Background

Non-alcoholic fatty liver disease (NAFLD) is a disorder of excessive fat accumulation (steatosis) in the liver of those who consume little to no alcohol [1, 2]. With the increase of rich foods in the diets of Western counties and develo** countries around the world, NAFLD has become a rising cause of hepatic steatosis since its first reported in 1983 [3,4,5]. Up to 25% of adults in the United States has NAFLD, with more than 80% of the obese population affected by the disease. Because there are often no initial symptoms for patients with NAFLD and the disease can only be treated by addressing the underlying condition, over time NAFLD patients often progress to severe liver fibrosis and cirrhosis [6, 7]. Because of this difficulty in treatment, liver disease is becoming one of the top leading causes of death in societies where rich diets are common. Liver disease is becoming an increasing problem in develo** countries as well. According to the Asia–Pacific Working Party guidelines on NAFLD, over-nutrition has increased the prevalence of NAFLD in the Asia Pacific regions from 23.3 to 31.9% in the past 30 years [8]. Due to increasing childhood obesity, NAFLD has also become the primary form of liver disease in both children and adolescents [5, 9, 10].

Only a few genetic variations and environmental factors have been contributed to the development of the complex metabolic associated syndrome that is NAFLD. Two variants of the triacylglycerol lipase gene PNPLA3 have been associated liver disease, one linked to severity of hepatic steatosis while the other affects hepatic triglyceride content by association with TM6SF2, a regulator of hepatic fat metabolism [14]. We have also previously reported a novel association of NG37 with high fat diet induced NAFLD [15]. Our data demonstrated that overexpression of NG37 in a transgenic mouse model results in liver enlargement and cardiac dysfunction in a high-fat diet dependent matter. This model provided a direct link between genetic predisposition and nutritional factors in hepatic lipid deposition [15].

Until now, the specific genetic causes of NAFLD were unclear, making it difficult to identify patients at high risk for severe NAFLD that is likely to progress to steatohepatitis, fibrosis, and cirrhosis. Recently, some researches have claimed that hepatic cholesterol crystals and crown-like structures distinguish Nonalcoholic steatohepatitis (NASH), an advanced subtype of NAFLD, from simple steatosis in humans [16]. These cholesterol crystal structures can also be detected in the fatty livers of thyroidectomized chickens and a murine NASH model [17, 18]. In our NG37 murine model, we occasionally found lipid droplets (LD) as optically active anisotropic liquid crystals (LC) in the hepatocytes of NAFLD mice. These anisotropic liquid crystal hepatic lipid droplets (LC-HLD) can transform into isotropic LD under the right condition. Both these LC-HLDs and isotropic HLDs could also transition into crystal structures. Based on increasing investigations on unveiling molecular mechanism of diseases with phase transition and separation [19,20,21], it is certain that the liquid crystalline could be important clue of NAFLD genesis. This phenomenon of LC to crystal transition in vitro indicates that the buildup of lipids inside hepatocytes from overnutrition could be being stored as liquid crystals. Thus we hypothesized that the crystal and crown-like structures previously identified in human hepatocytes of NASH patients are likely the final form of LC-HLD after further over-accumulation of cholesterols [16, 17, 21].

Materials and methods

Human subjects and animals maintenance

All animal care and experiment procedures were conducted in accordance with protocol approved by the Animal Care and Use Committee of Shaanxi Normal University. All human studies were conducted according to the principles of the Declaration of Helsinki, and the study protocol were approved by the Institutional Ethics Committee of the Shaanxi Normal University with written informed consents from Alenabio (** steatohepatitis, fibrosis, and cirrhosis.

In lipid-rich conditions, the apo-CIII protein is thought to function as a promotor of hepatocellular triglyceride uptake and triglyceride-rich VLDL assembly [42,43,44]. We hypothesize that during HiF diet over-nutrition, apo-CIII are distributed on the surface of liquid crystal HLD as they are on the surface of VLDL particles. From these surface locations, apo-CIII could collect and assimilates cholesterol and cholesterol derivatives into the HLD on which it resides. This gives the gene a critical role in lipid accumulation within LC-HLDs. And supports the strong association of promoter region polymorphism in apolipoprotein C3 (APOC3) (rs2854117 [− 482C>T] and rs2854116 [− 455T > C]) found to be strongly associated with NAFLD and its associated diseases, hypertriglyceridemia, metabolic syndrome, and coronary artery disease [45,46,47,48]. Thus, polymorphisms in the APOC3 gene resulting in greater than normal incorporation of cholesteric lipids into LC-HLDs would put the individual at greater risk of over saturating the HLDs liquid crystal buffering system and transitioning the HLD into its damaging crystalline form.

In addition to the APOC3 polymorphism described above, variations of PNPLA3 and TM6SF2 have also been linked to severe hepatic steatosis. Although the results are inconclusive, these genes are thought to contribute the disease by affecting hepatic triglyceride metabolism [11, 12]. Our previous study showing that liver-specific overexpression of NG37 induced fatty liver disease in a high fat diet dependent manner also put this new member of the von Willebrand A (vWA) super family on the map of metabolic diseases [15]. These high fat diet liver-specific overexpression of NG37 mice developed rapid hepatocellular liquid crystalline lipid accumulation that was greater than both high fat-diet wild type litter mates and normal diet NG37 mutant mice. In addition to liver enlargement and steatosis, these mice also developed cardiac arrhythmias commonly seen in NAFLD patients. This evidence clearly points at the importance of NG37 in hepatocellular lipid metabolism and NAFLD. Further studies are currently investigating the links between metabolism syndromes, cardiac function, and NG37. In this study, we found that MAP1LC3A a marker of lipophagy, lipid specific autophagy, is dramatically down-regulated in over-nutrition induced NAFLD liquid crystal hepatic lipid droplet accumulation. As exercise can improve lipid over accumulation by increasing lipophagy [49, 50], the next step is to investigate whether exercise can reactivate MAP1LC3A associated autophagy. Understanding the relationship between liquid crystal hepatic lipid droplets and the MAP1LC3A autophagy could provide a unique prospective towards understanding, preventing, and treating NAFLD.

Conclusions

Characterization of liquid crystal hepatic lipid droplets in mouse and patient NAFLD samples is a novel mechanism based on phase transition for evaluating the level of hepatic steatosis. Our discovery of hepatic lipid droplets with no core optical birefringence hints at the role cholesterol saturation plays in liquid crystal hepatic lipid droplet formation. By the laws of physics, as concentration of amphiphilic molecules increase it become energetically sensible to shift from a chaotic core of amphiphilic molecules protected by a layer of external facing hydrophilic molecules into a solid sphere of alternating hydrophilic and hydrophobic layers. This finding indicates that studying the degree of birefringence in patient hepatic lipid droplets may reveal disease severity long before patients become symptomatic. The greater the cholesterol content, the more thorough the droplet birefringence, and the closer a droplet comes to reaching full lipid saturation, at which point the liquid crystal droplet becomes crystalline. These less malleable droplets then cause hepatic damage, leading to steatohepatitis, fibrosis, and cirrhosis. Thus, identifying the degree of hepatic lipid droplet birefringence could be a novel diagnostic for NAFLD patients.