Original papers Internal Medicine 2019I4 vol. XV No. - www.srmi.ro 13 10.2478/inmed-2019-00 73

THE RELATIONSHIP BETWEEN HEPATIC STEATOSIS, AND INSULIN RESISTANCE IN TYPE 2 WITH METABOLIC IMBALANCE

Elena-Daniela Grigorescu11,21,3 , Mariana Floria , Cristina Mihaela Lăcătușu , Bogdan Mircea-Mihai1,3 , Ioana Creu 1,4 , Alina Delia Popa 1,3 , Alina Onofriescu 1,3 , Irina M. Jaba 1 , Victoria Șorodoc1,5 , Alexandr Ceasovschih 1,5 , Laureniu Șorodoc 1,5 1“Grigore T. Popa” University of Medicine and Pharmacy Iași, 23rd Medical Clinic,“St. Spiridon” Emergency Clinical Country Hospital Iaşi, 3Clinical Center for Diabetes, Nutrition and Metabolic Iași, 4Department Preventive Medicine and Interdisciplinarity, 52nd Medical Clinic,“St. Spiridon” Emergency Clinical Hospital Iaşi Correspondent author: Lecturer Dr. Victoria Șorodoc, E-mail: [email protected]

Abstract Aim. Non-alcoholic fatty liver (NAFLD) and type 2 diabetes mellitus (T2DM) are in a bidirectional relationship. This prospective study focused on associations between parameters common to the of insulin resistance, inflammation and hepatic steatosis in T2DM patients with metabolic imbalance. Methods. We used clinical data, insulin resistance and inflammation indices, and hepatic steatosis markers from 120 patients. Results. The patients (44% men, mean age 58) had a mean body mass index (BMI) of 32 kg/m2 and mean T2DM history of 6 years. With exceptions, significant correlations were found between metabolic, inflammatory and hepatic parameters. Conclusions. In T2DM patients with poor glycemic control, hepatic steatosis correlates significantly with insulin resistance and inflammation. Increased prevalence and poor prognosis of these diseases together justify the need for NAFLD screening of diabetic patients. Keywords: hepatic steatosis markers, type 2 diabetes, subclinical inflammation.

Rezumat Scop. Între boala ficatului gras non-alcoolic (NAFLD) și diabetul zaharat de tip 2 (DZ2) relaia este bidirecională. În acest studiu prospectiv am analizat asocierea parametrilor căilor comune de patogeneză insulinorezistenă-inflamaie-steatoză hepatică la pacienii cu DZ2 dezechilibrai metabolic. Material și metodă. Au fost utilizate date clinice, indici privind insulinorezistena și inflamaia, respectiv markeri surogat ai steatozei hepatice de la 120 de pacieni. 14

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Rezultate. Pacienii (44% bărbai, vârsta medie 58 ani) au avut media indicelui de masă corporală de 32 kg/m2 și 6 ani durata medie a DZ2. Cu excepii, observăm corelaii semnificative între parametrii metabolici, inflamatori și hepatici. Concluzii. La pacienii DZ2 cu control glicemic nesatisfăcător, markerii predictori ai steatozei hepatice se corelează semnificativ cu insulinorezistena și inflamaia. Prevalena crescută și evoluia nefavorabilă a asocierii acestor afeciuni justifică necesitatea screening-ului NAFLD în rândul pacienilor cu diabet zaharat. Cuvinte cheie: markeri hepatici ai steatozei, diabet zaharat tip 2, inflamaie subclinică.

