The role of autophagy in liver epithelial cells and its Impact on systemic homeostasis

Autophagy plays a role in several physiological and pathological processes as it controls the turnover rate of cellular components and influences cellular homeostasis. The liver plays a central role in controlling organisms’ metabolism, regulating glucose storage, plasma proteins and bile synthesis and the removal of toxic substances. Liver functions are particularly sensitive to autophagy modulation. In this review we summarize studies investigating how autophagy influences the hepatic metabolism, focusing on fat accumulation and lipids turnover. We also describe how autophagy affects bile production and the scavenger function within the complex homeostasis of the liver. We underline the role of hepatic autophagy in counteracting the metabolic syndrome and the associated cardiovascular risk. Finally, we highlight recent reports demonstrating how the autophagy occurring within the liver may affect skeletal muscle homeostasis as well as different extrahepatic solid tumors, such as melanoma

Different response to oleic acid administration in hepatocellular carcinoma vs non tumorigenic hepatocyte cell lines

Thanks to the presence of high levels of monounsaturated fatty acids, extra virgin olive oil is beneficial for health as it is associated with protection against liver steatosis, some types of cancer and cardiovascular diseases. Here we study different responses between hepatocarcinoma cell lines (Hep3B and Huh7.5) vs a healthy hepatocyte control (THLE-2) following the treatment with oleic acid (OA), in particular we have observed a dose-dependent response (50-300 M). First of all, we observed a different neutral lipid accumulation between the hepatocarcinoma cells and the healthy control. Moreover, we have found a differential viability rate; a reduction of hepatocarcinoma cell lines at 300 M OA dosage but unchanged vitality in healthy hepatocytes through Alamar Blue staining. Furthermore, we evaluated the apoptotic rate by western blot, we have found that the levels of cleaved PARP appear differently modulated in the hepatocarcinoma cell lines following the treatment with OA. We also found that, while hepatocarcinoma cell lines respond to dose-dependent OA treatment with autophagic flux reduction, healthy controls do not significantly modulate their autophagic flux, thus hypothesizing a protective role of autophagy in hepatocytes cell vitality. In order to better investigate the different autophagic response of Huh7.5 and Hep3B carcinoma cells as compared to healthy THLE-2 cells we analyzed the levels of perilipin-2 since it has been previously shown to have a crucial role in liver autophagy regulation. We interestingly found that in Huh7.5 and Hep3B carcinoma cells dose-dependent OA (50-300 M) treatment is associated to increasing perilipin-2 levels. Such results are in agreement with previous studies showing and inverse relationship between perilipin-2 and autophagy levels. Further experiments are ongoing to manipulate autophagy in order to investigate at molecular level the beneficial effects of OA in counteracting hepatocarcinoma

Mitophagy contributes to alpha-tocopheryl succinate toxicity in GSNOR-deficient hepatocellular carcinoma

The downregulation of the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR, EC:1.1.1.284), is a feature of hepatocellular carcinoma (HCC). This condition causes mitochondrial rearrangements that sensitize these tumors to mitochondrial toxins, in particular to the mitochondrial complex II inhibitor alpha-tocopheryl succinate (αTOS). It has also been reported the GSNOR depletion impairs the selective degradation of mitochondria through mitophagy; however, if this contributes to GSNOR-deficient HCC cell sensitivity to αTOS and can be applied to anticancer therapies, is still not known. Here, we provide evidence that GSNOR-deficient HCC cells show defective mitophagy which contributes to αTOS toxicity. Mitophagy inhibition by Parkin (EC: 2.3.2.31) depletion enhances αTOS anticancer effects, thus suggesting that this drug could be effective in treating mitophagy-defective tumors

Different autophagic response and viability effect in hepatocarcinoma cell lines vs healthy controls upon oleic acid stimulation

