A new mechanism of lipotoxicity: Calcium channel blockers as a treatment for nonalcoholic steatohepatitis?
Authors: Mark J. Czaja
Journal: Hepatology (2015)

Investigations into the mechanism of impaired autophagy in fatty liver disease have identified increased intracellular calcium as a mediator of this defect. Park et al.1 have demonstrated that saturated free fatty acids (FFAs) induce increased cytosolic calcium in hepatocytes that inhibits autophagic function. The decrease in autophagy can be reversed in obese mice by administration of a calcium-channel blocker, which results in a reduction in steatohepatitis. These findings suggest the possibility that a common class of medications extensively employed in humans for cardiovascular disease (CVD) may be efficacious in the therapy of nonalcoholic steatohepatitis (NASH). The finding that hepatocyte lipids are metabolized by the lysosomal degradative pathway of macroautophagy2 has generated growing interest in the mechanistic involvement of autophagy in NASH.3 Potential mechanisms by which hepatocyte autophagy may prevent NASH development and/or progression include not only the limitation of excessive hepatic lipid accumulation by increasing lipid metabolism, but also promotion of hepatocyte survival postinjury from oxidant stress or tumor necrosis factor.3 Macrophage autophagy may protect against NASH by down-regulating the innate immune response.4 These potentially beneficial effects of autophagy, together with the fact that autophagic function is decreased in hepatocytes and macrophages in steatotic livers,2, 4 suggest that a therapeutic approach directed at increasing hepatic autophagy may be an effective treatment for NASH. A better understanding of the mechanism(s) that underlies the defect in autophagy in hepatocytes and other cells in hepatic steatosis may lead to the identification of novel compounds to reverse this problem. Initial findings of a decrease in levels of critical autophagy proteins in the liver, such as autophagy-related protein 7, as a mechanism for the defect have not been reproduced.3 Subsequent studies have pointed to a problem not in autophagosome formation, but in the process of fusion between autophagosome and -lysosome or in lysosomal function and therefore degradation of the contents of the autolysosome.5 The mechanism of the fusion defect has been attributed to altered membrane lipid content secondary to the dyslipidemia associated with insulin resistance (IR) and obesity. One report implicated both the unsaturated fatty acid, oleate, and the saturated fatty acid, palmitate, in decreasing autophagy through defective fusion.6 Other studies have shown differential effects with oleate actually increasing autophagy. This finding would be consistent with the general concept that saturated, but not unsaturated, FFAs are toxic in NASH.7 The importance of the finding that decreased autophagy in steatotic hepatocytes is a failure of fusion is that agents that only target the autophagic pathway upstream of this block may be ineffective in increasing hepatic autophagy. One potential mechanism of saturated FFA toxicity in hepatocytes is endoplasmic reticulum (ER) stress.7 Palmitate-induced ER stress in hepatoma cells decreases activity of sarco-ER calcium ATPase (SERCA), which normally maintains cellular calcium homeostasis by sequestering calcium in the ER.8 Park et al. reasoned that saturated FFAs may impair autophagy through ER stress-induced SERCA inhibition given that the ER stress inducer, thapsigargin, which inhibits SERCA, also blocks autophagosome-lysosome fusion. Studying HepG2 cells, they found that the saturated FFAs, palmitate and stearate, but not oleate, induced cellular accumulation of ubiquitinated proteins and the autophagy-degraded protein, p62, along with autophagosomes. By numerous techniques, they demonstrated that saturated FFAs inhibit autophagic function primarily by blocking the fusion step. One limitation of this study is that the investigations were all performed in HepG2 cells, and it would be important to know whether the findings are reproducible in primary hepatocytes. However, these results, together with the previous study by Koga et al.,6 strongly suggest that FFAs inhibit hepatocyte autophagy at the level of fusion. To prove that saturated FFAs inhibit autophagy through SERCA and not nonspecific ER stress, Park et al. first showed that only ER stress inducers that also inhibit SERCA decrease autophagy. In addition, they demonstrated that genetic silencing of SERCA increased, whereas SERCA overexpression decreased, protein accumulation from saturated FFAs. Palmitate elevated cytosolic calcium levels, an effect prevented by the calcium-channel blockers, verapamil and nicardipine, which prevent extracellular calcium entry into the cytoplasm. Both agents also reversed inhibition of autophagy by palmitate. Genetic inhibition of the primary calcium channel present in HepG2 cells also partially blunted the palmitate-induced decrease in autophagy. Although these findings do not conclusively prove that saturated FFAs act directly on SERCA, the data do clearly indicate that the effect of palmitate on autophagy is mediated by increased cytosolic calcium. Other agents that increase hepatocyte calcium through the inositol triphosphate receptor, such as glucagon and vasopressin, also increased HepG2 protein accumulation, although not to the extent of saturated FFAs. The efficacy of verapamil in obese mice is also demonstrated by the study. Mice fed 2 months of high-fat diet and treated with verapamil for 10 days had marked decreases in hepatic steatosis (HS) and macrophage infiltration. Unfortunately, the effects on liver injury are not discussed. There was indirect evidence of reduced hepatic cytosolic calcium in the verapamil-treated mice, and the drug suppressed the increase in cytosolic calcium from palmitate in cultured mouse hepatocytes. Autophagic function, including autophagosome-lysosome fusion, was restored by verapamil in obese mice. The effects of verapamil were somewhat liver specific given that white adipose tissue mass and inflammatory cytokine production were unaffected. Whether decreased HS and inflammation resulted completely from direct hepatic effects is unclear, given that these mice also had slightly reduced body weights as well an almost complete reversal of their glucose intolerance and IR. The mechanism by which increased calcium inhibits fusion is not addressed, and, interestingly, calcium is essential for fusion to occur.6 The possibility exists that calcium-channel blockers may affect autophagy through mechanisms other than an effect on fusion. However, the findings do significantly expand our understanding of how saturated FFAs are potentially toxic in NASH by establishing a novel mechanism by which ER stress and calcium disequilibrium can inhibit autophagy (Fig. 1). The study raises the question of whether calcium-channel blockers, with a proven safety profile from extensive human use in CVD, can treat human NASH and, possibly, alcoholic fatty liver disease as well. It should be noted that whether autophagy is defective in human NASH remains unknown. Autophagic function is almost impossible to assess in human tissue, with the possible exception of studies of autophagic flux in tissue explants. Published reports of levels of autophagy in human liver disease typically only provide assessments of steady-state levels of autophagosomes in which increases or decreases may both reflect either elevated or reduced autophagic function. A number of studies have examined calcium-channel blockers in metabolic syndrome with mixed conclusions on whether these agents have beneficial effects on glucose tolerance, hyperinsulinemia, and hyperlipidemia. Studies specifically examining NASH are lacking, although one study demonstrated no effect of amiodipine.9 The findings of Park et al. provide a scientific basis for a closer examination of these drugs in NASH. The lack of data on autophagic function in human NASH makes it possible that only a subset of patients with this disease have impaired autophagy and will therefore benefit from autophagy-directed therapy. Only by identifying these patients, and through the use of new agents developed specifically to target autophagy, will the role of autophagy in human NASH be determined. Mark J. Czaja, M.D. Department of Medicine and Marion Bessin Liver Research Center Albert Einstein College of Medicine Bronx, NY Author names in bold designate shared co-first authorship.