Introduction clinical and biological parameters. Among these are the Fatty Liver Index (FLI), the With a prevalence of 24% in 2018, non- NAFLD-Liver Fatty Score (FLS), and the alcoholic fatty (NAFLD) is Hepatic Steatosis Index (HSI). Given that the contributing to the current pandemic surge liver biopsy, currently the golden standard of chronic illness. On one hand, NAFLD is a for diagnosing NAFLD, is invasive, costly and leading cause of liver disease in adults. On not without risk, the question is whether such the other, its progression shares the main models can provide sufficient accuracy and features of other noncommunicable diseases reliability for both the diagnostic stage and in the sense that it involves a wide spectrum the subsequent follow up of histological of entities which are clinicohistopatho- developments(1,4) . logically distinct, from the uncomplicated From the point of view of prevention, as well hepatic steatosis to non-alcoholic as for the deeper understanding of the . These range from benign complex patho-physiological mechanisms manifestations to the more severe involved, it makes sense to suspect NAFLD in and even hepatocarcinoma(1,2,3) . all the patients who present with metabolic The scientific literature already provides risk factors such as obesity, dyslipidemia, several assessment models which have been hypertension and insulin resistance(5,6) . proven accurate – or at least promising - in Moreover, the bidirectional relationship establishing the diagnosis of hepatic between NAFLD and type 2 diabetes mellitus steatosis using markers and indices based on (T2DM) is widely accepted, and we know that Original papers Internal Medicine 2019I4 vol. XV No. - www.srmi.ro 15

T2DM, too, is exacerbated by the same risk were not considered. Also excluded factors(7) . from the research were smokers and female In addition, recent research has pointed to patients who were pregnant or seeking to the progression from NAFLD toward NASH in procreate. The 84 patients selected for the tandem with the aggravation of pre-diabetes study group were those whose ongoing to full blown diabetes, and this should be therapy schemes required supplementing taken into consideration when diagnosing a with a GLP-1 receptor agonist (15 patients), a diabetic patient(3) . For instance, FLI has been DPP-4 inhibitor (69 patients), sulfonylurea or found a valid predictor of diabetes in pre- acarbose, according to the diabetologist's diabetics within as few as 3 years. In the clinical judgment. The other 36 patients same study, elevated FLI values (>60) formed the control group (30%). indicative of hepatic steatosis were After obtaining informed consent in writing, concurrently strong independent predictors the patients were asked to fill in a for the risk of developing diabetes (HR = questionnaire with general demographic and 4.97)(8) . medical information, including their history In light of the above, it is of both scientific and of diabetes, treatment and comorbidities, if clinical interest to assess the extent to which any. They were then examined clinically; the parameters and indices for insulin their weight, height, waist circumference resistance, inflammation and hepatic were also measured and their BMIs steatosis correlate. This prospective study calculated. aims to investigate such associations and Toprofile the patients' , glycemic levels, their implications in T2DM patients who are kidney and liver functions (AST, ALT, GGT), struggling to achieve glycemic control and blood samples were collected in the morning, are in a state of metabolic imbalance. after overnight fasting. These were immediately centrifuged for 5 min at 3 000 G Materials and methods to collect insulin and C-peptide data. Chemiluminiscence (IMMULITE 1000) was The present study employs data obtained used on the serum stored at -20o C in order to from 120 consenting diabetic outpatients measure hsCRP, IL-6, insulin and C-peptide with poor glycemic control, who were treated levels, while Ion-exchange high-performance at a Clinical Center of Diabetes, Nutrition and liquid was used to measure

Metabolic Diseases over the course of 20 HbA1c. months during 2016-2018. Their inclusion in Insulin resistance was assessed using the the study cohort was based on age (30-75), validated formulae HOMA-IR = (glycemia à diagnosis of type 2 diabetes unsatisfactorily jeun in mg/dl × insulinemia à jeun in μU/ml) / managed with metformin and/or 405, HOMA C-peptide = (glycemia à jeun in sulphonylurea. Patients with a known history mg/dl /18 × C-peptide × 3.003) / 22.5 and of atherosclerotic cardiovascular disease index C-peptide = 20/[(C- peptide × 3,003) × (myocardial infarction, angina, coronary (glicemia à jeun (mg/dl)/18)] (9). Also, for the revascularization, signs of , stroke, liver steatosis prediction markers, we transient ischemic attack and peripheral calculated the Fatty Liver Index (FLI), Hepatic arterial disease), severe liver or kidney Steatosis Index (HSI) and Non-Alcoholic Fatty disease, acute pancreatitis, or major chronic Liver Disease-Liver Fat Score (NAFLD-LFS) 16

