*3.5. Coffee Modulates Expression of lncRNAs Associated with Circadian Clock Regulation*

Figure 4 shows the expression of fatty liver-related lncRNA 1 (FLRL1) and fatty liver-related lncRNA 2 (FLRL2), two lncRNAs that are involved in circadian clock regulation and whose liver expression is changed by HFD in mice [26]. Overall, we found an up-regulation of both lncRNAs in the liver of mice fed HFD versus SD, whereas their expression was differently modulated by coffee intake. In detail, coffee consumption further increased FLRL1 (Figure 4, Panel A), while FLRL2 was decreased by coffee intake to levels lower than those of mice fed SD (Figure 4, Panel B). It has been reported that FLRL1 and FLRL2 target *period circadian protein homolog 3* (UniProtKB-O70361 encoded by Per3) and *aryl hydrocarbon receptor nuclear translocator-like protein 1* (UniProtKB-Q9WTL8 encoded by Arntl), respectively [27]. Therefore, we extended qPCR analysis also to these target genes. mRNA for Per3 showed the same expression trend observed for its regulator lncRNA FLRL1; thus, it was upregulated by HFD and further increased by coffee intake (Figure 4, Panel C). Arntl mRNA expression, which was downregulated by HFD, was unaffected by coffee administration (Figure 4, Panel D).

**Figure 4.** Hepatic expression of fatty liver-related lncRNA 1 (FLRL1) (Panel **A**), Fatty liver-related lncRNA 2 (FLRL2) (Panel **B**), period circadian protein homolog 3 (Per3) mRNA (Panel **C**) and Aryl hydrocarbon receptor nuclear translocatorlike protein 1 (Arntl) mRNA (Panel **D**), analyzed through qPCR, in mice fed standard diet (SD), high fat diet (HFD) and HFD plus decaffeinated coffee; *n* = 23: 8 SD, 8 HFD, 7 HFD+ coffee. Transcript statistical significance was evaluated with one-way ANOVA with Tukey post-hoc test for multiple comparisons (two-tailed *p*-value < 0.05); FC = fold change.

#### *3.6. lncRNAs Not Modified by Coffee Consumption*

Another lncRNA involved in the regulation of metabolic processes is *colorectal neoplasia differentially expressed* (CRNDE) [28]. Although CRNDE was upregulated about three-fold by HFD, its expression was not modified by coffee consumption (Supplementary Figure S1A). Similarly, *nuclear enriched abundant transcript 1* (NEAT1), that plays a role in LDL uptake [29], was downregulated by HFD but its expression was unchanged by coffee intake (Supplementary Figure S1B). A summary of lncRNAs modified by coffee consumption and relative targets is shown in Supplementary Table S4.

#### **4. Discussion**

Epidemiological studies indicate that coffee intake favourably impacts on NAFLD prevalence and severity [30], although without fully clarified mechanisms. In this study we provide the first evidence that hepatoprotection induced by coffee in a mouse model is associated with the modulation of selected lncRNAs known to be involved in mechanisms related to NAFLD onset and progression such as impairment of lipid metabolism and circadian clock, pro-inflammatory state and activation of hepatic stellate cells.

Among the mechanisms connected to lipid metabolism and steatogenesis, Gm16551 has recently reported as a liver specific lncRNA downregulated in mice subjected to 24-h or a 12-week HFD that, through a negative feedback loop, reduces SREBP-1c functional activity thus inhibiting *de novo* lipogenesis [22]. In our study, Gm16551 was downregulated by a 12-week HFD, whereas coffee administration induced its expression. In agreement with histological improvement of steatosis, the induction of Gm16551 reduced the transcript for acetyl-CoA carboxylase 1 (Acaca), the enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting step of *de novo* fatty acid biosynthesis [31]. Coffee intake reduced the mRNA level of Scd1, an enzyme that also contributes to steatogenesis [32]. Therefore, according to our data, a potential mechanism by which coffee reduces steatosis could be represented by Gm16551 expression induction.

It is known that NAFLD is associated with a chronic inflammatory state as evidenced in the liver of animal models and patients [33]. Although the 12-week HFD is a model of early NAFLD, as showed by histology, we found a slight increase of the lncRNA CARMN that is a pro-inflammatory mediator that is upregulated in macrophages treated in vitro with high glucose and palmitic acid and in macrophages isolated from diabetic mice and whose transient overexpression stimulates the expression of inflammatory genes and of CD36 [23]. This last aspect is relevant because in HepG2 treated with palmitate, lipid overload is exacerbated by the upregulation of the receptor involved in the uptake of lipids such as CD36 [16]. Thus, in our model, the downregulation of CARMN induced by coffee administration could contribute to the observed inflammation reduction, and to the reduction of lipid uptake and consequent steatosis grade. However, the downregulation of CARMN by coffee administration could explain the complete absence of inflammatory foci in coffee treated mice. Further studies are needed to confirm this hypothesis.

