In this study, we quantitatively profiled, for the first time, bile acids in the serum of T2DM patients with or without NAFL based on targeted metabolomics, and combined traditional clinical characteristics to predict the development of NAFL, then further validated the results by another independent cohort study. Finally, we defined 5 serum bile acids extracted from the whole metabolites and combined traditional clinical characteristics to predict the NAFL status in T2DM patients. Interestingly, serum bile acid profiles in T2DM patients with NAFL were significantly different from T2DM patients without NAFL, as characterized by the significant elevation of LCA, TLCA, CDCA24G, TUDCA and TCDCA, which may be potential biomarkers for diagnosis of NAFL in T2DM based on UPLC-TQ/MS and OPLS-DA. To explore the potential ability of the biomarkers to discriminate NAFL status in T2DM patients, model performances were compared and it was shown that the addition of five bile acid species (selected based on the exploration cohort, then validated in the validation cohort) to the clinical variables, improved the AUC, and categorical IRI, NRI for predicting the NAFL development in T2DM. Thus, the above evidences showed these biomarkers contributed to the improved model performance in predicting the development of NAFL in T2DM patients.
At the same time, these results suggest that the increase in bile acids may contribute to the formation of NAFL in T2DM patients, which could cause the high risk of occurrence of CVD and HCC. The increased five bile acids may cause the progression of NAFL in T2DM patients with insulin resistance. Our motivation for performing this study was that biomarkers that improve discrimination of NAFL in T2DM patients will not only support the prediction of NAFL development but also promote to further acknowledging the roles of bile acids. However, the detailed mechanisms have been unclear until now.
It is shown that recent increase in the incidence of HCC is driven by NAFLD, especially in Western Countries . Nevertheless, the morbidity and mortality of CVD in NAFLD patients are also very high. It is therefore crucial to prevent the progression of metabolic diseases, including T2DM, dyslipidemia and NAFLD; moreover, elucidation of the pathogenesis of NAFLD in T2DM patients has become increasingly more important.
Arab et al  summarized current available data on the relationships of BAs to NAFLD. Cyrielle et al found although serum total BAs were no significant differeces between NAFLD and non-NAFLD participants, the proportion of serum CA, CDCA conjugates were higher in NAFLD, while serum GHCA was lower than those non-NAFLD participants. Jiao et al  found that total serum bile acid levels in patients with NASH were about 3 times that of healthy controls. Total plasma bile acid levels were higher in obese and T2DM subjects than in healthy controls [28, 29]. However, there is no systematical analysis about serum BAs between T2DM with or without NAFL. Puri et al found that NAFLD was related to significantly changed circulating BA composition, unaffected by T2DM, and associated with histological features of NASH. These observations provided the foundation for future hypothesis-driven studies of specific effects of BAs on specific aspects of NASH.
In the present study we mainly found two classes of bile acids metabolites between the two groups including (A) CDCA and its metabolites (LCA, TLCA, CDCA24G and TCDCA) and (B) UDCA metabolite (TUDCA) as illustrated in Fig. 4.
(A) CDCA and its metabolites
CDCA plays an important role in controlling cholesterol homeostasis. Hydrophobic bile acids are known to induce hepatocyte injury experimentally, and the mechanisms are supposed as several different mechanisms, 1) mitochondrial and endoplasmic reticulum (ER) oxidative stress, 2) activate the death receptors FAS and tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) solubilization of the hepatocellular plasma membrane directly . Here we found serum levels of CDCA metabolites such as LCA, TLCA, CDCA24G and TCDCA were higher in T2DM with NAFL than those without NAFL.
LCA increased in patients with liver disease as the most potent endogenous chemical causing liver toxicity . LCA is converted from the primary BA-CDCA in the distal small intestine and colon after undergoing deconjugation and dehydroxylation. LCA can be reabsorbed passively and constitute a portion of the total BA pool in the enterohepatic circulation. Fickert et al  found that segmental bile duct obstruction, destructive cholangitis and periductal fibrosis in mice were caused by LCA feeding. LCA was reported to alter the levels of phospholipids, cholesterol, free fatty acids and TGs , and LCA disrupted phospholipid/sphingolipid homeostasis through TGF-β signaling . Duboc et al found that a decrease in serum LCA was implicated in the development of coronary atheromatous plaques. However it was shown significant difference of LCA between the two groups only in exploration cohort, the possible reason may be the small sample size of the validation cohort.