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using online and mobile interfaces provided of 32.75±5.65kg/m2 and the mean by MDCalc, a well known and widely used abdominal circumference of 109,48±12.62 medical reference and e-tool(10-12) . cm. These features are consistent with The database was compiled electronically known patterns of obesity among T2DM and, apart from the general descriptive patients worldwide. statistics, the following statistical analyses Also, most patients had dyslipidemia and/or were conducted in SPSS 17.0 for Windows hypertension as pathological antecedents (SPSS Inc., Chicago): the t-test, Spearman and comorbidities. Non-alcoholic hepatic correlations, linear regression, logistic steatosis was also noted in 15% of the cases, regression and area under the ROC curve, at while only 15 patients suffered exclusively p < 0.05 statistical significance thresholds. from type 2 diabetes mellitus. Treatment wise, 70% of the patients were undergoing Results monotherapy, of whom only 3 persons were on sulfonylurea, and the rest were taking The results hereby presented are part of metformin. The other 30% were prescribed a initial patient assessment data collected in a combination of oral metformin and still ongoing prospective research at doctoral sulfonylurea (23.3%) or metformin and level. The data were analyzed descriptively acarbose. and comparatively, establishing the With regard to the patients' metabolic, homogeneity of features across both the inflammatory and hepatic statuses, the study and the control groups. This, then, may mean values computed for the entire cohort facilitate adequate highlighting of treatment may be summarized as follows: effects onto the cardiac function and l Metabolic status (including glycemic systemic chronic inflammation, as they are and profile): glycemia = monitored and measured over time. 171. 60 ±43.24 mg/dl , HbA1c = The cohort was comprised of 120 patients (of 8.03±0.95%, total cholesterol = whom 44,3% men) with a mean history of 195.04±47.49 mg/dl, LDL-cholesterol T2DM of 6.04 ± 4.63 years. Their mean age = 103.9±40.68 mg/dl, HDL- was 58.16 ± 8.72, most patients (56.7%) cholesterol = 56.97±15.09 mg/dl, being 50-65 years old, 20% younger than 50, triglycerides = 205.19±93.4 mg/dl, and 23.3% older than 65. Also noteworthy is uric acid = 5.41±1.35 mg/dl that 66.7% were obese and 29.2% l Insulin resistance indices: HOMA-IR = overweight, as indicated by their mean BMI 6.08±4.0, HOMA C-peptide = Original papers Internal Medicine 2019I4 vol. XV No. - www.srmi.ro 17

Table 1. The baseline parameters in patients included in the study 18

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4.15±2.0, index C-peptide = hepatic steatosis were influenced in any way 0.26±0.16 by the degree of glycemic imbalance and l Inflammation markers: on average, severity of insulin resistance. The Kruskall hsCRP = 9.69±10.49 mg/l, IL-6 = Wallis test revealed that the difference 3.39±4.57 pg/ml between the average ranking for each of the l Routine liver function test results: 3 calculated markers reached the p <0.001 AST = 27.64±18.46 U/L, ALT = threshold of statistical significance for the 36.58±24.0 U/L, GGT = 46.19±46.76 degree of insulin resistance (HOMA-IR<2, 2- U/L. 5, >5). This was not so when the variable l Hepatic steatosis indices: FLI = used in the comparison were the degree of 92.93±16.18, NAFLD-LFS = glycemic imbalance (HbA1c<7.5, 7.5-8, >8) 1.65±1.74 and HSI = 42.13±6.24. (see table 3).

