Endocrine Abstracts

Non-alcoholic fatty liver disease is the hepatic manifestation of metabolic disease. 5β-reductase (AKR1D1) is highly expressed in human and rodent liver where it inactivates steroid hormones and catalyzes a fundamental step in bile acid synthesis. Steroid hormones, including glucocorticoids, as well as bile acids are established regulators of metabolic phenotype. We have hypothesized that AKR1D1 plays a crucial regulatory role in hepatic metabolic homeostasis.

Genetic manipulation of AKR1D1 (over-expression, siRNA knockdown) was performed in human liver HepG2 cells alongside the metabolic phenotyping of AKR1D1 KO mice. Gene expression changes in HepG2 cells were confirmed by real-time PCR. Functional activity, assessed using gas chromatography mass spectrometry to measure cortisone clearance and tetrahydrocortisone generation was paralleled by the anticipated changes in glucocorticoid receptor activation measured by luciferase reporter assays.

AKR1D1 knockdown increased glucose transporter mRNA expression (GLUT1: 0.47±0.08 vs. 1.07±0.15, P<0.01; GLUT9: 0.56±0.08 vs. 0.85±0.15, P<0.05). Extracellular glucose concentrations in the culture media decreased (15.3±1.5 vs. 12.1±0.9 umol/mg, P<0.05) as did intracellular glycogen (14.0±0.1 vs. 10.5±1.7 ug/ml, P<0.05). Endorsing our cellular observations, fed blood glucose levels in AKR1D1 KO mice were lower than wild type (WT) controls (15.1±0.5 (WT) vs. 12.8±0.6 mmol/L (KO), P<0.05). In addition, hepatic glycogen content was lower in KO mice (6.7±0.3 (WT) vs. 5.1±0.5 ug/gram of liver (KO), P<0.05).

AKR1D1 knockdown in HepG2 cells increased Acetyl CoA Carboxylase 1 expression, the rate-limiting step in de novo lipogenesis, DNL) (0.52±0.06 vs. 0.89±0.04, P<0.01), and increased intracellular triglyceride (54.3±12.7 vs. 73.3±11.0 nmol/mg, P<0.01). Furthermore, 3-hydroxybutyrate levels in the cell media were reduced, indicative of decreased fatty acid oxidation (18.7±2.3 vs. 11.4±2.7 nmol/mg, P<0.01). Mass spectrometry analysis of lipid composition demonstrated increased palmitic and palmitoleic acid production consistent with increased DNL and fatty acid saturation.

In conclusion, we have demonstrated that AKR1D1 activity limits steroid hormone availability and is potently able to regulate hepatic carbohydrate and lipid metabolism.