TLCA is capable of impairing BA flow and inducing cholestasis . It was shown that the contribution score of TLCA was high with high VIP score (Fig. 2; Supplementary Table S1). However, serum concentration of TLCA was quite low compared to primary BA (CA, CDCA and their conjugates) (Fig. 1; Supplementary Table S1). These results suggested that LCA and TLCA are key products for developing NAFLD in patients with T2DM. It was shown that TLCA upregulated AP-1 proteins cFos and JunB in HepG2-Ntcp hepatoma cells . Amonyingcharoen et al  showed that TLCA induced cholangiocarcinoma cell growth via the muscarinic acetylcholine receptor (mAChR) and epidermal growth factor receptor (EGFR) /extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. However, the detailed mechanisms of the increased serum TLCA level in T2DM patients with NAFL than those without NAFL were unclear. Marialena et al reported that patients with NASH had increased fecal BA synthesis and high fecal levels of CA and CDCA, which suggested a possible role for BAs in the progression of NAFL to NASH. Li et al found that children with NAFLD had higher levels of CDCAs, CDCA and CA, and lower levels of total DCA, DCA, TDCA, GDCA, GLCA, and total conjugated LCA (GLCA + TLCA) compared to subjects without NAFLD. Similar to the progression of NAFLD to NASH, the formation of NAFLD in T2DM patients likely involves not just one pathway but rather repeated injuries over time.
Trottie et al  concluded that UGT1A3 was the main UGT enzyme for the formation of hepatic CDCA-24G and glucuronidation inhibited the ability of CDCA to act as an FXR activator.
TCDCA as cytotoxic bile acids, changes in bile flow, biliary excretions of bile acids. It was shown serum levels of ALT and AST were highly elevated in all rats given TCDCA, while another study showed that TCDCA as a signaling molecule showed obvious anti-inflammatory and immune regulation properties via protein kinase C (PKC)/Jun N-terminal kinase (JNK)-dependent pathway. Song et al  quantitatively analyzed 15 biliary bile acids in cholangiocarcinoma (CCA) (n = 30), benign biliary disease (n = 57) and pancreatic cancer (n = 17) patients and discovered GCA and TCDCA as specific CCA biomarkers. However, until now, the roles of CDCA-24G and TCDCA in the process of NAFLD in T2DM patients are unclear.
(B) UDCA metabolite
UDCA, a hydrophilic bile acid, is used to treat a number of cholestatic disorders. There was a significant improvement in the levels of aminotransferases and steatosis in NAFLD patients treating with UDCA . However, it was shown that UDCA did not offer a histological benefit compared with placebo in NASH patients. Therefore, till now UDCA has not been recommended to treat NAFLD/NASH. Daniel et al found that 24-norursodeoxycholic acid (NorUDCA), a side-chain shorted derivative of UDCA, may be a promising new approach in the treatment of cholestatic and metabolic liver diseases with anti-fibrotic, anti-inflammatory and anti-lipotoxic properties. The detailed mechanism of the increased serum level of TUDCA in T2DM patients with NAFL than those without NAFL remains unclear. Beuers et al  investigated that UDCA conjugates may improve the impaired secretory abilities of cholestatic hepatocytes.
It was reported that in the liver TUDCA improves insulin sensitivity and have cardiovascular protective effects by reducing ER stress . It was reported that TUDCA attenuated hepatocarcinogenesis by suppressing carcinogen-induced ER stress-mediated cell death and inflammation without stimulating tumor progression . However, until now there is no related research describing the effect of TUDCA on liver diseases.
There are still several limitations in our study to be addressed in the future studies. First, the sample size of groups may not be sufficient to achieve more powerful data although we validated the results in an independent cohort study. Second, although fast serum samples were used for analysis, the information about diet, sedentary lifestyle was not available. Nevertheless, there is no information on other confounding factors that may influence bile acid concentration such as treatments (e. g. statins) or the degree of insulin resistance[54, 55]. Thus we could not entirely exclude the effect of those treatments on metabolism. Third, the diagnosis of NAFL in our study was based on ultrasonography but not confirmed by histological examination. The guidelines for NAFL in Europe, America and China all stipulate that liver histological examinations are only necessary in clinical trials of new drugs or clinical studies of NASH. In the present study, the patients are all NAFL patients, and it is not obligatory to perform histological examinations considering the risk and benefit of patients. However, it is better to perform histological examination than ultrasonography if condition permits. Nevertheless, it is better to further illustrate the mechanistic insight into the changes in the BA profiles in the pathogenesis of NAFL in T2DM from experimental studies using animal models and/or cell lines. In addition, an integrative omics method with other -omics, such as proteomics, transcriptomics and genomics, should be explored. We also intend to perform both in vivo and in vitro studies in the near future, to confirm the mechanisms of selected BAs, such as LCA, TLCA, TUDCA, CDCA24G and TCDCA, in the formation and development of NAFL in T2DM patients.