association of APRI and NAFLD

The relationship between APRI and NAFLD is summarized in Table 3. After controlling for confounding variables, APRI Q (2), APRI Q (3), and APRI Q (4) values were considerably higher in patients with NAFLD (OR:1.365 (1.046–1.781), 1.623 (1.234–2.135), and 3.373 (2.509–4.536), respectively. The duration of diabetes, fasting blood sugar level, sex, age, 2hpp, creatinine, and BMI were all corrected using a tertiary multivariate adjusted model.

Table 3

Association between incidence of NAFLD and APRI
	NAFLD
	Risk estimate	95% CI	P-value
APRI_Q(2)	1.365	1.046–1.781	0.022
APRI_Q(3)	1.623	1.234–2.135	0.001
APRI_Q(4)	3.373	2.509–4.536	< 0.001
Data was adjusted for age, sex, duration of diabetes, fasting blood sugar, and 2hpp in the tertiary multivariate adjusted model. APRI, aspartate aminotransferase to platelet ratio index. The reference category is APRI Q1
aspartate aminotransferase to platelet ratio index (APRI)

In this longitudinal study, we found that patients with T2D who had NAFLD had greater CVDs and there was a strong association between NAFLD and the incidence risk of CVD in diabetic population when adjusted for different variables in the tertiary multivariate adjusted model. There was no association between liver enzymes (ALT, AST, ALK-P, and GGT) and a higher incidence risk of CVD in T2D. Additionally, there was no relationship between AST, ALT, and GGT enzymes with the incidence risk of CVD subgroups such as PCI, MI, CABG, and CHF.

Previous studies assessed the association between NAFLD and the incidence of CVD in general or diabetic population via diagnosis of NAFLD based on the serum liver enzymes and fatty liver index, or liver ultrasonography, or liver biopsy.

Studies using liver biopsy to detect NAFLD showed that the most common cause of death was CVD in patients who had the evidence of hepatic fibrosis on their histopathology (30–32). Moreover, another study reported that a higher mortality rate was found in fibrosis stage of 3–4(33). Additionally, a study determined that there was a significant relationship between overt steatohepatitis at biopsy and cardiac diseases such as left atrium enlargement and increased left ventricular mass (34). The results of our study support these findings.

A group of different studies, in contrast to our results, determined that there was a strong association between the elevated level of γ-glutamyl transferase (GGT) and the presence and progression of CVD and sever cardiovascular outcomes leading to death(35, 36). In addition, there were some reports about the association between increasing the level of alanine aminotransferase (ALT) and a higher 10-year risk of CVD after adjustment for metabolic risk factors and alcohol use(37, 38). A possible explanation for different result in our study could be a fewer follow-up period compared to the previous studies.

Regarding NAFLD detected by liver ultrasonography, several previous studies like ours showed that moderate to severe form of NAFLD (hepatic steatosis on ultrasound) was strongly correlated with a higher risk of fatal and nonfatal CVD after adjustment for cardiometabolic risk factors in patients with and without T2D(34, 39, 40). The results of our study support these findings. Liver ultrasonography could detect patients at high incidence risk of CVD based on previous studies that showed a strong association between ultrasonographic severity of steatosis and coronary/carotid atherosclerotic disease (40).

APRI was found to have a significant relationship with NAFLD in this study. A recent systematic review found that APRI risk stratifies morbidity and death in individuals with NAFLD, which is consistent with our findings (42). APRI can also detect fibrosis in NAFLD, according to a recent cross-sectional study in Iran (43). In addition, in NAFLD patients, a retrospective cohort research in Canada compared the predictive efficacy of non-invasive diagnostic procedures like APRI to liver histology and hepatic venous pressure gradient (HVPG). Their findings revealed that APRI can predict NAFLD patient outcomes and that it can be used to monitor, risk stratify, and identify targeted therapies (44). Furthermore, prospective research in Brazil found that is particularly effective in detecting NAFLD (45). In cohort research in South Korea, Koo et al. (46) found a strong link between APRI advancement and NAFLD, with the Q4 group having a greater hazard ratio for developing liver fibrosis than the Q1 group. Furthermore, in another prospective cohort study in Korea, the baseline HOMA-IR value was found to be an important risk factor for liver fibrosis advancement (47).

