Acute Phase Inflammatory Proteins, Gut Microbiota Dysbiosis and Severity of Nonalcoholic Fatty Liver Disease in Patients with Type 2 Diabetes Mellitus

Since obesity and type 2 diabetes mellitus (DM) became almost pandemic, incidence and prevalence of associated nonalcoholic fatty liver disease (NAFLD) rose accordingly. Aim of the study was assessing whether there is a link between circulating C-reactive protein(CRP), gut microbiota dysbiosis and severity of liver disease in patients with NAFLD and confirmed type 2 DM. 50 patients diagnosed with type 2 DM were consecutively enrolled in this cross-sectional, pilot study, being evenly divided in two groups, as matched pairs, on the basis of presence or absence of NAFLD confirmed by abdominal and CT exam, after ruling out a lot of diseases and conditions. Patients underwent measurements of waist circumference, blood pressure (BP), body mass index (BMI) and thoroughly physical examination. Complete blood count (CBC), liver tests, CRP, fasting plasma glucose (FPG), low density lipoprotein(LDL) and high-density lipoprotein(HDL) cholesterol, triglycerides, creatinin and uric acid, micro proteinuria as well as stool microbiology were run, using standardized methods. Fatty liver index (FLI) was also calculated. Fibromax (BioPredictive) was performed in NAFLD (+) patients. NAFLD (+) group displayed significant differences of age and BMI, as well as lab biochemistry: ALT, GGT, LDL-cholesterol and triglycerides, CRP, microprotienuria and stool microbiology.. Strong positive correlation of CRP with gut dysbiosis (DB) severity, microproteinuria, as well as with nonalcoholic steatohepatitis (NASH) score, were observed. DB range correlated positively in NAFLD (+) group with age, BMI, BP, dyspepsia as well as with NASH scores. In conclusion patients with NAFLD and associated type 2 DM displayed a specific profile with significant differences of age, BMI, FLI, diabetic treatment, biochemistry and gut microbiological charts. Strong positive correlations between CRP, gut DB and NASH score suggest the possibility of link between inflammation, gut microbiota DB and severity of liver disease.


1.Introduction
Nonalcoholic fatty liver disease (NAFLD) is a complex entity characterized by ≥5% accumulation of fat in liver cells, in the absence of excessive alcohol consumption or any other secondary causes, ranging from simple steatosis to inflammation and hepatocytes necrosis known as NASH (nonalcoholic steatohepatitis), or end stage liver disease (NASH-induced cirrhosis): fibrosis and architectural liver changes [1,2]. Since obesity and type 2 DM became almost pandemic, incidence and prevalence of NAFLD rose accordingly. It is estimated that in Asia and USA, between 2005 and 2010, the prevalence of NAFLD increased exponentially [3].

Patients and methods
2.1 Study design and methodology 2.1.1Inclusion criteria: 50 patients diagnosed with type 2 diabetes mellitus were consecutively enrolled in this cross-sectional, pilot study, being evenly divided in two groups, as matched pairs, on the basis of presence or absence of NAFLD confirmed by abdominal ultrasonography and CT exam. Study was approved by the Ethics Committee of Scientific Research of the University of Medicine and Pharmacy "Victor Babes" from Timisoara, Romania and was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent before the beginning of the study.

Exclusion criteria:
heavy consumption of alcohol: more than 40gr/day in men and more than 30gr/day in women, in past 10 years, significant exposure to industrial toxic substances or to medication with liver toxicity, hemochromatosis, alpha1 antitripsine deficiency, Wilson disease, autoimmune hepatitis, infection with viral B or viral C hepatitis, thyroid diseases, various organ failure (cardio-pulmonary, kidney or liver), oncologic conditions, long parenteral nutrition, associated inflammatory bowel disease, celiac disease or gluten sensitivity, various infectious diseases, malnutrition syndromes, as well as recent treatment with antibiotics or probiotics.

C-reactive protein (CRP)
is a nonglycosilated plasma protein, with an annular shape and pentameric structure, consisting of five conformational protein subunits, with high resistance to proteolysis, that migrates near the gamma zone on electrophoresis. It is synthesized by many immune cells with various locations, predominantly in the liver, as a reaction of proinflammatory interleukins and cytokines. Occasionally could be produced de novo in extrahepatic sites. Because its levels rise and fall depending on the amount of inflammation or tissue lesions in the body, CRP is also known as an acute phase reactant. To determine CRP, venous blood samples were collected in the morning, in fasting patients, within vacutainers and processed after centrifugation, using immunoturbidimetry assay. The cutoffs were 0.5 mg/dL [12].

Non invasive assessment of NAFLD 2.4.1.
Abdominal helical CT, as well as real-time high resolution ultrasonography were used to confirm or rule out fatty liver disease in study participants. Lipid accumulation determines decrease of liver density, which becomes less dense when compared to spleen. It is considered that a decrease more than 10 Hounsfield units (HU) on a native CT is consistent with diagnosis of liver steatosis, while using contrast the difference between liver and spleen augments to more than 20 HU. Abdominal ultrasonography was performed in all study participants, with semiquantitative assessment of steatosis, when observed, as follows: mild, moderate and severe [13].

2.4.2.
Fibromax, developed by BioPredictive®, consistent with 5 different noninvasive tests, from which: FibroTest®, SteatoTest® and NashTest® were actually used in this study, in the evaluation of NAFLD (+) group. This complex test is based on validated mathematical application, that uses some biochemical blood variables, such as: alpha-2 macroglobulin, haptoglobin, apolipoprotein A1, total bilirubin, gamaglutamiltranspeptidase( GGT), alanin -aminotransferase (ALT), aspartataminotransferase (AST), fasting plasma glucose (FPG), cholesterol and triglycerides, as well as some clinical data such as: age, gender, weight and height, in order to determine the severity of liver disease [14].

