Non-alcoholic fatty liver disease and type 2 diabetes mellitus: The liver disease of our age?

Table 1 Prevalence of non-alcoholic fatty liver disease, non-alcoholic fatty liver and non-alcoholic steatohepatitis

NAFLD/NAFL prevalence	NASH prevalence	Population studied	Population size	Method of diagnosis	Remark	Ref.
46 (40)% NAFLD in the entire US cohort	12.2% in the US entire cohort	18-70 yr aged US cohort	328	Liver biopsy (in 134 ultrasound pre-screend patients)		[6]
	29.9% in patients with NAFLD				Greatest risk for both NAFLD and NASH in Hispanics and with diabetes
NAFL: 49.3%-mild, moderate, and severe NAFL in 38.9%, 9.0% and 1.4% donor candidates respectively (mild steatosis was defined as fatty changes in 5%-30% of hepatocytes, moderate steatosis in 30% to 60% of hepatocytes, and severe steatosis in > 60% of hepatocytes without significant inflammation on liver histology)	2.2% (Asian population)	Korean living liver donor candidates	589	589 US guided liver biopsy		[126]
NAFL: 4.2% in pediatric European population	1% (pediatric population)	European children (6 mo-18 yr old)	342 medicolegal autopsy reports/265 children died from trauma	Histopathology at autopsy (and typical macroscopic imaging)	Excess body weight was observed in 55.6% of children with NAFL	[11]
NAFLD 9.6% in pediatric US population fatty liver was defined as > or = 5% of hepatocytes containing macrovesicular fat	NR	United States children (2-19 yr old)	742 children (2-19 yr old) who had autopsy (form 1993 to 2003)	Histopathology at autopsy	Different prevalence according to subpopulation (Asians: 10.2%; Black: 1.5%; Hispanic: 11.8%; White: 8.6%). The highest rate of NAFLD was seen in obese children (38%)	[12]
NAFLD 31% in adult Urban US population	NR	Large, ethnically diverse, probability-based adult population sample from Dallas, Texas, United States -participants in the Dallas Heart Study	2349	1H-MRS of the liver to quantify HTGC	79% of patients with hepatic steatosis had normal levels of serum alanine aminotransferase. Different prevalence of hepatic steatosis in different sub-populations: 45% in Hispanics, 33% in Whites and 24% in Blacks	[4]

Based on histological examination or proton magnetic resonance spectroscopy (¹H-MRS) measurement in Hallmark Studies. HTGC: Hepatic triglyceride content; NAFLD: Non-alcoholic fatty liver disease; NAFL: Non-alcoholic fatty liver; NASH: Non-alcoholic steatohepatitis; NR: Not reported.

Table 2 Adipokine hormones, cytokines, growth factors and other mediators that play important role in non-alcoholic fatty liver disease pathology

Mediator/pathway	Observation /proposed mechanism	Ref.
Adiponectin	Shown to have anti-inflammatory and antifibrotic activity, serum levels were found to be decreased in NAFLD and NASH patients. Plasma adiponectin in NAFLD is related to hepatic insulin resistance and hepatic lipid content - not to liver disease severity	[127]
Ghrelin	Serum levels were found to be diminished in NAFLD and NASH patients - no correlation with histological grade. Ghrelin administration attenuated oxidative stress, inflammation and apoptosis in high fat diet induced NAFLD animal model	[128,129]
Leptin	Leptin levels are generally known to be higher in the sera of NASH patients, except for a subgroup; serum levels were shown to negatively correlate with AST/ALT levels. The livers of leptin-deficient mice were found to be unusually sensitive to LPS-induced injury. Recombinant leptin therapy was in clinical trial in patients with NASH and low leptin levels - no results were posted	[128] and ClinicalTrials.gov Identifier: NCT00596934
Resistin	Serum levels were shown to be significantly higher in patients with NAFLD and NASH	[130]
Small bowel bacterial overgrowth (SIBO)	Increased gut permeability and tight junction alterations in NAFLD. Higher prevalence of small intestinal bacterial overgrowth in NAFLD patients - correlated with the severity of liver steatosis	[131]
Toll-like receptor-4 (TLR4)	Both the TLR4 (endotoxin-receptor) protein and RNA levels were found to be elevated in liver in NASH	[132]
Nuclear factor-κB (NF-κB)	Increased activation of NF-κB was found in NASH	[132]
Tumor necrosis factor-α (TNF-α)	Key mediator of inflammation, serum levels are elevated in NASH. TNF-α expression in adipose tissue is upregulated in several models of obesity. In patients, TNF-α levels were shown to be higher in obese than in lean individuals, and were correlated with insulin resistance	[133]
Interleukin-6 (IL-6)	Increased plasma levels and hepatic expression was described in NASH patients. Increased hepatic IL-6 production may play an important role in NASH, insulin resistance and diabetes development	[134]
Transforming growth factor-β (TGF-β)	A key growth factor and a major inductor of hepatic stellate cell activation and therefore hepatic fibrosis, TGF-β signaling in hepatocytes may contribute to hepatocyte death and lipid accumulation via Smad signaling and ROS production	[135]
Th17 cells and IL-17	In the livers of mice on a high fat diet and also NASH patients an increased number of hepatic Th17 cells could be detected. In mice Il-17 neutralization ameliorated LPS induced liver injury	[136]
	Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease
Notch-mTOR pathway	Liver-specific ablation of Notch signaling, or its acute inhibition with a decoy Notch1 receptor, prevents hepatosteatosis by blocking mTor complex 1 (mTorc1) activity. Notch gain of function induces NAFL through constitutive activation of mTorc1	[137]
Mastocyte chymase	This enzyme is important in the convertion of angiotenzin-I to angiotenzin-II and the activation of matrix metalloproteinase-9, which both are involved in the development of liver fibrosis. Chymase inhibitor prevents the nonalcoholic steatohepatitis in a hamster model	[138]
Galectin 3	Galectin 3 is a β-galactoside-binding lectin with a multiple functions. It is also a receptor of advanced lipoxidation endproducts and plays important role in inflammation, fibrosis and carcinogenesis. Its role is suspected in NASH. Regression of fibrosis by galectin inhibitors in thioacetamide-induced liver disease animal model. Phase 1 Study with a Galectin inhibitor GR-MD-02 in patients with NASH and advanced fibrosis	[139,140], ClinicalTrials.gov Identifier: NCT01899859
Fibroblast geowth factor -19 (FGF19)	Both the intestinal FGF19 production and the hepatic response is impaired in NAFLD patients. A decrease in fasting FGF19 levels is associated with the development of non-alcoholic fatty liver disease in obese adolescents	[141]