Pathophysiology of NAFLD and CVD

Although the exact mechanism of the relationship between NAFLD and CVD is not well understood, there are some possible explanations for the role of NAFLD in increasing the prevalence and incidence of cardiovascular events as follows.

The first possible explanation is due to insulin resistance and abdominal obesity in NAFLD patients. The elevated insulin level leads to change in the pathway of cellular free fatty acid (FFA) storage resulting in increasing its storage in skeletal muscle and liver rather than adipose tissue. This alternation in FFA pathway leads to fat accumulation into liver cells causing insulin resistance resulting in increasing oxidative stress in hepatocytes that causes abnormal adipocytokine profile and endothelial dysfunction and activation of further cardio-metabolic dysfunctional cascade(41, 42). Additionally, some studies showed that elevated FFA and very low-density lipoprotein had a role in producing more plasminogen activator inhibitor 1 that lead to endothelial dysfunction and stimulation of atherosclerosis resulting in adverse cardiovascular events (43, 44).

The second possible mechanism could be increased inflammatory mediators due to liver dysfunction and chronic disease. A study by Biddinger et al. showed that there was a strong relationship between the liver injury severity and the presence of atherosclerosis (45). Also based on a group of studies, NAFLD patients had a higher level of inflammatory mediators and prothrombotic including high-sensitive C-reactive protein, tumor necrosis factor α, and interleukin 6, fibrinogen, and plasminogen activator inhibitor that stimulated the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathway(46, 47). Increasing nuclear factor-κB (NF-κB) plays a role in activation of more genes in hepatocytes (e.g. intercellular adhesion molecule-1 and monocyte chemoattractant protein-1) related to production of pro-inflammatory and atherogenic factors and aggregation of systemic inflammatory mediators(48). Moreover, excess c-Jun N-terminal kinase(JNK) can lead to insulin resistance via its effect on intracellular signaling pathway causing deactivation of insulin receptor(49). However, some studies determined that there was no association between endothelial dysfunction and NAFLD that may be related to considering mild form of NAFLD as the other studies revealed more cardiovascular events and endothelial dysfunction in the sever form of NAFLD(50, 51). Furthermore, there are some evidences that showed larger intima-media thickness and more calcified and non-calcified coronary plaques in NAFLD patients explaining the higher incidence and prevalence of CVD in this group compared to patients without NAFLD (52, 53).

The role of NAFLD in CVD subtypes

Based on clinical studies, NAFLD has an important role in the presence and progression of different CV manifestations, such as left ventricular dysfunction, atherosclerotic CV disease, coronary heart disease(54); structural cardiac abnormalities(valve dysfunction, myocardial hypertrophy, heart failure), arrhythmias(atrial fibrillation, premature ventricular beats, and non-sustained ventricular tachycardia(35)), cerebrovascular and thromboembolic events (ischemic and hemorrhagic stroke)(55, 56), suggesting that its contribution may be independent of the presence of traditional CV risk factors(57–59). However, there was no relationship between NAFLD and liver enzymes (AST, ALT, and GGT) with the subgroups of cardiovascular disease such as CHF, PCI, MI, and CABG in our study except for ALK-P which had significant association with CHF. This could be because the patients with incident CVD were prescribed high dose statins after diagnosis, preventing the more serious cardiovascular complications such as MI and CHF.

Treatment of NAFLD

Although the effectiveness of current treatment for NAFLD patients has not yet been approved, there are some recommended strategy to avoid further adverse cardiovascular risk in NAFLD patients such as; lifestyle changes (e.g. an appropriate and healthy diet such as Mediterranean diet(60), regular physical activity, and smoking cessation), pharmacotherapy to control metabolic syndrome components such as antidiabetic drugs (e.g. Pioglitazon(61), Glucagon-like Peptide-1 Receptor Agonists(62), and Dipeptidyl Peptidase-4 Inhibitors(63)), anti-hypertensive drugs(e.g. Angiotensin Receptor Blockers(64)), and lipid lowering agents(e.g. Statins(65, 66) and Omega-3 Polyunsaturated Fatty Acids(67)), and pharmacotherapy to control liver disease in sever NAFLD patients to avoid cirrhosis and its consequences. It is recommended to give some treatments such as; orlistat, vitamin E(68), and bariatric surgery(69, 70)to manage NAFLD in patients with current cardiometabolic disease based on a group of studies.

This study had several strengths. Firstly, to the best of our knowledge, it is the first prospective longitudinal study that assessed the association between NAFLD and liver enzymes (AST, ALT, ALK-P, and GGT) with the incidence risk of CVD complications in patients with T2D and the prospective design of this study can establish a causal relationship between the measured variables. Secondly, previous studies showed the association between NAFLD and the prevalence of CVD in general population, however, only a few have considered their possible role as a risk factor and a contributor to the development of CVD complications in patients with diabetes. Thirdly, the adequate sample size and the exclusion of other liver disease has increased the representativeness of our results. Nonetheless, this study also had some limitations. We considered the NAFLD in the present study according to definite signs of hepatic steatosis (grade 1–3 hepatic steatosis on abdominal ultrasound), our results may not apply to patients with earlier hepatic steatosis stages on ultrasound or those individuals with sonographically undetectable NAFLD. Further long-term, prospective, observational studies on various ethnic groups in all stages of NAFLD are recommended to determine NAFLD's possible role as a predictive factor for developing CVD complications of T2D.

In the present study, although after adjustment for different variables, only NAFLD increased the incidence risk of CVD in T2D after 5 years, there was a positive association between NAFLD and liver enzymes (ALT, AST, ALK-P, and GGT) in increasing the incidence risk of CVD in T2D.

Ethics approval and consent to participate

Approval of the research protocol:

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The studies involving human participants were reviewed and approved by Ethics Committee of Tehran University of Medical Sciences.

Informed Consent:

Informed consent was obtained from all individual to participate in the study.

Consent to publication:

Not applicable.

Acknowledgements:

Authors wish to thank patients for their participation and kind cooperation.

Funding Statement:

Research is reported in this publication was supported by Elite Researcher Grant Committee under award number 996449 from the National Institutes for Medical Research Development (NIMAD), Tehran, Iran.

Conflict of interest disclosure:

The authors declare that they have no competing interests.

Authors contributions:

M.D.F and A.P contributed in drafting. A.S and F.D.F contributed in data analysis. F.M and S.R contributed in data gathering. S.M contributed in resources. M.N contributed in draft revision and study design. A.E contributed in supervision, study design, and draft revision. All authors read and approved the final manuscript.

Data availability: All data generated or analysed during this study are included in this submitted article.

Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat reviews Gastroenterol Hepatol. 2018;15(1):11–20.
Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat reviews Gastroenterol Hepatol. 2013;10(6):330–44.
Babaniamansour S, Aliniagerdroudbari E, Niroomand M. Glycemic control and associated factors among Iranian population with type 2 diabetes mellitus: a cross-sectional study. J Diabetes Metabolic Disorders. 2020;19(2):933–40.
Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology. 1999;116(6):1413–9.
Marchesini G, Brizi M, Morselli-Labate AM, Bianchi G, Bugianesi E, McCullough AJ, et al. Association of nonalcoholic fatty liver disease with insulin resistance. Am J Med. 1999;107(5):450–5.
Hamaguchi M, Kojima T, Takeda N, Nakagawa T, Taniguchi H, Fujii K, et al. The metabolic syndrome as a predictor of nonalcoholic fatty liver disease. Ann Intern Med. 2005;143(10):722–8.
Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui JM, Fung C, et al. NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology (Baltimore MD). 2002;35(2):373–9.
Niroomand M, Babaniamansour S, Aliniagerdroudbari E, Golshaian A, Meibodi AM, Absalan A. Distress and depression among patients with diabetes mellitus: prevalence and associated factors: a cross-sectional study. Journal of Diabetes & Metabolic Disorders; 2021.
Unamuno X, Gómez-Ambrosi J, Rodríguez A, Becerril S, Frühbeck G, Catalán V. Adipokine dysregulation and adipose tissue inflammation in human obesity. Eur J Clin Invest. 2018;48(9):e12997.
Buzzetti E, Pinzani M, Tsochatzis EA. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metab Clin Exp. 2016;65(8):1038–48.
Kitade H, Chen G, Ni Y, Ota T. Nonalcoholic Fatty Liver Disease and Insulin Resistance: New Insights and Potential New Treatments. Nutrients. 2017;9(4).
Babaniamansour P, Mohammadi M, Babaniamansour S, Aliniagerdroudbari E. The Relation between Atherosclerosis Plaque Composition and Plaque Rupture. J Med signals Sens. 2020;10(4):267–73.
Wong VW, Wong GL, Yip GW, Lo AO, Limquiaco J, Chu WC, et al. Coronary artery disease and cardiovascular outcomes in patients with non-alcoholic fatty liver disease. Gut. 2011;60(12):1721–7.
Targher G, Day CP, Bonora E. Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease. N Engl J Med. 2010;363(14):1341–50.
Moosaie F, Firouzabadi FD, Abouhamzeh K, Esteghamati S, Meysamie A, Rabizadeh S, et al. Lp(a) and Apo-lipoproteins as predictors for micro- and macrovascular complications of diabetes: A case-cohort study. Nutr Metabolism Cardiovasc Dis. 2020;30(10):1723–31.
Targher G, Bertolini L, Padovani R, Rodella S, Tessari R, Zenari L, et al. Prevalence of Nonalcoholic Fatty Liver Disease and Its Association With Cardiovascular Disease Among Type 2 Diabetic Patients. Diabetes Care. 2007;30(5):1212.
Hamaguchi M, Kojima T, Takeda N, Nagata C, Takeda J, Sarui H, et al. Nonalcoholic fatty liver disease is a novel predictor of cardiovascular disease. World J Gastroenterol. 2007;13(10):1579–84.
Liu H-H, Cao Y-X, Sun D, Jin J-L, Guo Y-L, Wu N-Q, et al. Impact of Non-Alcoholic Fatty Liver Disease on Cardiovascular Outcomes in Patients With Stable Coronary Artery Disease: A Matched Case-Control Study. Clin Transl Gastroenterol. 2019;10(2):e00011–e.
Targher G, Bertolini L, Rodella S, Tessari R, Zenari L, Lippi G, et al. Nonalcoholic fatty liver disease is independently associated with an increased incidence of cardiovascular events in type 2 diabetic patients. Diabetes Care. 2007;30(8):2119–21.
Petit JM, Guiu B, Terriat B, Loffroy R, Robin I, Petit V, et al. Nonalcoholic fatty liver is not associated with carotid intima-media thickness in type 2 diabetic patients. J Clin Endocrinol Metab. 2009;94(10):4103–6.
McKimmie RL, Daniel KR, Carr JJ, Bowden DW, Freedman BI, Register TC, et al. Hepatic steatosis and subclinical cardiovascular disease in a cohort enriched for type 2 diabetes: the Diabetes Heart Study. Am J Gastroenterol. 2008;103(12):3029–35.
Sun W, Cui H, Li N, Wei Y, Lai S, Yang Y, et al. Comparison of FIB-4 index, NAFLD fibrosis score and BARD score for prediction of advanced fibrosis in adult patients with non‐alcoholic fatty liver disease: a meta‐analysis study. Hepatol Res. 2016;46(9):862–70.
Wada T, Zeniya M. Background of the FIB-4 index in Japanese non-alcoholic fatty liver disease. Intern Med. 2015;54(2):127–32.
Afarideh M, Ghajar A, Noshad S, Saadat M, Khajeh E, Esteghamati A. Serum 25-hydroxyvitamin D, non-alcoholic fatty liver disease and type 2 diabetes. Nutr Metabolism Cardiovasc Dis. 2017;27(1):93–5.
Afarideh M, Aryan Z, Ghajar A, Noshad S, Nakhjavani M, Baber U, et al. Complex association of serum alanine aminotransferase with the risk of future cardiovascular disease in type 2 diabetes. Atherosclerosis. 2016;254:42–51.
Esteghamati A, Ismail-Beigi F, Khaloo P, Moosaie F, Alemi H, Mansournia MA, et al. Determinants of glycemic control: Phase 2 analysis from nationwide diabetes report of National Program for Prevention and Control of Diabetes (NPPCD-2018). Prim Care Diabetes. 2020;14(3):222–31.
Levey AS, Coresh J, Greene T, Marsh J, Stevens LA, Kusek JW, et al. Expressing the Modification of Diet in Renal Disease Study equation for estimating glomerular filtration rate with standardized serum creatinine values. Clin Chem. 2007;53(4):766–72.
Schumann G, Klauke R, Canalias F, Bossert-Reuther S, Franck PF, Gella FJ, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C. Part 9: reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE) (1)). Clin Chem Lab Med. 2011;49(9):1439–46.
Thomas L, Müller M, Schumann G, Weidemann G, Klein G, Lunau S, Pick K. Consensus of DGKL and VDGH for interim reference intervals on enzymes in serum Konsensus von DGKL und VDGH zu vorläufigen Referenzbereichen für Serumenzyme. J Lab Med. 2005;29(5):301–8.
Valbusa F, Agnoletti D, Scala L, Grillo C, Arduini P, Bonapace S, et al. Non-alcoholic fatty liver disease and increased risk of all-cause mortality in elderly patients admitted for acute heart failure. Int J Cardiol. 2018;265:162–8.
Yoshihisa A, Sato Y, Yokokawa T, Sato T, Suzuki S, Oikawa M, et al. Liver fibrosis score predicts mortality in heart failure patients with preserved ejection fraction. ESC heart failure. 2018;5(2):262–70.
Mantovani A, Dauriz M, Sandri D, Bonapace S, Zoppini G, Tilg H, et al. Association between non-alcoholic fatty liver disease and risk of atrial fibrillation in adult individuals: An updated meta-analysis. Liver international: official journal of the International Association for the Study of the Liver. 2019;39(4):758–69.
Tana C, Ballestri S, Ricci F, Di Vincenzo A, Ticinesi A, Gallina S, et al. Cardiovascular Risk in Non-Alcoholic Fatty Liver Disease: Mechanisms and Therapeutic Implications. Int J Environ Res Public Health. 2019;16(17):3104.
Simon TG, Bamira DG, Chung RT, Weiner RB, Corey KE. Nonalcoholic Steatohepatitis is Associated with Cardiac Remodeling and Dysfunction. Obes (Silver Spring Md). 2017;25(8):1313–6.
Mantovani A, Rigolon R, Pichiri I, Bonapace S, Morani G, Zoppini G, et al. Nonalcoholic fatty liver disease is associated with an increased risk of heart block in hospitalized patients with type 2 diabetes mellitus. PLoS ONE. 2017;12(10):e0185459–e.
Kim SU, Song D, Heo JH, Yoo J, Kim BK, Park JY, et al. Liver fibrosis assessed with transient elastography is an independent risk factor for ischemic stroke. Atherosclerosis. 2017;260:156–62.