Figure 3C. Correlations of gut DB to CRP and microproteinuria in NAFLD (+) group
In accordance with others, we observed that NAFLD+ group displayed significant older age by comparison to NAFLD-participants. It seems that incidence and prevalence of NAFLD increase with growing old, also resulting in more severe biochemical and histological features [17].
As others reported, we also observed that FLI showed utility and reliability in the assessment of risk for liver steatosis, while all study participants with confirmed NAFLD exhibited levels above 60 units [18].
Whether current, DZ treatment influenced results of this study, in respect to presence and severity of fatty liver, it is however not clear. Patients with the second group (NAFLD negative), were mainly treated with oral antidiabetic drugs (OAD) (metformin), while the group displaying NAFLD did not further receive OAD, patients becoming insulin necessitant. In this regard many multicenter studies and meta-analysis investigated relation between various OAD and NAFLD, but results are neither coherent nor homogeneous, and rather contradictory. However, to date, European Association for Study of the Liver (EASL) did not yet introduce any specific recommendation of drugs in the guidelines for NAFLD treatment [19]. To record, all patients in this study with NAFLD received lipid lowering agents, basically statins, in a significant higher perecentage, by comparison to those without NAFLD and a few of them combination of statins with fibrates. Even NAFLD group was basically treated with antidiabetics and statins, they showed elevated transaminases amd GGT level. However, there is yet no conclusive evidence that dyslipidemia treatment with any kind of agent either statins, fibrates, fish oil, etc,, could significantly improve liver histology severity or outcome in NAFLD patients [20]. https://doi.org/10.37358/RC.20.5.8136 Gut microbiota DB, obesity and metabolic syndrome are close related, as confirmed by many recent studies, including ours, conducted both in animal and humans, by modulation of host nutritional and energy metabolic pathways. No less important seems to be so called metabolic endotoxinemia, as a part of this complex process, related to overgrowth of some microbial gut species and reduction of local flora biodiversity, with increased ratio Firmicutes to Bacteroidetes. These aspects correlate with adiposity, insulin resistance and proinflammatory status. [21] Interestingly, metformin seems to stimulate bacteria species that produce butyrate and consecutively promote normobiosis or a healthy microbiome. [22] In accordance with these observations we have recorded significant lower rates of dysbiosis severity in patients without NAFLD, treated by OAD-metformin.
Inflammation, gut DB and NAFLD seem to be linked, as many studies support this hypothesis. The bioenergetic pathways related to mithocondria activity, immunometabolism with pivotal, biochemical reactions, performed by numerous immune cells effectors, could be reprogrammed under particular stimuli. One of these stimuli that can drive host immunological functions is represented by gut mibrobiota DB. DB could definitively affect the ability of adaptative, inflammatory responses and trigger proinflammatory cells with release of various interleukins and cytokines, eventually altering the host metabolism. [23] In this regard, we have noticed a positively strong correlation not only between the levels of CRP and range of DB severity, as well as NASH scores, but also directly between DB severity itself and NASH scores.
Results of the present study have also highlighted strong positive correlations between range of DB and several clinical data, such as: age, BMI and SBP. Many recent studies advocated the role of DB in the setting of cardio-vascular risk, via trimethylamine N-oxide,short chain fatty acids and secondary bile acids, that interfere various signaling and communication pathways, resulting in hypertension, atherosclerosis and coronary artery disease. Otherwise cardio-vascular comorbidities represent another important issue to manage in NAFLD population, as we also observed in present study [24].
There is an increased risk of associated microproteinuria and chronic kidney disease (CKD) in patients with NAFLD, as a recently meta-analysis concluded.
[25] Our study identified significant increased incidence of microalbuminuria in NAFLD (+) group, suggesting the importance of screening for CKD in population already affected by NAFLD, not to mention the risk for cardio-vascular diseases. We also observed a strong correlation between microalbuminuria levels and DB severity.
Given data from present study and many others, inflammation is a fundamental process, implicated in many conditions, orchestrated by gut microbiota DB, that triggers other participants either local or distant, which further aggravates the initial condition. [26] Since the understanding of this basic process associates DB with inflammation, a lot of scientists embraced the idea of natural remedies , such as probiotics, in order to influence this pathophysiological pathway and the occurrence or development of many disorders. [27] In this regard, other potential natural antiinflammatory formulas could be taken into account, based on their promising preliminary results [28,29]. No less important will be to improve our possibility to predict the risk for DM and its complications, including NAFLD. [30] Limitations This observational study has a few limitations. Even if it is a pilot study, though the sample size of study population is relatively small, with consecutive potential statistical draw-backs. Another limitation is related to methodology of NAFLD diagnostic. Although the diagnosis protocol was homogenous, it did include only noninvasive methods, which could trigger ambiguity, considering that liver biopsy remains the gold standard in liver diseases diagnosis. Not least, given the crosssectional study, a lot of statistical bias could also intervene, related to sources of information bias and possible confounders.

Conclusions
Patients with type 2 DM that developed NAFLD displayed a profile characterized by older age, higher BMI and FLI range, necessity of insulin therapy, modified biological charts that included acute inflammatory phase protein-CRP, liver tests, serum circulating lipids, microproteinuria, as well as https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (5) higher range of gut DB. Strong positive correlations observed between CRP, gut DB and NASH and also between DB, BMI and NASH suggest the possibility of link between inflammation, gut microbiota DB and severity of liver disease.