A few of these molecules are therapeutic targets (e.g., galectin 3) in early phase clinical trials. NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.

Table 3 Single nucleotide polymorphisms associated with non-alcoholic fatty liver disease in genome wide association studies

Region	SNPs	Reported gene(s)	Mapped gene	OR	Risk allele frequency in controls	P value	Gene product function	Context	Initial sample size	Replication sample size	Platform (SNPs passing QC)	Ref.
22q13.3	rs738409 (I148M), rs2896019-G	PNPLA3	PNPLA3	2.02	0.450	2 × 10-20	Adiponutrin (PNPLA3)–nutritionally regulated lysophosphatidic acyltransferase: expressed in liver and adipose tissue. High CH diet increases expression. Has TAG hydrolase and DG transacylase activity. Strong predictor of steatosis, inflammation and fibrosis. Dysfunctional PNPLA3 promotes accumulation of lipotoxic substrates. I148M–association with HCC in severely obese individuals	Intron	392 Japanese cases, 934 Japanese controls	172 Japanese cases, 1012 Japanese control	Illumina [261540]	[119,120]
	rs738491, rs3761472, rs2143571	SAMM50	SAMM50
	rs6006473, rs5764455, rs6006611	PARVB	PARVB
1p35	rs6691847-C	PTPRU	PTPRU - MATN1	1.32	0.770	7 × 10-6	Member of the protein tyrosine phosphatase (PTP) family. PTPs are signaling molecules that regulate cell growth, differentiation, mitotic cycle, and oncogenic transformation
4q13.3	rs222054-C	GC	LDHAL6EP - GC	2.54	0.301	1 × 10-6	Albumin gene family. Multifunctional protein found in plasma, ascitic fluid, cerebrospinal fluid and on the surface of many cell types. It binds to vitamin D and its plasma metabolites and transports them to target tissues		126 European adolescent cases - 802 European adolescent controls	NR	Illumina [2078805] (imputed)	[142]
16q23	rs11864146-A	SLC38, A8	SLC38A8	3.14	0.100	2 × 10-6	Amino acid transport: Putative sodium-coupled neutral amino acid transporter 8	Intron
13q14.1	rs7324845-A	LCP1	LCP1	3.29	0.096	3 × 10-6	L-plastin, an actin binding protein expressed in hemopoetic cell lineages. It is expressed by many solid tumor types of non-hemopoetic origin, suggesting its role in tumorigenesis	Intron
1p21	rs12743824-C	LPPR4	LPPR4 - PALMD	2.30	0.441	5 × 10-6	LPPR4: a lipid phosphate phosphatase, catalyzes the dephosphorylation of a number of bioactive lipid mediators, found to be important for axonal outgrowth; PALMD: cytosolic isoform of paralemmin-1, a lipid raft-associated protein implicated in cell shape control	NR				PALMD gene product - [143]
7p14	rs343064-A	Intergenic	TBX20-HERPUD2	1.31	0.400	3 × 10-8	TBX20: transcription factor; HERPUD2: not reported		236 non-Hispanic Caucasian women	NR	Illumina [324623]	SNPs of other genes were also associated with qulatitative traits (e.g. NAFLD activity score and FDFT1 - rs2645424) as discussed in the text [123]
2q31	rs1529093-A	Intergenic	RPL29P8 - KRT8P40	4.13	0.410	2 × 10-6	RPL29P8, KRT8P40: pseudogenes			NR	Illumina [324623]
4p15.2	rs959903-A	23231	SEL1L3	3.81	0.290	7 × 10-6		Intron		NR	Illumina [324623]

Not all GWAS studies were replicated. Risk gene variants with OR higher than 1.31 are indicated in the table. Based on the National Human Genome Research. SNPs: Single nucleotide polymorphisms; NR: Not reported.