Table 1. is a summary of the baseline ROC Curve characteristics of the subjects included in the Source study and control groups. The Spearman of the Curve correlation formula was employed to assess FLI the relationship between the inflammation Hepatic Steatosis markers, insulin resistance and hepatic Index NAFLDLFS steatosis predictor markers. The results are 1.0 Reference Line summarized in Table2 below. 0.8 The preliminary results with regard to the

relationship between insulin resistance and 0.6 inflammation in the same patients have Sensitivity already been presented in national 0.4 congresses(13) ; statistically significant 0.2 correlations were noted between HOMA-IR and the inflammation markers IL-6 (r=0.22, 0 p=0.012) and hsCRP (r=0.29, p=0.001). At 0 0,2 0,4 0,6 0,8 1,0

the same time HOMA C-peptide correlated 1 - Specificity weakly only with hsCRP levels (r=0.22, p=0.01). We were also interested in Figure 1. AUROCs for diagnostic accuracy of establishing if the predictor markers for severe insulin resistance Original papers Internal Medicine 2019I4 vol. XV No. - www.srmi.ro 19

Table 2. The correlations between hepatic steatosis, inflammation and insulin resistance markers

Table 3. Difference of steatosis marker values depending on the insulin resistance and glycemic control 20

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The diagnostic performance of the indices IR at p=0.02 justifying incretinic therapy for was tested by the area under the receiver the 64 patients whose values were >5). Also, operating characteristic curve (AUROC). This regarding the lipid profile, the total showed that all three surrogate markers for cholesterol of 42.5% of patients exceeded hepatic steatosis also act as predictors of 200 ml/dl and only 17.9% had their LDL- severe insulin resistance expressed as cholesterol under 70 ml/dl. At the same time, HOMA-IR>5. the triglycerides in two thirds of the patients In our study, the AUROC for FLI was 0.78 were higher than 150 mg/dl. [95% CI (0.70, 0.86)]. For HSI it was 0.70 The appropriate correlation formulae were [95% CI (0.60, 0.79)], and for NAFLDLFS it selected in order to analyse any associations was 0.87 [95% CI (0.81, 0.94]. bearing statistical significance between the various parameters. Thus, we identified that Discussion insulin resistance levels expressed as HOMA- IR correlated significantly with the patients' NAFLD is recognized as a public health HDL-cholesterol values (r=-0.24 at problem of epidemic proportions, and its p=0.007). Similarly, HOMA C-peptide values prevalence among T2DM patients reaches a correlated with the patients' triglycerides staggering 56.8 – 70%(14-15). The two medical (r=0.19 at p=0.03) and with uric acid levels conditions combined add to already elevated (r=0.28, p=0.001). levels of cardiovascular risk in these Although the coefficient points to only a weak patients. This substantiates our interest in positive correlation, it suggests that the clinical and biological data we collected, outpatients, too, may be resistant to insulin with a view to unravelling the pathophy- to a certain degree, even if this is not siological mechanisms of insulin resistance, specifically measured in their case. The subclinical inflammation and hepatic practical usefulness of this observation lies in steatosis. providing more convincing explanations and Overall, the baseline features of the patients increasing the patient's adherence to weight included in our study as calculated by means and lipid control measures. As a central of validated formulae point to metabolic feature of metabolic syndrome, insulin imbalance, poor long-term glycemic control resistance accelerates multiple damaging (HbA1c 8.03%), and substantial insulin processes (inflammation, thrombosis, resistance (HOMA-IR 6.08±4 and C-peptide oxidation) which, in turn, add to the level of 4,15±2; the significant difference for HOMA- cardiovascular risk (16). Original papers Internal Medicine 2019I4 vol. XV No. - www.srmi.ro 21

