Benjamin L. Farah

Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice

Caffeine is one of the worlds most consumed drugs. Recently, several studies showed that its consumption is associated with lower risk for nonalcoholic fatty liver disease (NAFLD), an obesity‐related condition that recently has become the major cause of liver disease worldwide. Although caffeine is known to stimulate hepatic fat oxidation, its mechanism of action on lipid metabolism is still not clear. Here, we show that caffeine surprisingly is a potent stimulator of hepatic autophagic flux. Using genetic, pharmacological, and metabolomic approaches, we demonstrate that caffeine reduces intrahepatic lipid content and stimulates β‐oxidation in hepatic cells and liver by an autophagy‐lysosomal pathway. Furthermore, caffeine‐induced autophagy involved down‐regulation of mammalian target of rapamycin signaling and alteration in hepatic amino acids and sphingolipid levels. In mice fed a high‐fat diet, caffeine markedly reduces hepatosteatosis and concomitantly increases autophagy and lipid uptake in lysosomes. Conclusion: These results provide novel insight into caffeines lipolytic actions through autophagy in mammalian liver and its potential beneficial effects in NAFLD. (Hepatology 2014;59:1366‐1380)

Epigallocatechin-3-Gallate (EGCG), a Green Tea Polyphenol, Stimulates Hepatic Autophagy and Lipid Clearance

Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has been shown to have anti-inflammatory, anti-cancer, anti-steatotic effects on the liver. Autophagy also mediates similar effects; however, it is not currently known whether EGCG can regulate hepatic autophagy. Here, we show that EGCG increases hepatic autophagy by promoting the formation of autophagosomes, increasing lysosomal acidification, and stimulating autophagic flux in hepatic cells and in vivo. EGCG also increases phosphorylation of AMPK, one of the major regulators of autophagy. Importantly, siRNA knockdown of AMPK abrogated autophagy induced by EGCG. Interestingly, we observed lipid droplet within autophagosomes and autolysosomes and increased lipid clearance by EGCG, suggesting it promotes lipid metabolism by increasing autophagy. In mice fed with high-fat/western style diet (HFW; 60% energy as fat, reduced levels of calcium, vitamin D3, choline, folate, and fiber), EGCG treatment reduces hepatosteatosis and concomitantly increases autophagy. In summary, we have used genetic and pharmacological approaches to demonstrate EGCG induction of hepatic autophagy, and this may contribute to its beneficial effects in reducing hepatosteatosis and potentially some other pathological liver conditions.

Thyroid hormone induction of mitochondrial activity is coupled to mitophagy via ROS-AMPK-ULK1 signaling

Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T3) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T3 is coupled to oxidative phosphorylation and ROS production. We show that T3 induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) mediated phosphorylation of PRKAA1/AMPK (5′ AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T3-treated cells impairs both mitophagy as well as OXPHOS without affecting T3 induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T3-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.

Induction of autophagy improves hepatic lipid metabolism in glucose-6-phosphatase deficiency.

BACKGROUND & AIMS Glucose-6-phosphatase (G6Pase α, G6PC) deficiency, also known as von Gierkes disease or GSDIa, is the most common glycogen storage disorder. It is characterized by a decreased ability of the liver to convert glucose-6-phosphate (G6P) to glucose leading to glycogen and lipid over-accumulation progressing to liver failure and/or hepatomas and carcinomas. Autophagy of intracellular lipid stores (lipophagy) has been shown to stimulate fatty acid β-oxidation in hepatic cells. Thus, we examined autophagy and its effects on reducing hepatic lipid over-accumulation in several cell culture and animal models of GSDIa. METHODS Autophagy in G6PC-deficient hepatic cell lines, mice, and dogs was measured by Western blotting for key autophagy markers. Pro-autophagic Unc51-like kinase 1 (ULK1/ATG1) was overexpressed in G6PC-deficient hepatic cells, and lipid clearance and oxidative phosphorylation measured. G6PC(-/-) mice and GSDIa dogs were treated with rapamycin and assessed for liver function. RESULTS Autophagy was impaired in the cell culture, mouse, and canine models of GSDIa. Stimulation of the anti-autophagic mTOR, and inhibition of the pro-autophagic AMPK pathways occurred both in vitro and in vivo. Induction of autophagy by ULK1/ATG1 overexpression decreased lipid accumulation and increased oxidative phosphorylation in G6PC-deficient hepatic cells. Rapamycin treatment induced autophagy and decreased hepatic triglyceride and glycogen content in G6PC(-/-) mice, as well as reduced liver size and improved circulating markers of liver damage in GSDIa dogs. CONCLUSIONS Autophagy is impaired in GSDIa. Pharmacological induction of autophagy corrects hepatic lipid over-accumulation and may represent a new therapeutic strategy for GSDIa.

ETHANOL CAUSES THE REDISTRIBUTION OF L1 CELL ADHESION MOLECULE IN LIPID RAFTS

J. Neurochem. (2011) 119, 859–867.

β-Adrenergic Agonist and Antagonist Regulation of Autophagy in HepG2 Cells, Primary Mouse Hepatocytes, and Mouse Liver

Autophagy recently has been shown to be involved in normal hepatic function and in pathological conditions such as non-alcoholic fatty liver disease. Adrenergic signalling also is an important regulator of hepatic metabolism and function. However, currently little is known about the potential role of adrenergic signaling on hepatic autophagy, and whether the β-adrenergic receptor itself may be a key regulator of autophagy. To address these issues, we investigated the actions of the β2-adrenergic receptor agonist, clenbuterol on hepatic autophagy. Surprisingly, we found that clenbuterol stimulated autophagy and autophagic flux in hepatoma cells, primary hepatocytes and in vivo. Similar effects also were observed with epinephrine treatment. Interestingly, propranolol caused a late block in autophagy in the absence and presence of clenbuterol, both in cell culture and in vivo. Thus, our results demonstrate that the β2- adrenergic receptor is a key regulator of hepatic autophagy, and that the β-blocker propranolol can independently induce a late block in autophagy.

Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease

Enzyme or gene replacement therapy with acid α‐glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation‐independent mannose‐6‐phosphate receptor (CI‐MPR)‐mediated uptake and intracellular trafficking of GAA during muscle‐specific GAA expression with an adeno‐associated virus (AAV) vector in GAA‐knockout (KO) mice. Clenbuterol, which increases expression of CI‐MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA‐KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI‐MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA‐KO mice that lacked CI‐MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI‐MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2‐agonist treatment enhanced CI‐MPR‐mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.—Farah, B. L., Madden, L., Li, S., Nance, S., Bird, A., Bursac, N., Yen, P. M., Young, S. P., Koeberl, D. D. Adjunctive β2‐agonist treatment reduces glycogen independently of receptor‐mediated acid α‐glucosidase uptake in the limb muscles of mice with Pompe disease. FASEB J. 28, 2272–2280 (2014). www.fasebj.org

Hepatic mitochondrial dysfunction is a feature of Glycogen Storage Disease Type Ia (GSDIa)

Glycogen storage disease type Ia (GSDIa, von Gierke disease) is the most common glycogen storage disorder. It is caused by the deficiency of glucose-6-phosphatase, an enzyme which catalyses the final step of gluconeogenesis and glycogenolysis. Clinically, GSDIa is characterized by fasting hypoglycaemia and hepatic glycogen and triglyceride overaccumulation. The latter leads to steatohepatitis, cirrhosis, and the formation of hepatic adenomas and carcinomas. Currently, little is known about the function of various organelles and their impact on metabolism in GSDIa. Accordingly, we investigated mitochondrial function in cell culture and mouse models of GSDIa. We found impairments in oxidative phosphorylation and changes in TCA cycle metabolites, as well as decreased mitochondrial membrane potential and deranged mitochondrial ultra-structure in these model systems. Mitochondrial content also was decreased, likely secondary to decreased mitochondrial biogenesis. These deleterious effects culminated in the activation of the mitochondrial apoptosis pathway. Taken together, our results demonstrate a role for mitochondrial dysfunction in the pathogenesis of GSDIa, and identify a new potential target for the treatment of this disease. They also provide new insight into the role of carbohydrate overload on mitochondrial function in other hepatic diseases, such as non-alcoholic fatty liver disease.

Loss of ULK1 increases RPS6KB1-NCOR1 repression of NR1H/LXR-mediated Scd1 transcription and augments lipotoxicity in hepatic cells

ABSTRACT Lipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of Scd1 in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced Scd1 expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of Ulk1 blocked the induction of Scd1 gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the Scd1 promoter. These events abrogated the stimulation of Scd1 gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, Scd1 expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment.

Renal endoplasmic reticulum stress is coupled to impaired autophagy in a mouse model of GSD Ia

GSD Ia (von Gierke Disease, Glycogen Storage Disease Type Ia) is a devastating genetic disorder with long-term sequelae, such as non-alcoholic fatty liver disease and renal failure. Down-regulated autophagy is involved in the development of hepatic metabolic dysfunction in GSD Ia; however, the role of autophagy in the renal pathology is unknown. Here we show that autophagy is impaired and endoplasmic reticulum (ER) stress is increased in the kidneys of a mouse model of GSD Ia. Induction of autophagy by rapamycin also reduces this ER stress. Taken together, these results show an additional role for autophagy down-regulation in the pathogenesis of GSD Ia, and provide further justification for the use of autophagy modulators in GSD Ia.