Distinct nuclear lipid droplet characterized in liver biopsies
The patient characteristics are summarized in Table 1. Out of 80 patients, 35 were males and 45 females. Their mean age was 58, ranging from 24 to 89 years old. Liver biopsy-proven liver diseases included nonalcoholic steatohepatitis (NASH) in 12 patients, drug-induced liver injury (DILI) in 11 patients, malignant tumor in 22 patients, autoimmune hepatitis (AIH) in 7 patients, and others in 28 patients. According to the METAVIR classifications of liver fibrosis, the scores were F0 in 39 patients, F1 in 26 patients, F2 in 3 patients, and F3 in 5 patients, respectively. Liver steatosis assessed by H&E staining was less than 5% in 46 patients, 5–33% in 21 patients, 33–66% in 4 patients, and more than 66% in 2 patients. Blood profiling of the patients is summarized in Table 2. Although the levels of liver enzymes, alkaline phosphatase, and gamma-glutamyl transpeptidase were elevated, that of plasma lipids were within normal range.
Electron microscopy observations of liver biopsy samples were conducted independently from clinical diagnosis by light microscopy. Six patients were excluded from further analysis because the electron microscopy sample did not contain the corresponding background liver specimen. Surprisingly, 76% of the liver biopsy specimen presented lipid droplets in the hepatocyte nucleus.
Case assessment toward liver diseases and patient profile
Three representative cases are as shown in Figure 1C–E. The first case is a female in her 70s showing mild increase in aminotransferase levels (aspartate aminotransferase; AST 125 U/L and alanine aminotransferase; ALT 106 U/L), who was diagnosed with NASH. In her liver biopsy, hepatocyte ballooning and mild inflammation were observed with 40% steatosis and F2 fibrosis liver tissue (Figure 1C). nLDs were found in 5.6% of nuclei whereas cLD in NR was not found in nuclei (Figure 1C). The second case is a male in his 20s with elevated AST (211 U/L) and ALT (546 U/L) levels, who was diagnosed with DILI. Mild inflammation in the portal area was observed with 0% steatosis and F0 fibrosis liver tissue (Figure 1D). nLDs were found in 4.2% of the nuclei and cLDs in NR were found in 1.2% of the nuclei in the liver biopsy sample (Figure 1D). The third case is a male in his 40s with mild elevations in AST (134 U/L) and ALT (250 U/L), who was diagnosed with AIH. Severe inflammation in the portal area was observed with 0% steatosis and F1 fibrosis liver tissue (Figure 1E). nLDs were found in 5.7% of nuclei and cLDs in NR were found in 5.7% of nuclei of the liver biopsy sample (Figure 1E). Overall, nLDs were found in 69% of liver biopsy samples and cLDs in NR were found in 32%, less frequent than nLDs (Figure 2A, B). Notably, there was a significant positive correlation between the frequencies of nLD and cLD in NR (Figure 2C). Although both nLD and cLD in NR are most frequently detected in AIH (Table 1), no disease-specific distributions were observed in their frequencies (Figure 2D). These results revealed that two types of nuclear lipid droplets are found in the human liver.
Correlation analysis of lipid droplets and clinical parameters
Next, we studied the correlations between these two types of lipid droplets in the nucleus and their clinical characteristics. Although patients who presented with nLD showed higher plasma liver enzymes than those who without nLD, there were no significant differences in tests depending on the presence of nLD (Table 3). Conversely, patients who presented cLD in NR showed significantly lower levels of total cholesterol and low-density lipoprotein (LDL) cholesterol compared to patients without cLD in NR (Table 3). Thus, the two types of lipid droplets in the nucleus were clinically different. Further, nLD was frequently found in hepatocytes of NASH, however, there was no correlation between nLD and hepatic steatosis, indicating that nLD does not reflect only cytoplasmic lipid accumulation (Figure 3A). Moreover, positive correlations were found between nLD and ER expansion or liver enzymes (Figure 3B, C), suggesting nLD formation in the nucleus under ER stress or liver damage. In contrast, cLD in NR showed a negative correlation with steatosis, implying that it is formed in lipid-restricted circumstances (Figure 3D). Furthermore, no correlations were found between cLD in NR and ER expansion or liver enzyme (Figure 3E, F). Unlike our expectations, no correlation was found between these two types of nuclear lipid droplets and plasma lipids (Suppl Figure 2A–F). These results unveiled two distinctly possible pathophysiological roles of nLD and cLD in NR in liver diseases (Figure 4).