Another possible contribution in this direction may rely on lncRNA SRA. In fact, it has been reported that SRA genetic knockout protects against high fat diet-induced obesity [34] and hepatic steatosis [24]. In accordance with this evidence, HFD induces the expression of lncRNA SRA, while coffee co-administration decreases its expression level with respect to HFD. Thus, SRA downregulation could contribute to the observed reduced steatosis levels.

Since fibrosis is the main predictor of mortality in patients with NAFLD [35], it is relevant to identify molecular determinants of fibrogenesis. In this respect, experimental studies have reported the important role of the lncRNA H19. Zhu J et al. showed that H19 is overexpressed in the liver and primary hepatic stellate cells (HSCs) of mice with CCl4-induced liver fibrosis and demonstrated that the stable H19 overexpression induces the upregulation of α-SMA and Col1a1 both in vitro and in vivo [36]. Cholangiocytederived exosomal H19 stimulates trans-differentiation of mouse primary HSCs and induces proliferation and collagen production in HSC-derived fibroblasts [25]. In our study, we showed an up-regulation of H19 by HFD and a downregulation of H19, Col1a1 and αSMA by coffee intake. A main limitation in the interpretation of these results lies in the fact that we studied a model of early NAFLD that does not display fibrosis at H&E staining, although we cannot exclude the presence of small amount of pericellular or perisinusoidal fibrosis that could have been detected by Sirius Red. However, it is reliable to consider the upregulation of α-SMA as a marker of onset of the fibrogenesis process since in mice fed

steatogenic diets the increase of α-SMA expression is confined at hepatic stellate cell level as showed by immunohistochemical analysis [37,38].

As concerns the circadian clock lncRNAs, Yi Chen et al., after performing a whole transcriptome analysis in an eight-week HFD mouse model, identified 266 differentially expressed lncRNA, among which they validated the expression of eight lncRNA through real time PCR [26]. To gain further insights into the molecular mechanisms regulated by such lncRNAs they performed a computational analysis that led to the identification of two fatty liver related lncRNAs associated with clock gene regulation, FLRL1 and FLRL2. They identified Per3 as a molecular target of FLRL1 computationally. The role of FLRL2 was investigated through transient inhibition in a cellular model of NAFLD; the authors demonstrated that FLRL2 downregulation is associated with Arnt downregulation at protein level [26]. However, physiological and pathophysiological functions of FLRL1 and FLRL2 and of their targets have not been elucidated so far and thus we cannot speculate on this aspect, although it deserves further exploration.

#### **5. Conclusions**

In this study we observed that decaffeinated coffee modulates expression of lncRNAs involved in key pathways of NAFLD onset and progression. Our data extend the knowledge concerning the molecular mechanism underlying beneficial effects exerted by coffee consumption against NAFLD.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/nu13092952/s1, Table S1: Diet composition, Table S2: Primer sequences of selected mouse lncRNAs and relative targets, Table S3: Liver histology scores, Table S4: List of analyzed lncRNAs modulated or not modulated by coffee supplementation and relative targets, Figure S1: Dot plots of CRNDE lncRNA and NEAT1 lncRNA, analyzed through qPCR in mice fed with Standard Diet (SD), High Fat Diet (HFD) and HFD plus decaffeinate coffee *n* = 23: 8 SD, 8 HFD, 7 HFD+ coffee. Transcript statistical significance of DE transcripts was evaluated with one-way ANOVA with Tukey post-hoc test for multiple comparisons (two-tailed *p*-value < 0.05); FC= Fold Change.

**Author Contributions:** S.D.M. Investigation, formal analysis, methodology, writing and visualization. F.S. Conceptualization, investigation, formal analysis, methodology writing and visualization. A.S. Investigation, formal analysis, methodology and visualization. A.F. Investigation and methodology. F.M. Investigation and resources. M.G. Investigation, methodology and formal analysis. V.L. Investigation. V.C. Investigation. R.M.P. Resources. S.G. Resources. R.M. Supervision. F.P. Resources and supervision. S.P. Project administration, supervision, writing—review. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** All experiments were performed according to the Ethics Committee of the University of Naples and approved by the Italian Minister of Scientific Research (Code 2014/0013808).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Material.

**Acknowledgments:** We wish to thank the Scientific Bureau of the University of Catania for language support. This study was in keeping with the objectives of the project "DEGENER-action", Department of Clinical and Experimental Medicine University of Catania.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

