Studies on the regulation of hepatic and intestinal lipid metabolism

Disturbances in the lipid homeostasis can result in a wide variety of different diseases and symptoms. This thesis is focused on the regulation of cholesterol metabolism and its relation to atherosclerosis development, and the effect of mitochondrial (mt)DNA depletion on hepatic β-oxidation of fatty acids. ApoE deficient mice spontaneously develop atherosclerosis due to accumulation of chylomicron remnants in the circulation, a process highly dependent on intestinal cholesterol absorption. The lack of functional ApoE prevents receptor mediated uptake of lipoproteins, leading to very high levels of circulating cholesterol. We used two approaches to test the influence of plasma cholesterol levels on atherosclerosis development in ApoE deficient mice.

In paper I we disrupted the Cyp8b1 gene to disable the endogenous synthesis of cholic acid and thereby reduce the intestinal absorption of cholesterol. Cyp8b1/ApoE double knockout mice displayed reduced atherosclerotic lesions in aorta, reduced cholesterol levels in plasma and liver and reduced intestinal cholesterol absorption compared to ApoE knockout mice.

In paper II we treated ApoE deficient mice with the thyroid receptor βmodulator GC-1. Supplementation with thyroid hormone reduces plasma cholesterol in humans and rodents; this effect is mediatedby the thyroid receptor isoform β(TRβ). We found that oral administration of GC-1 reduced atherosclerosis after 20 weeks of treatment. This reduction was accompanied by increased hepatic LDL-receptorexpression, bile acid synthesis and fecal bile acid excretion, butserum cholesterol levels were not reduced. The onset and development of atherosclerosis was likely delayed by short-term (one to ten weeks) treatment effects, such as increased fecal excretion of neutral sterols, reduced intestinal cholesterol absorption and reduced plasma cholesterol levels, which were not significant after long-term (20 weeks) treatment. Increased HDL-cholesterol and reducedintestinal cholesterol absorption may lead to improved anti-atherosclerotic effects on cholesterol metabolism, but the LXR-induced increase in HDL-cholesterol may depend on the intestinal cholesterol absorption.

In paper III we investigated thisby oral administration of the LXR agonist GW3965 and the cholesterol absorption inhibitor ezetimibe. The combined treatment effectively reduced the intestinal cholesterol absorption and increased serum HDL-cholesterol and ApoAI. Neither intestinal nor hepatic ABCA1 protein expression increased, but hepatic ApoaI expression was induced. The LXR-driven increase in HDL-cholesterol and ApoAI was thus independent of both intestinal cholesterol absorption and increased intestinal or hepatic ABCA1 protein. The combined treatment effectively increased fecal excretion of cholesterol. Thymidine kinase (TK) 2 deficiency leads to mtDNA depletion. TK2 deficient mice have been developed and display symptoms of defect fat metabolism.

In paper IV we studied the lipid phenotypeof TK2 deficient mice in search for treatment options. TK2 deficient mice displayed elevated plasma levels of cholesterol, free fatty acids and long-chain acylcarnitines. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced hepatic mitochondrial β-oxidation and CPT activity, indicating reduced capacity to metabolize long-chain acylcarnitines. Hepatic ATP synthesis rates were unaltered in 14 days old TK2 deficient mice compared to controls.

List of scientific papers

I. Slätis K., Gåfvels M., Kannisto K., Ovchinnikova O., Paulsson-Berne G., Parini P., Jiang Z-Y., Eggertsen G. Abolished synthesis of cholic acid reduces atherosclerotic development in ApoE knockout mice. Journal of Lipid Research. 2010 51(11):3289-98.
https://doi.org/10.1194/jlr.M009308

II. Kannisto K., Rehnmark S., Slätis K., Webb P., Larsson L., Eggertsen G., Gåfvels M. and Parini P. The thyroid receptor β modulator GC-1 reduces atherosclerosis in ApoE deficient mice. [Manuscript]

III. Kannisto K., Gåfvels M., Jiang Z-Y., Slätis K., Hu X., Steffensen KR. and Eggertsen G. LXR driven induction of HDL-cholesterol is independent of intestinal cholesterol absorption and ABCA1 protein expression. [Manuscript]

IV. Zhou X., Kannisto K., Curbo S., von Döbeln U., Hultenby K., Isetun S., Gåfvels M., Karlsson A. Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation. PLoS ONE. 2013 8(3): e58843.
https://doi.org/10.1371/journal.pone.0058843