Non-Alcoholic Fatty Liver Disease

The medical world has not done any clinical studies in humans regarding NAFLD and rapamycin. However, there is robust body of basic science studies involving anmal models of NAFLD and rapamycin. In the following studies  rapamycin ameliorated or prevent disease in animal models.

This study (Ref 2) done 2014 is entitled "Fructose leads to hepatic steatosis in Zebrafish that is reversed by mTOR Inhibition" (Rapamycin). The study done at Children's Hospital of Philadelphia Research Institute is an excellent study and presented in detail.

Of special interest is they used fructose to  induce hepatic steatosis in Zebrafish; but that equal calories of glucose did not induce disease. The use of fructose has increased from 16% of all added sweeteners in 1978 to 42% of all added sweeteners in 1998. Fructose consumption has been linked with the development and progression of NAFLD. Patients with NAFLD were reported to have greater fructose consumption. In rodent models fructose consumption has been associated with increased lipogenesis, decreased fatty acid oxidation, increased periportal fibrosis, and impaired endoplasmic reticulum (ER) stress, and impaired insulin signaling. Fructose consumption is thus implicated in the development of NAFLD."  

A number of studies showed that zebrafish develop hepatic steatosis in response to similar triggers in mammals.

"Using our NASH models, we identified mTOR signaling as a potential pathway important in NASH and demonstrate that rapamycin, an inhibitor of mTOR signaling, reverses NASH in all our models. Moreover, we show that there is activation of mTOR signaling in patients with NASH, suggesting that this pathway may be important in NASH patients, and thus perhaps rapamycin or related compounds could reverse NASH in patients."

Results

1. Fructose Treatment Induces NASH in Larval Zebrafish

Zebrafish larva were maintained in 4% fructose or 4% glucose. Larva treated with fructose developed significantly more hepatic steatosis, (Over 20% exposed to fructose and less than 10% exposed to isocaloric glucose). 

2. TOR1 Axis is Up-Regulated in Zebrafish models of Hepatic Steatosis.

All the gene pathways downstream of TOR were upregulated in fatty liver including mitochondrial dysfunction and TCA cycle (citric acid cycle).

3. Rapamycin treatment reverses Fructose-Induced Steatosis.

Rapamycin treatment rescued the steatosis and reversed the abnormal genes upregulated by steatosis.

"These results demonstrate that TOR1 inhibition reverses steatosis, as demonstrated both by lipid deposition and gene expression, caused by fructose exposure in zebrafish larvae. 

Of special note: Treatment 100 nM zebrafish did not develop hepatic steatosis. In contrast, treatment with 400 nM demonstrated ATTENUATED RESCUE.  As typical of rapamycin treatment, higher dose causes worse results. [Right dose lowers activity TOR1; but too high dose with complete inhibition of TOR1 triggers negative feedback through AKT which blunts rescue effect.]

4. Changes in TOR1 activity in patients with NASH

They examined liver biopsies of patients with NAFLD. They found high levels of phosphorylated S6, the active downstream signaling molecule of TOR1. "The increased phosphorylated (S6) appeared to be most robust around the large lipid droplets present in biopsies from patients with hepatic steatosis. These studies demonstrate that there is an increase inTOR1 signaling in patients with hepatic steatosis and NASH."

Discussion

"Here we have demonstrated that fructose treatment of larval zebrafish induces a phenotype similar to NASH, characterized by hepatic steatosis and gene expression changes consistent with oxidative stress and activation of inflammation. We also confirmed that induction of ER stress or oxidative stress independently induces hepatic lipid accumulation. We used adult animal models of NASH to identify the TOR1 pathway as a conserved pathway mediating hepatic steatosis. We then demonstrated that the TOR1 inhibitor rapamycin reverses hepatic lipid acccumulation and associated gene expression changes induced by fructose, oxidative stress, or ER stress, indicating that TOR1 signaling is necessary for hepatic lipid accumulation in multiple models of NASH. Finally, we found that TOR1 signaling was increased in liver biopsies from patients with hepatic steatosis."

"Our results indicate that rapamycin alleviates steatosis in zebrafish models through diverse downstream mechanisms...We postulate that TOR1 mediates hepatic lipid accumulation induced by fructose or oxidative stress mainly through regulation of lipogenesis. 

"Our study here demonstrates the utility of zebrafish as a model organism for studying NAFLD. We were able to generate parallel models, including nutritional models, and identify increased TOR1 signaling as a common mechanism in NAFLD. We then confirmed the relevance of this finding to liver samples from NAFLD patients, which showed increased TOR1 activity. Taken together, our results indicate that increased TOR1 signaling is a conserved mechanism in the development of hepatic steatosis."

Conclusion [Abstract]

"Fructose treatment of larval zebrafish induces hepatic lipid accumulation, inflammation and oxidative stress. Our results indicate that TOR1 activation is required for hepatic lipid accumulation across models of NAFLD, and in patients."

Study entitled "Rapamycin decreases proinflammarory and profibrogenic gene expression in experimental NASH," (Ref 3). They had 2 groups of mice, control group and group fed high fat diet (17% by adding lard and cocoa butter). The high fat diet increased liver to body weight ratio from 4.5% to 9%. (big fatty liver).

Liver injury indicated by high ALT serum levels (400 nl 30). Liver levels high TNF, IL1, TIMP1 collagen-mRNA. 

Rapamycin significantly decreased ALT.

Rapamycin significantly blunted high fat induced elevation of IL1 (pro-inflammatory), TIMP1 (inhibits proteinases) inhibits collagen I-mRNA (production of collagen and fibrosis).

Conclusion: "Rapamycin prevents proinflammatory and profibrogenic gene expression in a new model of experimental NASH. Rapamycin might therefore be a promising anti-inflammatory and anti-fibrotic long-term therapy." 

"Pharmacological Promotion of Autophagy Alleviates Steatosis and Injury in Alcoholic and Non-alcoholic Fatty Liver Conditions in Mice"; 2013 done at University Pittsburgh Medical School. (Ref 4 )

Autophagy is an extremely important cellular degradation process that can remove subcellular organelles including lipid droplets. Autophagy is part of clean up and good house keeping. Since steatosis involves excess fat droplets within cell, it is natural to suspect that removal of excess lipid droplets by autophagy should be beneficial.

Elevated TOR1 reduces autophagy. Reducing TOR1 increases autophagy.

In this study, they investigated two autophagy enhancer drugs: rapamycin and Carbamazepine. Rapamycin increase autophagy by reducing activity of TOR1. Carbamazazepine (Tegretol) is  commonly used for epilepsy, peripheral neuropathy and bipolar disorder; the drug  increases autophagy by indirect pathways not related to TOR.

In this study they induced fatty liver by either acute alcohol intoxication, chronic alcohol ingestion or high fat diet (HFD) using C57BL/6 mice (standard lab mouse).

Results Summary

Both Rapamycin and Carbamazepine induced autophagy in hepatocytes both in vivo and in vitro by both acute and chronic alcohol intake and by high fat diet.  Hepatic injury was alleviated by both rapamycin and Carbamazepine. In high fat diet both drugs reduced steatosis and improved insulin sensitivity and prevented NAFLD.

Conclusion

"Pharmacological modulation of autophagy in the liver can be an effective strategy for reducing fatty liver condition and liver injury."

"We found that the autophagy-enhancing drugs alleviated liver steatosis, liver injury, and dyslpidemia in both alcohol-fed and HFD fed mice. These finding indicate that the autophagy-promoting drugs have important implications as a new genre of therapeutic agents for fatty livers."

Results

"Enhanced autophagy could improve fatty liver condition in HFD-fed mice. Mice were fed a high fat diet for 12 weeks. In last week rapamycin group given 3 injection, every other day at dose of 2 mg/kg. [Note: this is low intermittent dose for mouse which caused no side effects.]

"We found that this short-term treatment could significantly reduce hepatic steatosis and hepatic and blood TG (tryglyceride) levels. The plasma ALT (liver enzyme showing liver damage) was also noticeably reduced. Interestingly, insulin resistance was improved as well. as measured by level of blood glucose and insulin."

Discussion

"Emerging evidence now indictes that the progression of NAFLD is associated with a reduced function of autophagy."..."Our present study provides the first example of achieving results using available pharmacological agents that stimulate autophagy, indicating that this strategy could be consiered as a novel approach for alleviating the liver condition in NAFLD as well as in AFLD (alcoholic).

"Frank Mallory described cytoplasmic hyaline inclusions in hepatocytes of patients with alcoholic hepatitis in 1911."  (Ref 5) These inclusions were recently renamed Mallory-Denk bodies (MDB). "MDBs are large eosinophic hepatocellular cytoplasmic aggregates which are characteristic hallmarks of alcoholic steatohepatis and NASH They are associated with fat droplet accumulation combined with failure of autophagy combined with misfolding proteins. They are histologic marker of progression of NASH to hepatic fibrosis (cirrhosis)."(Ref 6)

In the study noted (Ref 7) they used mice overexpressing keratin (K8 mice) which made the mice vulnerable to liver damage and formation of MBDs. They treated the mice with bortezomid, a drug which impairs the proteasomal and lysosomal/autophagy pathway. The mice developed MBDs. 

"Activation of autophage by rapamycin decreased the number of MBD inclusions."

"Rapamycin also led to resorption of spontaneously formed MBDs in aging K8 overexpressing mice."

Conclusion "Rapamycin-mediated activation of autophagy prevents MBD formation and causes MBD resorption."

"Autophagy, or cellular self-digestion, is a catabolic process that targets cell constituent, such as damaged organlles, unfolded proteins, and intracellular pathogens, to lysosomes for degradation" Autophagy is crucial for survival. Autophagy increases in starvation and decreases with nutrient abundance. Autophagy also decreases with age and with increased activity of mTOR1. 

Autophagy plays a major role in many age-related diseases. The reason autophagy is of such importance is that many substances if not properly removed become toxic. The most toxic substance is degenerating mitochondria which contain DNA of bacterial origin and thus can cause inflammation and death of the cell. As seen in this discussion of NAFLD, excess lipids are also toxic to liver cells. Autophagy is the key player in NAFLD. This places autophagy and the liver at the center of insulin resistance, diabetes type 2 and metabolic syndrome.

In simple terms autophagy is a 4 step process: form a sac, engulf stuff in the sac, add a packet of acid to the sac and then dissolve contents of the sac.

18 different autophagy related proteins (Atg) participate in process.

The sac is called a phagophore and is a double-membranes structure, derived from endoplasmic reticulum (ER).  The first step called "Initiation" is controlled by mTOR1 and inhibited by high activity of mTOR1. The second step involves Beclin, PI3K , Atg7, LC3-11 as major players and is called Nucleation step and is engulfment of substance.

The sac with enclosed particles is called an autophagosome.

Next step is Fusion, which consists of adding a vesicle called Lysosome,  filled wth "acid" called Hydrolases to the autophagosome which after adding lysosome is called Autolysosome. The final step is Degradation, which is dissolving stuff in autolysosome.

TFEB (transcription factor EB) is exercises the master control of the autophagosome-lysosome process and TFEB is under control of mTOR1. High activity of mTOR1 inhibits activity of TFEB. 

[The discussion of Parkinson disease under Neurodegeneration has major discussion of TFEB.]

The obesity complications of liver disease fall under broad category of Non-alcoholic fatty liver disease (NAFLD). There are 4 stages of disease:

Steatosis: Intrahepatic triglyceride accumulation (a reversible disease)

Non-alcoholic steatohepatitis (NASH): Inflammation + hepatocellular ballooning/Mallory-Denk bodies + hepatocyte death.

Fibrosis/Cirrhosis: Fibrosis sometimes leads to cirrhosis

Hepatocellular Carcinoma (HCC): a potential complication 

NAFLD could be considered a "2 hit disease". "Peripheral insulin resistance may represent the "first hit" in the pathogenesis of NAFLD, which leads to hepatic steatosis (fatty liver). Combined hyperglycemia and hyperinsulinemia promote de novo lipid synthesis and structural defects in mitochondria within hepatocytes. Moreover, insulin resistance of adipose tissue leads to an enhanced free fatty acid influx to the liver that contributes to steatosis."

Steatotic hepatocytes may be vulnerable to a "second hit" induced by cytokines (such as TNFa) and oxidative/ER stress, which lead to the development of NASH and fibrosis. Apoptotic hepatocytes are engulfed by kupffer cells, which results in their activation and inflammation. Activate kupffer cells leads to transformation into stellate cells which produce liver fibrosis.

Rapamycin can suppress both "first hit" and "second hit". Insulin resistance and elevated activity of mTOR are at center of molecular disease process

In obesity, the level of autophagy is decreased. Several mechanisms may account for this decline.

1. Autophagy is inhibited by the mTOR-insulin-amino acid signaling pathway. Short-term inhibition can be produced by mTOR1. Long-term regulation occurs via transcription factors FoxO and TFEB. FoxoO can be inhibited by insulin through Akt/PKB pathway. mTOR1 can inhibit TFEB, which is master regulator of autophagy/liposomal pathway.

"In obese mice with hepatic steatosi, the autophagy inhibitor mTOR1 is overactivated  in the liver,presumably as a result of an increasedaminoacid concentration following overnutrition. It has been shown that  the overactivation of mTOR by infusion of amino aci mixture can resultin liver and muscle insulin resistance because of phosphorylation and inhibition of IRS1 by S6 kinase, a downstream target of mTOR1. "

2. Hyperinsulinemia can cause downregulation of autophagy hroug Akt/PKB, a key molecule in insulin pathway which decrease autophagy.

3. Calpain-2/Atg7. Increased levels of Calpain-2, a protease are produced in obesity. Calpain 2 inactivates Atg7 and Atg7 is a key regulator of level of autophagy. 

In 2010 paper by Yang, Ref 12 entitled, "Defective Hepatic autophagy in Obesity promotes ER Stress (endoplasmic reticulum) and causes Insulin Resistance" they demonstrated following:

a. Severe downregulation of Autophagy in obesity.

b. Severe downregulation of Atg7.

c. Inverse relationship between Calpain 2, produced by adipose tissue and Atg7 (calpain 2 inactivates Atg7)

d. Suppression of Autophagy result in insulin resistance and ER stress.

e.Restoration of hepatic autophagy in obese mice restores glucose homeostasis, insulin acion and ameliorated ER stress. 

f. Restoration of Atg7 restored insulin sensitivity.

This paper showed how obesity, acting through action of calpain-2 was directly controlling Atg7, thereby level of autophagy and insulin resistance. (Ref 12)

4. Cathepsin. Cathepsin is a protease, it causes proteolysis (dissolves proteins). Cathepsin is present in lysosomes (bags of "acid") which when added to autophagosome forms autolysosomes and dissolves contents.

In hepatic steatosis (in contrast to other liver diseases) cathepsin production is reduced causing impairment of autophagosomal acidification and cathepsin expression. [autolysosomes don't work)

5. Defective autophagosome-lysosome fusion. There is change in membrane lipid composition in livers on high fat diet. This results in defection fusion of autophagosome and lysosome. Result is defect in hepatic autophagy. This results in increase in ER stress and increase in insulin resistance.

All these defects in obesity/fatty liver act together to cause a vicious cycle which exacerbates situation. 

"In response to a moderate increase in lipid availability or during nutrient deprivation, hepatic autophagy degrades lipid droplets to provide FFA for ATP production.In contrast, a sustained availability of lipids, induced by a long-lasting HFD (high fat diet) challenge, inhibits autophagic turnover. This ability of autophagy to degrade lipid droplets in hepatocytes has been termed lipophagy."

An important paper is, "Autophagy regulates lipid metabolism" (ref 13) from Albert Einstein College of Medicine (2009). 

