Ezetimibe in the balance: can cholesterol-lowering drugs alone be an effective therapy for NAFLD?
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- Musso, G. Diabetologia (2014) 57: 850. doi:10.1007/s00125-014-3192-1
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KeywordsCholesterol absorption NAFLD NASH NPC1L1
ATP-binding cassette transporter A1
Hepatic stellate cell
Lectin-like oxidised LDL receptor-1
Liver X receptor
Non-alcoholic fatty liver disease
Niemann–Pick C1 like 1
Sterol regulatory element binding factor
Sterol regulatory element binding protein
Transforming growth factor
Tumor necrosis factor
Non-alcoholic fatty liver disease (NAFLD) affects 30% of the general adult population and 70–80% of diabetic and obese patients . NAFLD confers a 1.8-fold higher mortality, largely accounted for by liver-related complications occurring in non-alcoholic steatohepatitis (NASH), the progressive form of NAFLD; accordingly, the frequency of NASH as the primary indication for liver transplantation increased from 1.2% to 9.7% in the last decade, becoming the third most common indication for liver transplantation in Western countries . In addition to liver-related complications, NAFLD is also an emerging risk factor for diabetes and cardiovascular disease .
There is no established treatment for NASH: lifestyle-induced weight loss is effective in less than 50% of individuals and the long-term safety of pioglitazone and vitamin E, suggested as possible therapeutic options by current guidelines , is far from being demonstrated; furthermore, there is no established treatment able to reverse hepatic fibrosis, the strongest predictor of future development of cirrhosis .
In 32 biopsy-diagnosed NAFLD patients, treatment for 6 months with the Niemann–Pick C1-like 1 (NPC1L1) inhibitor ezetimibe ameliorated histological features of fibrosis and hepatocyte ballooning, while there was no significant difference in steatosis, inflammation or NAFLD activity score (NAS) compared with controls (see electronic supplementary material [ESM] Table 2 of the Takeshita et al article ).
Ezetimibe treatment was associated with an increase in HbA1c and a worsening in whole-body, muscle and adipose tissue insulin sensitivity (the latter with borderline significance), while plasma pro-/anti-inflammatory markers and cytokines, including C-reactive protein, TNF-α, leptin, plasminogen activator inhibitor (PAI)-1 and adiponectin were unaffected by treatment (Table 2 of the article ).
Ezetimibe treatment was associated with hepatic downregulation of genes involved in skeletal muscle development, cell adhesion and long chain NEFA β-oxidation (including the rate-limiting enzyme, carnitine palmitoyltransferase 1A) and with upregulation of genes involved in cholesterol biosynthesis (including sterol regulatory binding factor [SREBF]-2) and immune response/phagocytosis (Table 3 of the article ).
These changes were accompanied by a significant increase in the hepatic fraction of several saturated fatty acids, including margaric (C17:0) and stearic (C18:0) acids (Table 6 of the article ).
What messages can be drawn from this small, proof-of-concept study?
As well as evaluating the effects of ezetimibe on liver fibrosis, this RCT also disclosed the shortcomings of a strategy using cholesterol absorption inhibition as the only therapeutic tool in NAFLD treatment: HbA1c and insulin resistance increased, hepatic steatosis was unaffected, and systemic proinflammatory and oxidative markers did not change or even tended to increase (TNF-α, leptin, 8-isoprostanes) with ezetimibe treatment. Most of these consequences could be anticipated by understanding the changes evoked by inhibition of cholesterol uptake in different cell types, including enterocytes, hepatocytes and adipocytes, and could be adequately prevented by proper combination therapy.
The inhibition of cholesterol uptake evokes a compensatory increase in hepatic cholesterol synthesis, driven by upregulation of the gene encoding sterol regulatory element binding factor (SREBF)-2. In addition to its main product (the key regulatory nuclear transcription factor sterol regulatory element binding protein [SREBP]-2), the SREBF2 locus also generates the microRNA miRNA-33a, which is processed from an intron within the SREBF2 primary transcript : miRNA-33a inhibits ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol export and reduces the expression of several enzymes involved in mitochondrial fatty acid β-oxidation, thereby further promoting cellular cholesterol retention and NEFA accumulation (Fig. 1). While physiological activation of SREBP-2/miRNA33a under low cellular cholesterol conditions increases the availability of cholesterol and fatty acids to promote storage of neutral cholesteryl esters, in patients with NASH this physiological feedback is disrupted and the hepatic SREBP-2/miRNA33a axis is inappropriately activated despite cellular cholesterol overload , due to several reasons: direct stimulatory effects of hyperinsulinaemia [5, 15] and proinflammatory cytokines [5, 16], downregulation of hepatic miRNA122, and functional genetic polymorphisms in SREBF-2  (Fig. 1). The net result is a cellular toxic non-esterified cholesterol and NEFA overload, promoting steatosis and insulin resistance in the liver, a proinflammatory pattern of adipokine secretion and enhanced release of toxic NEFA by insulin-resistant adipocytes (alterations collectively referred to as ‘adipocyte dysfunction’) . Therefore, it is not surprising that the lack of a consistent benefit of ezetimibe on inflammation, insulin resistance and glucose homeostasis observed in this study  has been replicated in clinical and experimental contexts other than NAFLD [19, 20, 21, 22], suggesting that other therapeutic approaches may be usefully combined with ezetimibe. The combination of ezetimibe with statins ensures a more consistent cellular cholesterol depletion, further reducing cholesterol toxicity, and it will be important to analyse the metabolic and liver-related effects of ezetimibe taken together with simvastatin in the NAFLD subgroup of the IMProved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT) . However, aggressive cholesterol reduction may not relieve lipotoxic NEFA accumulation, which results from reduced NEFA β-oxidation (induced by miRNA33a activation) and from stimulated de novo lipogenesis through liver X receptor (LXR)-α/SREBP-1c axis activation: consistently, neither atorvastatin nor ezetimibe, nor their combination, improved hepatic saturated fatty acid infiltration, insulin resistance or plasma adiponectin levels in an experimental model of NASH .
Several other therapeutic strategies may be usefully combined with cholesterol inhibitors to enhance cholesterol unloading and improve inflammatory/metabolic variables in NAFLD. The role of dietary caloric restriction and weight loss cannot be overemphasised. while excessive cholesterol intake has also been documented in non-obese NAFLD patients , resembling the much higher dietary cholesterol intake (0.2–1.25%) adopted in most experimental animal models, excessive caloric and carbohydrate consumption is common in overweight and obese NAFLD patients, which represent the vast majority of this population. Excessive dietary intake of simple carbohydrates, especially fructose, may play an important role in the pathogenesis of NAFLD-associated abnormalities [26, 27]: consistently, ezetimibe did not prevent hepatic and metabolic abnormalities induced by a high fructose diet, which triggered intestinal and hepatic de novo lipogenesis and insulin resistance independently of the cholesterol metabolic pathway .
The importance of caloric restriction and weight loss as an adjunct to ezetimibe emerges when comparing the study by Takeshita et al  with the RCT carried out by Chan et al : while in the former there was no caloric restriction or weight loss in either arm, the addition of a weight-loss regimen to ezetimibe treatment significantly improved hepatic steatosis, VLDL secretion rate and systemic inflammatory markers/cytokines compared with either weight loss or ezetimibe alone. Therefore, the synergism of caloric restriction and weight loss with cholesterol depletion could potentially fully reverse NASH-related hepatic, inflammatory and metabolic abnormalities.
Other synergisms awaiting exploration in human trials include cholesterol depletion plus gut microbiota modulation, as it has been shown that cholesterol overload sensitises HSCs to the effect of lipopolysaccharide (LPS)/TLR-4 axis activation; and cholesterol-lowering drugs plus acarbose/glucagon-like peptide (GLP)-1 analogues, since ezetimibe stimulates GLP-1 secretion by enterochromaffin cells and, conversely, GLP-1 reduced hepatic cholesterol synthesis in experimental models [30, 31, 32].
Potential synergisms of ezetimibe with other therapeutic strategies in NAFLD
Mechanisms of action/rationale
Ezetimibe + gut microbiota modulation
Intracellular non-esterified cholesterol accumulation in HSCs inhibited endosomal–lysosomal TLR4 protein degradation → ↑ TRL4 membrane expression → sensitisation of HSCs to LPS-mediated TLR4 pathway activation 
Fibrogenic effects of non-esterified cholesterol diet were independent of Kupffer cell activation or recruitment of circulating T cells/monocytes/neutrophils into the liver
Ezetimibe + statins
↓ Cholesterol accumulation in hepatocytes, Kupffer cells and HSCs 
↓ hepatic inflammation and fibrosis
Ezetimibe + curcumin
↓ hepatic fibrogenesis
Ezetimibe + caloric (fructose) restriction
↓ intestinal and hepatic de novo lipogenesis, dyslipidaemia and insulin resistance
Ezetimibe + GLP-1 analogues
GLP-1 analogues →↓ hepatic cholesterol synthesis 
↓ hepatic cholesterol overload
Ezetimibe + acarbose
Restoration of metabolic and anti-inflammatory GLP-1 action
Duality of interest
The author declares that there is no duality of interest associated with this manuscript.
GM was the sole contributor to this article.