Abdeldyem SM, Goda T, Khodeir SA, Abou Saif S, Abd-Elsalam S. Nonalcoholic fatty liver disease in patients with acute ischemic stroke is associated with more severe stroke and worse outcome. J Clin Lipidol. 2017;11(4):915–9.
Tripodi A, Fracanzani AL, Primignani M, Chantarangkul V, Clerici M, Mannucci PM, et al. Procoagulant imbalance in patients with non-alcoholic fatty liver disease. J Hepatol. 2014;61(1):148–54.
EASL-EASD-EASO. Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016;64(6):1388–402.
Ballestri S, Romagnoli D, Nascimbeni F, Francica G, Lonardo A. Role of ultrasound in the diagnosis and treatment of nonalcoholic fatty liver disease and its complications. Expert Rev Gastroenterol Hepatol. 2015;9(5):603–27.
Pereira E, Silvares RR, Flores EEI, Rodrigues KL, Ramos IP, da Silva IJ, et al. Hepatic microvascular dysfunction and increased advanced glycation end products are components of non-alcoholic fatty liver disease. PLoS ONE. 2017;12(6):e0179654.
Jaruvongvanich V, Chenbhanich J, Sanguankeo A, Rattanawong P, Wijarnpreecha K, Upala S. Increased arterial stiffness in nonalcoholic fatty liver disease: a systematic review and meta-analysis. Eur J Gastroenterol Hepatol. 2017;29(9):e28–e35.
Steffen H, Demir M, Lang S, Schulte S, Töx U. HIGH RATE OF UNDETECTED ARTERIAL HYPERTENSION IN PATIENTS WITH NON-ALCOHOLIC STEATOHEPATITIS (NASH): PP.34.356. J Hypertens. 2010;28.
Tana C, Tana M, Rossi S, Silingardi M, Schiavone C. Hepatic artery resistive index (HARI) and non-alcoholic fatty liver disease (NAFLD) fibrosis score in NAFLD patients: cut-off suggestive of non-alcoholic steatohepatitis (NASH) evolution. J Ultrasound. 2016;19(3):183–9.
Biddinger SB, Hernandez-Ono A, Rask-Madsen C, Haas JT, Alemán JO, Suzuki R, et al. Hepatic insulin resistance is sufficient to produce dyslipidemia and susceptibility to atherosclerosis. Cell Metab. 2008;7(2):125–34.
Dowman JK, Tomlinson JW, Newsome PN. Pathogenesis of non-alcoholic fatty liver disease. QJM. 2010;103(2):71–83.
Stefan N, Kantartzis K, Häring HU. Causes and metabolic consequences of Fatty liver. Endocr Rev. 2008;29(7):939–60.
Shoelson SE, Lee J, Goldfine AB. Inflammation and insulin resistance. J Clin Investig. 2006;116(7):1793–801.
Villanova N, Moscatiello S, Ramilli S, Bugianesi E, Magalotti D, Vanni E, et al. Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease. Hepatology (Baltimore MD). 2005;42(2):473–80.
Tilg H, Moschen AR. Insulin resistance, inflammation, and non-alcoholic fatty liver disease. Trends Endocrinol Metab. 2008;19(10):371–9.
Volzke H, Robinson D-M, Kleine V, Deutscher R, Hoffmann W, Ludemann J, et al. Hepatic steatosis is associated with an increased risk of carotid atherosclerosis. World J Gastroenterol. 2005;11(12):1848–53.
Assy N, Djibre A, Farah R, Grosovski M, Marmor A. Presence of coronary plaques in patients with nonalcoholic fatty liver disease. Radiology. 2010;254(2):393–400.
Lazo M, Hernaez R, Bonekamp S, Kamel IR, Brancati FL, Guallar E, et al. Non-alcoholic fatty liver disease and mortality among US adults: prospective cohort study. BMJ (Clinical research ed). 2011;343:d6891.
Hu J, Xu Y, He Z, Zhang H, Lian X, Zhu T, et al. Increased risk of cerebrovascular accident related to non-alcoholic fatty liver disease: a meta-analysis. Oncotarget. 2017;9(2):2752–60.
Stine JG, Niccum BA, Zimmet AN, Intagliata N, Caldwell SH, Argo CK, et al. Increased risk of venous thromboembolism in hospitalized patients with cirrhosis due to non-alcoholic steatohepatitis. Clin Transl Gastroenterol. 2018;9(3):140.
Ma J, Hwang SJ, Pedley A, Massaro JM, Hoffmann U, Chung RT, et al. Bi-directional analysis between fatty liver and cardiovascular disease risk factors. J Hepatol. 2017;66(2):390–7.