Extra virgin olive oil is beneficial for health as it is associated with protection against liver steatosis, cardiovascular diseases and some types of cancer. These features are related to the presence of high levels of monounsaturated fatty acids including oleic acid (1). Here we study different responses between hepatocarcinoma cell lines (Hep3B and Huh7.5) vs a healthy hepatocyte control (THLE-2) following oleic acid treatment (OA; 50-300 μM). First of all, we observed a different neutral lipid accumulation between the hepatocarcinoma cells and the healthy control. We also found that, while hepatocarcinoma cell lines respond to dose-dependent OA treatment with autophagic flux reduction, healthy controls do not significantly modulate their autophagic flux. In order to better investigate the different autophagic response of Hep3B and Huh7.5 carcinoma cells as compared to THLE-2 cells, we analyzed the levels of perilipin-2 since it has been previously shown to have a crucial role in liver autophagy regulation. We interestingly found that in Hep3B and Huh7.5 carcinoma cells, but not in THLE-2 cells, dose-dependent OA treatment is associated to significant increasing perilipin-2 levels. Such results are in agreement with previous studies showing and inverse relationship between perilipin-2 and autophagy levels (2). Moreover, we found a differential viability rate: a reduction of hepatocarcinoma cell lines at 300 μM OA dosage but unchanged vitality in healthy hepatocytes through Alamar Blue staining. Furthermore, in preliminary experiments aimed at evaluating the apoptotic rate by western blot, we found increased levels of cleaved caspase-3 in Huh7.5 but not in Hep3B and THLE-2. Further experiments are ongoing to i) investigate cell death mechanism in Hep3B and ii) manipulate autophagy in order to investigate at molecular level the beneficial effects of OA in counteracting hepatocarcinoma

Anti-tumor effect of oleic acid in hepatocellular carcinoma cell lines via autophagy reduction

Oleic acid (OA) is a component of the olive oil. Beneficial health effects of olive oil are well-known, such as protection against liver steatosis and against some cancer types. In the present study, we focused on OA effects in hepatocellular carcinoma (HCC), investigating responses to OA treatment (50–300µM) in HCC cell lines (Hep3B and Huh7.5) and in a healthy liver-derived human cell line (THLE-2). Upon OA administration higher lipid accumulation, perilipin-2 increase, and autophagy reduction were observed in HCC cells as compared to healthy cells. OA in the presence of 10% FBS significantly reduced viability of HCC cell lines at 300µM through Alamar Blue staining evaluation, and reduced cyclin D1 expression in a dose-dependent manner while it was ineffective on healthy hepatocytes. Furthermore, OA increased cell death by about 30%, inducing apoptosis and necrosis in HCC cells but not in healthy hepatocytes at 300µM dosage. Moreover, OA induced senescence in Hep3B, reduced P-ERK in both HCC cell lines and significantly inhibited the antiapoptotic proteins c-Flip and Bcl-2 in HCC cells but not in healthy hepatocytes. All these results led us to conclude that different cell death processes occur in these two HCC cell lines upon OA treatment. Furthermore, 300µM OA significantly reduced the migration and invasion of both HCC cell lines, while it has no effects on healthy cells. Finally, we investigated autophagy role in OA-dependent effects by using the autophagy inducer torin-1. Combined OA/torin-1 treatment reduced lipid accumulation and cell death as compared to single OA treatment. We therefore concluded that OA effects in HCC cells lines are, at least, in part dependent on OA-induced autophagy reduction. In conclusion, we report for the first time an autophagy dependent relevant anti-cancer effect of OA in human hepatocellular carcinoma cell lines

Evaluation of autophagy in lymphocyte populations during atherosclerotic plaque progression with flow cytometry

Atherosclerosis is a chronic inflammatory disorder of the large arteries and represents the primary cause of heart disease and stroke. The exact cause of atherosclerosis is not known. A variety of studies show that autophagy deficiency may be pro-atherogenic and the role of autophagy in smooth muscle cells, macrophages and endothelial cells has been investigated. However, to date no studies addressed the effect of autophagy on lymphocyte subsets playing a role in plaque formation and development. The present project aims to better clarify the role played by autophagy in lymphocytes homeostasis in human atherosclerotic plaques. We characterized lymphocyte populations in different types of lesion by using flow cytometry. In particular, we detected OX40 as marker for conventional T cells promoting division and survival of effector and memory populations and pS6, a marker for an active mTOR pathway and autophagy detection. The understanding of the role of autophagy as a further mechanism underlying lymphocytes stability may open new therapeutic avenues for atherosclerosis