[The hepatic lipophagy system is perfectly designed to provide energy from stored fat during starvation. It is a very bad system to handle prolonged excess calories and fat. The reason being that high levels of lipophagy is required to handle lipid load into liver. During starvation mTOR activity goes down and lipophagy goes up. In contrast during abundance of energy, calories, fat; mTOR goes up and lipophagy goes down. ]

"Free fatty acids (FFAs) are taken up by hepatocytes and converted into triglycerides (TGs) for storage with cholesterol in lipid droplets (LDs). LD-sequestered TGs continually undergo hydrolysis, generating FFAs that are predominantly re-esterified back into TGs for storage. Nutrient deprivation upregulates TG hydrolysis to supply FFAs for oxidation to meet cellular energy demands. Both autophagy and lipolysis are regulated hormonally by insulin.

The main points are:

a. Inhibition of autophagy increases lipid storage.

b. Autophagy is required for lipid droplet breakdown

c. Autophagy regulates hepatic lipid storage. 

Their finding summarized as follows:

"Autophagy regulates lipid content because: inhibition of autophagy increased TGs and lipid droplets; loss of autophagy decreased TG breakdown.

"Moreover, a reverse relationship exists in which abnormal increase in intracellular lipid impairs autophagic clearance." (Ref 13)

"This interrelationship may trap hepatocytes in a harmful cycle in which decreased autophagy promotes lipid accumulation that then further suppresses autophagic function, thereby additionally increasing lipid retention."

"Decreased autophagy in the liver with ageing may contribute to hepatic lipid accumulation that occurs along with an increased incidence of the metabolic syndrome in aged humans. The ability of increased  lipid content to impair autophagy also indicates that lipid accumulation could contribute to the decrease in autophagic function with ageing. Therapeutic stategies to increase autophagic function may therefore provide a new approach to prevent the metabolic syndrome and its associated pathologoes." (Ref 13)

Activation of autophagy in hepatocytes could constitute a therapeutic approach against hepatic complications". This is shown by 4 different lines of evidence.

1. Genetic: "Overexpression of ATg7 (which increases Autophagy) in HFD-fed mice improved fatty liver and insulin resistance."

2. Starvation: Starvation increases the delivery of FFA from adipose tissue to the liver. However, starvation also reduces mTOR1, which increases autophagy. The liver of starved mice displayed an increase in lipid droplets, plus autophagosomes, lysosomes and autophagolysosomes. These mice were protected against fatty liver disease. In contrast, hepatocyte-specific Atg7- deficient mice with a defect in autophagy developed fat-induced liver disease in starvation.

3. Pharmacologic studies: Enhancers of autophagy such as rapamycin and carbamazepine (see above study Ref 6) protected HFD-obese mice from fat induced liver disease.

4. Epidemiologic studies (Coffee). A large number epidiologic studies have shown a remarkable beneficial effect of coffee in protction against fat induced liver disease. The mechanism is that coffee (caffeine) protects against fatty liver disease bu coordinatin induction of lipophagy and increasing mitochondrial B-oxidation which burn FFAs.

"A vicious cycle takes place: hyper-insulinemia negatively regulates hepatic autophagy in the steatotic liver and the decline in hepatic autophagy enhances ER stress and insulin resistance."

"Recent studies have suggested that ER (endoplasmic reticulum) stress could be the link between obesity, insulin resistance, and type 2 diabetes. The inhibition of hepatic ER stress reduced liver steatosis. Defective hepatic autophagy in obesity could promote ER stress and cause insulin resistance. Since autophagy is known to eliminate mis-/unfolded proteins, and impairment in hepatic autophagy could lead to accumulation of mis-/unfolded proteins and induction of ER stress. In the liver of obese mice, it has been reported that a decrease in autophagy promotes ER stress leading to insulin resistance." Increase in autophagy in the liver of obese mice significantly reduced ER stress, decreased the triglyceride content, and improved glucose tolerance and insulin sensitivity."

[The above is excellent description of the vicious cycle of increasing insulin resistance and at the center of this vicious cycle is increased activity of mTOR1.]