Lonardo A, Ballestri S, Marchesini G, Angulo P, Loria P. Nonalcoholic fatty liver disease: a precursor of the metabolic syndrome. Digestive and liver disease: official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2015;47(3):181–90.
Ballestri S, Lonardo A, Bonapace S, Byrne CD, Loria P, Targher G. Risk of cardiovascular, cardiac and arrhythmic complications in patients with non-alcoholic fatty liver disease. World J Gastroenterol. 2014;20(7):1724–45.
Estruch R, Ros E, Salas-Salvadó J, Covas MI, Corella D, Arós F, et al. Primary Prevention of Cardiovascular Disease with a Mediterranean Diet Supplemented with Extra-Virgin Olive Oil or Nuts. N Engl J Med. 2018;378(25):e34.
Lincoff AM, Wolski K, Nicholls SJ, Nissen SE. Pioglitazone and Risk of Cardiovascular Events in Patients With Type 2 Diabetes MellitusA Meta-analysis of Randomized Trials. JAMA. 2007;298(10):1180–8.
Armstrong MJ, Gaunt P, Aithal GP, Barton D, Hull D, Parker R, et al. Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): a multicentre, double-blind, randomised, placebo-controlled phase 2 study. Lancet (London England). 2016;387(10019):679–90.
Cui J, Philo L, Nguyen P, Hofflich H, Hernandez C, Bettencourt R, et al. Sitagliptin vs. placebo for non-alcoholic fatty liver disease: A randomized controlled trial. J Hepatol. 2016;65(2):369–76.
Al-Mallah M, Khawaja O, Sinno M, Alzohaili O, Samra AB. Do angiotensin converting enzyme inhibitors or angiotensin receptor blockers prevent diabetes mellitus? A meta-analysis. Cardiol J. 2010;17(5):448–56.
Athyros VG, Tziomalos K, Gossios TD, Griva T, Anagnostis P, Kargiotis K, et al. Safety and efficacy of long-term statin treatment for cardiovascular events in patients with coronary heart disease and abnormal liver tests in the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) Study: a post-hoc analysis. Lancet (London England). 2010;376(9756):1916–22.
Tikkanen MJ, Fayyad R, Faergeman O, Olsson AG, Wun CC, Laskey R, et al. Effect of intensive lipid lowering with atorvastatin on cardiovascular outcomes in coronary heart disease patients with mild-to-moderate baseline elevations in alanine aminotransferase levels. Int J Cardiol. 2013;168(4):3846–52.
Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology (Baltimore MD). 2018;67(1):328–57.
Singh S, Khera R, Allen AM, Murad MH, Loomba R. Comparative effectiveness of pharmacological interventions for nonalcoholic steatohepatitis: A systematic review and network meta-analysis. Hepatology (Baltimore MD). 2015;62(5):1417–32.
Mechanick JI, Farkouh ME, Newman JD, Garvey WT. Cardiometabolic-Based Chronic Disease, Addressing Knowledge and Clinical Practice Gaps. J Am Coll Cardiol. 2020;75(5):539.
Mechanick JI, Farkouh ME, Newman JD, Garvey WT. Cardiometabolic-Based Chronic Disease, Adiposity and Dysglycemia Drivers. J Am Coll Cardiol. 2020;75(5):525.

No competing interests reported.

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Severe liver steatosis as potential risk factor of cardiovascular disease in patients with type 2 diabetes: a prospective cohort study

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Abstract

Background and Aims:

Methods and results

Conclusion

Figures

Introduction

Methods

1. Study Population:

2.Examination:

3.Laboratory Investigations:

4. Assessment of CVD:

5.Statistical Analysis:

Results

Baseline characteristics of the study population:

NAFLD and liver enzymes and incidence of CVD:

NAFLD and liver enzymes and incidence of CVD subgroups:

A. Association of APRI and NAFLD

Discussion

Pathophysiology of NAFLD and CVD

The role of NAFLD in CVD subtypes

Treatment of NAFLD

Conclusion

Declarations

References

Additional Declarations

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