Urbanised diets and the consequent rise in obesity amongst Black South Africans has led to gallstones becoming more prevalent amongst this population group in recent years. Established risk factors for gallstones – over forty years of age, female, pregnancy, overweight, high-fat diet, and being sedentary, are all lifestyle or demographic dependent (10). Little is known on the implications of chronic infectious diseases like HIV on gallstone disease risk. Our unpublished data evaluating risk factors in HIV positive females (n = 18) showed that these patients do not conform to these risk factors, with this population being significantly younger with lower BMI relative to uninfected patients (26). The present study sought to investigate molecular events that may contribute to this phenotype by measuring the hepatic expression of cholesterol metabolism genes that influence gallstone formation. Biological response to a high fat diet, the rate of cholesterol absorption, hepatic cholesterol metabolism and the concentration of cholesterol excreted in bile differs between patients with and without gallstones (27). Raised LDL-c in circulation is strongly implicated in lithogenic states. Lowered HDL-c levels are also implicated in gallstone formation but data regarding this finding has been conflicting in reports (28).
Mounting evidence suggests the resultant abnormalities are linked to dietary and genetic factors. LDL-c is cholesterol that is available for delivery and cellular uptake; the circulating concentration is linked directly to dietary cholesterol consumption and associated with both cardiovascular disease and cholesterol gallstone formation (11, 29). HDL-c, excess cholesterol transported from cells to the liver for excretion in a process called reverse cholesterol transport, is considered the “healthier” cholesterol due to protective properties against atherosclerosis. Lower HDL-c is linked to poorer cardiovascular outcomes and is also implicated in cholesterol gallstone formation (30). Elevated activity of cholesteryl ester transfer protein (CETP) in patients with gallstones may lower HDL-c and increase LDL-c by transferring the lipoprotein from HDL to LDL (31).
A high circulating LDL-c will result in higher concentrations of cholesterol being delivered to the liver for excretion resulting in supersaturation of bile, inevitably leading to gallstone formation. This is best explained by Admirand’s triangular relationship between cholesterol, bile salts and lecithin, where biliary cholesterol hypersecretion supersedes the concentration of bile salts and lecithin with resultant precipitation and formation of cholesterol stones (9). The cholesterol hypersecretion is due to excess free cholesterol pooling in the liver either due to increased HMGCR activity or larger volumes of cholesterol returning to the liver. Cholesterol is returned to the liver via one of 4 pathways; via the LDLr, via apoE-rich lipoproteins through the LDL receptor-like protein (LRP), via HDL2 free cholesterol "exchange," or via nonreceptor-mediated LDL uptake (32).
Altered cholesterol metabolism, favouring LDL-c is complicit in gallstone formation, among other metabolic disorders such as cardiovascular disease. Although HIV and ART are linked to metabolic disorders leaning toward this phenotype, little is known of the effects of HIV on gallstone formation. In HIV positive patients, low levels of HDL-c are congruent with high viral loads, linking this metabolic profile directly to the virus itself independent of ART (33). The use of ART reverses these effects. LDL-c levels however appear similar to HIV negative female patients, but rises dependant on the use of PI-ART (34). In male patients however, there is an inverse relation with LDL-c and HIV RNA levels independent of ART, and this may well be a major contributing factor to males being relatively protective against gallstones as indicative of the low incidence of gallstones in HIV positive males (35).
These findings are not dissimilar to that in our study group where all patients were female and all on ART. The use of ART may explain the significantly higher LDL-c, and the slight increase in HDL-C in the HIV group compared to the control (Table 1). Altered circulating LDL-c would prompt cellular homeostatic responses. As HIV positive women in our study displayed higher LDL-c, we evaluated the first line of hepatic LDL-c uptake. Located on the cell membrane, LDLr is responsible for hepatic absorption of LDL, with higher expression reducing circulating LDL-c levels. The expression of LDLr is regulated by various regulatory mechanisms including transcription factors (SREBP2 and LXR) and epigenetic regulators like miR-148a. SREBP transcription factors regulate the biosynthetic pathway of cholesterol and LDLr by stimulating transcriptional genes containing sterol response elements (36). There are 3 isoforms; SREBP1a, SREBP1c and SREBP2. SREBP2 is highly expressed in liver amongst other cells. Abnormalities in these regulatory elements (decreased SREBP2 and LXR) results in decreased LDLr expression and decreased LDL catabolism resulting in raised LDL-c serum levels (37).