Anti-inflammatory Role of Carotenoids in Endothelial Cells Derived from Umbilical Cord of Women Affected by Gestational Diabetes Mellitus

Diabetes is associated with vascular inflammation, endothelial dysfunction, and oxidative stress, promoting the development of cardiovascular diseases (CVD). Several studies showed that a carotenoid-rich diet is associated to a reduced cardiovascular risk in healthy and diabetic subjects, although the mechanisms of action are still unknown. Here, the potential role of β-carotene (BC) and lycopene (Lyc) in human endothelial cells isolated from human umbilical cord vein (HUVECs) of women with gestational diabetes (GD) and respective controls (C) has been investigated. Results showed that BC and Lyc reduced the tumor necrosis factor alpha- (TNF-α-) stimulated monocyte-endothelium interaction (adhesion assay), membrane exposure (flow cytometry), and total expression levels (Western blot) of VCAM-1 and ICAM-1 in both cell types. Moreover, the treatment with BC and Lyc reduced the TNF-α-induced nuclear translocation of NF-κB (image flow cytometry) by preserving bioavailability of nitric oxide (NO, flow cytometry, and cGMP EIA kit assay), a key vasoactive molecule. Notably, BC and Lyc pretreatment significantly reduced peroxynitrite levels (flow cytometry), contributing to the redox balance protection. These results suggest a new mechanism of action of carotenoids which exert vascular protective action in diabetic condition, thus reinforcing the importance of a carotenoid-rich diet in the prevention of diabetes cardiovascular complications

Calcimimetic R-568 vasodilatory effect on mesenteric vascular beds from normotensive (WKY) and spontaneously hypertensive (SHR) rats. Potential involvement of vascular smooth muscle cells (vSMCs).

The potential role of calcimimetics as vasculotropic agents has been suggested since the discovery that calcium sensing receptors (CaSRs) are expressed in cardiovascular tissues. However, whether this effect is CaSR-dependent or -independent is still unclear. In the present study the vascular activity of calcimimetic R-568 was investigated in mesenteric vascular beds (MVBs) isolated from Spontaneously Hypertensive rats (SHR) and the relative age-matched Wistar-Kyoto (WKY) control rats. Pre-constricted MBVs were perfused with increasing concentrations of R-568 (10 nM- 30 μM) resulting in a rapid dose-dependent vasodilatation. However, in MVBs from SHR this was preceded by a small but significant vasoconstriction at lowest nanomolar concentrations used (10-300 nM). Pre-treatment with pharmacological inhibitors of nitric oxide (NO) synthase (NOS, L-NAME), KCa channels (CTX), cyclo-oxygenase (INDO) and CaSR (Calhex) or the endothelium removal suggest that NO, CaSR and the endothelium itself contribute to the R-568 vasodilatory/vasoconstrictor effects observed respectively in WKY/SHR MVBs. Conversely, the vasodilatory effects resulted by highest R-568 concentration were independent of these factors. Then, the ability of lower R-568 doses (0.1-1 μM) to activate endothelial-NOS (eNOS) pathway in MVBs homogenates was evaluated. The Akt and eNOS phosphorylation levels resulted increased in WKY homogenates and Calhex significantly blocked this effect. Notably, this did not occur in the SHR. Similarly, vascular smooth muscle cells (vSMCs) stimulation with lower R-568 doses resulted in Akt activation and increased NO production in WKY but not in SHR cells. Interestingly, in these cells this was associated with the absence of the biologically active dimeric form of the CaSR thus potentially contributing to explain the impaired vasorelaxant effect observed in response to R-568 in MVB from SHR compared to WKY. Overall, these findings provide new insight on the mechanisms of action of the calcimimetic R-568 in modulating vascular tone both in physiological and pathological conditions such as hypertension

Altered tregs differentiation and impaired autophagy correlate to atherosclerotic disease

Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy—a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress—may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-β (TGFβ). TGFβ is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategie