Deleterious liver-adipose crosstalk in obesity: Hydroethanolic extract of Lampaya medicinalis Phil. (Verbenaceae) counteracts fatty acid-induced fibrotic marker expression in human hepatocytes

Obesity, a pathology characterized by an excessive accumulation of fat, represents a great health concern since it is associated with several conditions such as dyslipidemia, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease (NAFLD) and cancer (Blüher, 2019). The prevalence of NAFLD is around 20%–30% in Western countries (Byrne and Targher, 2015; Mitra et al., 2020), and one of the main characteristics of the disease is the accumulation of fat, primarily as triglycerides, in the liver (Polyzos et al., 2019). Such phenomena, known as steatosis, is aggravated by increased circulating fatty acids occurring in obesity as the result of impaired adipose tissue (AT) function and insulin resistance (Byrne and Targher, 2015; Cobbina and Akhlaghi, 2017). Specifically, excess circulating free fatty acids (FFA) resulting in part from accelerated AT lipolysis may lead to excess liver fat uptake and lipotoxicity characterized by hepatic inflammation and fibrosis (Byrne and Targher, 2015; Polyzos et al., 2019; Kral et al., 1977). In turn, hepatic factors such as fatty acids from de novo lipogenesis and hepatocyte-secreted products (hepatokines) may reach AT impairing local metabolic processes (Blüher, 2019; Meex and Watt, 2017a). The cross-talk between these metabolically relevant tissues (liver and AT) is mediated by hepatokines and adipokines, which are bioactive peptides with positive and negative metabolic functions that can act via endocrine, paracrine and autocrine pathways. A transcriptional reprogramming occurs during hepatic steatosis with an impact on hepatokine secretion. Interestingly, these steatosis-induced changes are associated with impaired insulin action and activated pro-inflammatory pathways (Meex and Watt, 2017b). In this regard, the crosstalk between the liver and AT plays an important role in obesity-induced cardiometabolic disorders (Ott et al., 2022).

Liver fibrosis and its progression are the most important predictors of negative NAFLD outcomes and mortality (Dulai et al., 2017). Fibrosis is a reversible wound-healing response to either acute or chronic cellular injury. During chronic injury, such as the case of liver damage in NAFLD, there is progressive substitution of the liver parenchyma by scar tissue (Lee and Friedman, 2011). Collagen and other extracellular matrix (ECM) components are deposited as part of the liver wound-healing response, however hepatocyte function becomes impaired along this encapsulation process (Lee and Friedman, 2011; Aydin and Akcali, 2018). Interestingly, AT fibrosis has also been described as pathogenic in obesity-related diseases (Debari and Abbott, 2020).

The ECM is a complex network of macromolecules which determines the histoarchitecture of the liver (Zhang et al., 2016), as well as AT. Among its constituents are collagens, glycoproteins, proteoglycans and ECM-bound molecules such as matrix metalloproteinases (MMPs), a family of enzymes which catalyze the degradation of the ECM under physiological or pathological conditions (Benyon and Arthur, 2001). Altered ECM composition occurring during liver fibrosis is associated with increased protein synthesis and deposition of fibronectin (FN), collagen type I and alpha-smooth muscle actin (α-SMA) (McQuitty et al., 2020). As in liver fibrotic disease, AT fibrosis is characterized by the accumulation and increased production of ECM proteins (Buechler et al., 2015). Likewise, connective tissue growth factor (CTGF), a protein with various biological functions including ECM production, is known to be upregulated in multiple chronic diseases associated with tissue fibrosis and congestive heart failure (Choi et al., 2020). In addition, MMPs are induced to remodel the ECM during fibrogenic processes (Duarte et al., 2015).

The two most prevalent dietary fatty acids are palmitic acid (PA; C16:0) and oleic acid (OA; C18:1). Western-style diets are rich in PA, which have been linked to increased risk of cardiovascular disease, while the fat derived from animal products is high in OA (Kien et al., 2013, 2014). Classically, obesity has been associated with increased AT lipolysis and excess FFA release that results in ectopic accumulation of lipids in other tissues (Berger and Barnard, 1999). Such deposition of fatty acids will be a function of both intake and metabolism (Kien et al., 2014). Interestingly, OA and PA are some of the most abundant fatty acids in liver triglycerides of individuals with NAFLD (Araya et al., 2004), and have been used in vitro to induce lipid accumulation in hepatocyte cell models (Gómez-Lechón et al., 2007; Sanhueza et al., 2021; Ormzabal et al., 2021).

Metabolic homeostasis is achieved by the correct function of complex endocrine interactions that sustain inter-organ crosstalk, which has received increased attention in obesity research, with particular focus on adipose-liver interactions (Meex and Watt, 2017a; Debari and Abbott, 2020; Zhang et al., 2018a). Hepatic steatosis can induce changes in hepatokine secretion, promoting insulin resistance and resulting in impaired crosstalk between organs (Meex and Watt, 2017a). Interestingly, the liver can signal to AT to modulate lipolysis and fibrosis (Azzu et al., 2020; Mandard et al., 2006; Chen et al., 2017), with important metabolic consequences in visceral AT, a fat depot that plays an important role in obesity-associated inflammation and cardiometabolic complications (Tchernof and Després, 2013). To our knowledge, there are no studies that have addressed the effect of hepatocyte-secreted products on human visceral AT explants obtained from subjects with obesity, particularly on lipolysis and fibrosis-associated gene expression.

Lampaya medicinalis Phill. (Verbenaceae) is a small bush that grows in Northern Chile. Its leaves are used in Chilean folk medicine to prepare infusions for kidney, urinary and liver conditions (Mellado et al., 1977; Castro, 1995; Monterrey, 1994; Gómez et al., 1997). The hydroethanolic extract of lampaya (HEL) shows antioxidant and anti-inflammatory activities in vivo and in vitro, which may be explained by its phytochemical composition consisting of large amounts of phenols and flavonoids, and a minor fraction phenolic acid, p-hydroxyacetophenones derivatives, naphthalenic and iridoid glycosides (Supplementary Table 1) (Morales et al., 2014; Ormazabal et al., 2020a, 2020b; Morales and Paredes, 2014). HEL has been shown to ameliorate the fatty acid-induced proinflammatory response in cultured macrophages, and to have beneficial effects on lipid metabolism and markers of inflammation in both HepG2 hepatocytes exposed to OA/PA and Drosophila melanogaster fed a high-fat diet (Sanhueza et al., 2021; Ormzabal et al., 2021; Ormazabal et al., 2020b). Interestingly, HEL has been shown to prevent OA/PA-induced TNF-α and IL-6 production (RNA expression and protein secretion) in HepG2 hepatocytes (Sanhueza et al., 2021). However, it is unknown whether HEL has a beneficial effect on hepatic markers of fibrosis. Thus, the objective of this study was to evaluate the effect of exposure to HepG2 secretion products on lipolysis and expression of fibrosis markers in human visceral AT explants, as well as the impact of HEL pre-treatment on the expression of pro-fibrotic markers in HepG2 hepatocytes exposed to a fatty-acid rich environment (OA/PA).

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