The other element in the chain linking insulin potential consequences are twofold: on one resistance to cardiovascular risk is the hand, insulin resistance and the onset of presence of subclinical inflammation. We diabetes mellitus per se, and, on the other, a investigated it by assessing the serum levels build up of fat in the liver in the form of of IL-6 and hsCRP, two markers of particular atherogenic dyslipidemia (elevated LDL- relevance in type 2 diabetic patients given cholesterol and triglycerides, low HDL- how inflammatory cytokines alter cholesterol) and increased oxidative stress cardiomyocytes as another form of chronic (7). (17). These intervene further by Also, we analyzed the levels of hsCRP in augmenting the effects of insulin resistance: conjunction with the patients' BMI and the free fatty acids resulting from the learned that inflammation levels differ activation of intracellular kinases lead to significantly between patients who are serine phosphorylation at the level of insulin normal weight and those who are overweight receptors and, in this way, undermine the (1.6 mg/l versus 9.01 mg/l, p =0.01) or obese signalling pathway of insulin(18). In light of groups (1.6 mg/l versus 10.52 mg/l, p <0.01). these insights, the relationship between The fact that the significance threshold was insulin resistance parameters and not reached when comparing overweight inflammatory markers fell within the scope of and obese patients (p =0.48) demonstrates our analysis. that any patient with a BMI > 25 kg/m2 is As discussed elsewhere, the degree to which likely to develop subclinical inflammation. our T2DM patients are subject to subtle The evaluation of the subclinical inflammatory processes is of material clinical inflammatory status based on the mean interest, given the wide-ranging damage values of the 2 markers in relation to the that inflammation exerts on the body(19). The patients' weight status (normal, overweight, hsCRP values recorded suggest that no less obese) indicated the only statistically than 75% of the patients were at high risk of significant difference in the case of hsCRP (p experiencing adverse cardiovascular events, =0.04). as hsCRP > 3 mg/l in 88 cases and the median Upon subgroup analysis, as between normal value of hsCRP was as high as 5.40 mg/l weight and overweight patients, we noted (interquartile range 8.92) even after that the statistical significance threshold was excluding two extreme cases > 50 mg/l. reached for hsCRP (1.6±0.93 mg/l vs. The patients' BMIs correlated positively with 9.01±11.21 mg/l, p=0.001). their levels of hsCRP (r=0.33, p=0.00), as This was not seen in the case of IL-6, for well as their IL-6 values (r=0.244, p=0.007). which p=0.3 and the mean values for the The same trend was noted when abdominal patients with normal weight compared to the circumference was taken into consideration overweight ones were 2±0 pg/ml vs. (r=0.403 at p=0.00, and r=0.206 at 3.29±2.77 pg/ml. Also, while the results for p=0.024, respectively). This reinforces the normal weight vs obese patients were knowledge that abdominal weight surplus significant with regard to hsCRP (1.6 vs. leads to dysfunction by 10.52, p=0.00), there was no statistical increasing the flow of free fatty acids in the significance when comparing the overweight liver, the synthesis of inflammatory patients with either normal weight or obese, cytokines and a decrease in adiponectin. The whatever the marker taken into 22

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consideration (p=0.81 for IL-6, and p=0.48 correlation between inflammation and for hsCRP). When assessing the patients' insulin resistance. By applying the insulin poor glycemic control (no exceptions to resistance grading scale HOMA-IR <2, 2-5, HbA1c >7) in conjunction with inflammation and >5 to the inflammation results, we found markers, HbA1c correlated positively with that both IL-6 and hsCRP levels accurately hsCRP(r=0.18,p=0.042), but less than in confirmed the presence of a higher degree of other studies(20) . HbA1c also correlated with insulin resistance defined as HOMA-IR >5 for IL-6 (r=0.41, p<0.001). These findings 64 subjects. The AUROC was 0.63 for IL-6 (CI: suggest that the link between glycemic 0.53-0.74;p=0.01)and0.68forhsCRP(CI: imbalance and subclinical inflammation is 0.58-0.78;p=0.01). Given the inhibitive not necessarily a strong one, but may require costs and poor availability of some biological additional analyses to address potential markers profiling the metabolism of diabetic confounding factors which may diminish the patients, the inclusion of hsCRP in routine strength of the relationship (e.g. years of tests could facilitate the early identification diabetes history, other comorbidities, age, of insulin resistance in patients with no gender, treatment). Also, note should be clinical signs of inflammation, and justify the made that our cohort was made exclusively subsequent prescription of medication to of diabetic subjects whereas other studies address the subtle build up of insulin included non-diabetics as well as diabetics, resistance. which might explain the difference. The vicious cycle between inflammation and The average duration of disease progression insulin resistance in diabetic patients is was of 7 years, which is less than recorded by further augmented by hepatic steatosis. others(21,22) . The patient distribution based on Research has shown that being at high risk of their T2DM evolution (<5, 5-10, >10 years) developing T2DM also multiplies the risk of reveals that the serum levels of the studied NAFLD fivefold(23) . Moreover, NAFLD is not inflammation markers did not differ necessarily a consequence, but rather one of significantly.Glycemic imbalance is known to the factors causing the metabolic syndrome aggravate the degree of subclinical or even type 2 diabetes mellitus, worsening inflammation. However, our analysis of its progression(5) . For instance, patients patient data indicative of moderate and poor suffering from both T2DM and NAFLD glycemic control did not yield any struggle harder to achieve satisfactory statistically significant differences either. glycemic control compared to diabetic Equally interesting is the significant positive persons without NAFLD(24) . Original papers Internal Medicine 2019I4 vol. XV No. - www.srmi.ro 23

The cut-off values for the hepatic steatosis effects of liver fat accumulation on the liver markers evaluated in our study are FLI > 60, itself and on other organs. HIS > 36 and NAFLD-LFS > -0.64. The first With regard to the limits of research – ours as two were reached by 91% of the patients and well as generally on this topic – the data the latter by 100%(10-12). featured in this study does not allow us to Findings reported in the literature with establish the relationship of causality in the regard to the 3 markers predicting hepatic observed associations. Predicting hepatic steatosis are promising, e.g. their steatosis using the studied markers may be association with abnormal insulin sensitiviy inexpensive and straightforward(28) , but its and secretion in 92 non-diabetics of normal reliability has yet to be confirmed by means weight(25) , or how FLI values higher than 60 of hepatic ultrasound. Although it requires appeared to predict the risk of developing specialized equipment and training, T2DM in Korean subjects monitored over a ultrasonography is currently a common period of 2.6 years (HR = 2.84)(26) . Also, in method for diagnosing a fatty liver and, in another study enrolling type 1 diabetic addition, it allows for the semi-quantitative patients, FLI was shown as a predictor of ultrasonographic scoring of mild, moredate metabolic syndrome and liver fat content in and severe hepatic steatosis. For some of the 41 of 201 patients assessed using magnetic 15% of patients diagnosed with hepatic resonance, and in the case of those with steatosis in our study, this diagnosis was confirmed hepatic steatosis, FLI was as high maintained based on prior medical records as 83.5 and HSI was 35.58(27) . and altered transaminase levels. The analysis of trends and patterns in our Our position in favor of calculating these data revealed that hepatic steatosis surrogate parameters for hepatic steatosis is predictor markers correlated positively with also informed by situations when patients both inflammation markers and insulin could suffer from underlying NAFLD without resistance indices. The most significant of concurrently having abnormal levels of coefficients was that between HOMA-IR and serum liver enzymes and/or ultrasound NAFLD-LFS (r=0.77, p<0.001), possibly due confirmation. Apart from screening to the fact that both formulae include plasma applications, these markers could be used to insulin values. In another study aiming to research and/or diagnose hepatic steatosis assess the performance and limitations of (retrospectively) without having to resort to markers predicting hepating steatosis by the liver ultrasound as routine examination. comparing them with liver biopsy results in a Furthermore, diabetologists were proven to cohort of 320 patients (of whom 41% with grossly underestimate the prevalence and T2DM and 50% with metabolic syndrome), severity of advanced fibrotic NAFLD in their the three markers pointed reliably to the diabetic patients (only 5% correctly chose diagnosis of hepatic steatosis, but without the 50-75% interval). facilitating accurate grading of severity(4) . Also, few reported using or intending to use For instance, moderate hepatic steatosis non-invasive staging algorithms(29) . Such could not be told from the more severe one issues related to clinician perceptions and based on FLI and NAFLD-LFS values alone. practices provide an interesting avenue for Such a distinction, however, would be further research in conjunction with instrumental in addressing the harming understanding the pathways by which NAFLD 24

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