HIV-1 infection of CD4+ T cells stimulates cholesterol biosynthesis via SREBP2 sterol response gene (protein TFII-I) activation for enhanced HIV-1 transcription and HIV-1 replication (38). HIV-1 transcription is thus modulated by LDL-c, since uptake of LDL-c results in feedback inhibition of SREBP2- dependent proteins such as TFII-I (38). The hepatic impact of this in HIV patients with gallstones in not known. However, LDLr levels in mononuclear cells of both the blood and liver have been found to be down-regulated in HIV positive patients with lipodystrophy compared to HIV negative controls and positive patients without lipodystrophy, independent of PI-ART (39). The pathogenesis of this lipodystrophy by down-regulation of LDLr may not be dissimilar to that of gallstone pathogenesis in HIV positive patients as these mimic our findings in this study. Further to this we demonstrated that the downregulation of LDLr (Fig. 1D) was due to a decrease in SREBP2 mRNA (Fig. 2) with overall significantly raised LDL-c levels in the HIV group.
Cholesterol reverse transport is also an important determinant in cholesterol homeostasis. In this regard, the ABC family of efflux pumps plays a significant role in cellular efflux of cholesterol. ABCA1 is a major regulator of cellular reverse cholesterol transport by transporting cholesterol, mainly the lipid poor apoA1, out of the cell and converts it into mature HDL for transport back to the liver. Overexpression of ABCA1 in transgenic mice results in a lithogenic state by increasing plasma HDL-c levels, hepatic delivery of HDL cholesteryl-esters and biliary cholesterol concentrations (40). Its lithogenic role is further accentuated in gallbladder epithelial cells, where ABCA1 is regulated by LXR and RXR and modulates biliary cholesterol concentrations and its excretion (41).
ABCA1 expression can be epigenetically regulated by miR-148a. Inhibition of miR-148a increases ABCA1 mRNA levels resulting in increased cholesterol efflux to ApoA1, thus increasing plasma HDL-c (20). The inverse relationship between ABCA1 by miR-148a is evident in HIV positive patients with gallstones compared to their negative counterparts as demonstrated in our study (Fig. 1B, C). The effect on plasma HDL-c however was negligible in our study (Table 1).
The effect of HIV on hepatic ABCA1 expression has not been studied, however in order for survival and replication of the virus within lymphocytes, it requires large amounts of cholesterol within the cell. It achieves this by directly inhibiting ABCA1. The level of inhibition of cholesterol efflux is directly proportional to the level of viral replication within the cell. It achieves this by encoding a small protein called Negative Regulatory Factor (Nef) which binds to ABCA1 and down regulates it thus preventing the efflux of apoA1 cholesterol to HDL (5).
Besides its regulatory role of ABCA1, miR-148a is considered a central miRNA in cholesterol and fat metabolism. MicroRNA-148a is located in a gene-poor intergenic region of human chromosome 7 and is predominantly expressed in liver (20). Notably, the expression of miR-148a is significantly increased in the liver of high-fat diet (HFD)-fed mice (20). Out of 159 miRNAs identified to be highly expressed in human liver and modulated by dietary lipids, miR-148a emerged as the strongest with highest liver activity and expression in livers of HFD fed mice (42). Lastly, we assessed cholesterol biosynthesis in HIV positive patients via HMGCR levels. HMGCR, the rate limiting enzyme in the mevalonate pathway is regulated by SREPB2 which up-regulates cholesterol synthesis genes when cholesterol levels are low (43). Our study showed the HIV group having lower HMGCR mRNA levels (Fig. 3) which may be due to cholesterol levels being higher in this group.