The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism

Abstract

Reduced expression of the Indy ("I am Not Dead, Yet") gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane-associated citrate transporter expressed highly in the liver, protects mice from high-fat diet-induced and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We studied a possible role of mIndy in human hepatic fat metabolism. In obese, insulin-resistant patients with nonalcoholic fatty liver disease, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In nonhuman primates, a 2-year high-fat, high-sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription through the IL-6 receptor and activation of the transcription factor signal transducer and activator of transcription 3, and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-signal transducer and activator of transcription 3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx, and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and nonhuman primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 through mINDY.

Conclusion: Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450. (Hepatology 2017;66:616-630).

Figure 1. Human mIndy tissue distribution and functional characteristics

A) Human mIndy mRNA tissue expression in different tissues. B and C) mINDY transport kinetics for citrate (B) and succinate (C) were carried out in HEK293 cells transfected with either the pIndy-human.31 plasmid or empty expression vector pcDNA3.1(+). The net uptake is expressed as the difference between the uptake of substrates into HEK293 cells overexpressing human mINDY and pcDNA-transfected controls (n=3–6 for each concentration). K_m values were determined by fitting the data to a non-linear regression curve fit.

Figure 2. mIndy expression in the liver of patients with different BMI and liver fat contents

A) mIndy expression was positively associated with BMI, B) waist circumference, C) body fat, D) histologically assessed liver fat content. The degree of liver histology-determined steatosis remained significantly associated with mIndy expression after adjustment for several confounders (Model in Table 1), identifying mIndy as an independent risk factor for NAFLD in our cohort of patients.

Figure 3. mIndy expression in liver tissue from non-human primates

Rhesus monkeys were maintained on a standard diet (SD, n=4) or a high fat, high sucrose diet (HFS, n=10) for 24 months. Monkeys on HFS diet had A) increased BMI, B) larger waist circumference, C) elevated serum IL-6 levels, and D) increased hepatic mIndy expression compared to SD-fed animals. E) In non fasted male 16 week high fat diet fed C57BL/6 mice, hepatic mIndy expression was induced compared to the SD-fed group (n=4–5) F). In a murine NASH model (high fat/methionine low-choline deficient diet=HFD-CD), hepatic mIndy expression was markedly increased after 6 weeks of the intervention.

Figure 4. mIndy interacts with IL-6

A) IL-6 is a predictor of hepatic steatosis at cut-off level 4.81pg/ml (29, 30). Patients were divided into two groups with IL-6 levels below or above this threshold and mIndy expression in liver samples were determined. B) In human primary hepatocytes, IL-6 induced mIndy expression in a time-dependent manner (n=6). C) Blockade of the IL-6R with the monoclonal IL-6R antibody tocilizumab, completely abolished IL-6-mediated induction of mIndy (n=6). D) I.v. injection of IL-6 into C57BL/6 wildtype mice leads to an increase in mIndy mRNA levels (n=4). E) Targeted, liver-specific deletion of the IL-6R (IL-6R^L-KO) (n=3) in mice leads to a decrease in hepatic mIndy expression. F) Human non-parenchymal cells (including Kupffer cells) were co-cultivated with human primary hepatocytes and stimulated with lipopolysaccharide (LPS) with or without tocilizumab (n=3).

Figure 5. Characterization of the human mIndy promoter

The putative start site and upstream promoter region of the human mIndy gene were determined by 5′ RACE (A) The most frequent transcription start site (bold) was located 40 bp upstream of the ATG start codon. (B) Reporter gene constructs were generated by cloning the 621 bp (prom-621, blue top) or 379 bp (prom-379, red top) sequence upstream of the ATG start codon of human mIndy in front of a luciferase reporter. C) Site directed mutagenesis strategy in 376 bp and 618 bp mIndy promoter fragments. D) Site directed mutagenesis at STAT3-elements 376 bp and 618 bp in the mIndy promoter did not reduce promoter activity. Luciferase activity was determined and compared to empty vector control.

Figure 6. Cytoplasmic citrate uptake and lipid synthesis

A,B) In human primary hepatocytes, IL-6 increased cytoplasmic influx of citrate (A) and lipid synthesis from citrate (B). C and D) IL-6 increased citrate uptake (C) and lipid synthesis from citrate (D) in a primary culture of hepatocytes from wildtype (WT) mice, but not from mice with deletion of mIndy (mINDY-KO). E) In liver specific mINDY KO mice (mINDY-KO) and mINDY fl/fl control mice, IL-6 was infused for 14 days via miniosmotic pumps. ¹⁴C-citrate was then administered iv for 4 hours and fatty acid synthesis was determined in liver of these mice. mINDY fl/fl mice without IL-6 infusion were used as negative controls. IL-6 increased fatty acid synthesis from citrate, an effect prevented when mIndy was deleted in the liver. F) ¹⁴C Citrate uptake into primary mouse, rat and human hepatocytes incubated for 24h hours with citrate. Data represent values+/- SEM from primary hepatocytes from independent assays from three different mice, 5 different rats and 5 different human donors.