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. 2018 Nov 12;3(1):84-98.
doi: 10.1002/hep4.1279. eCollection 2019 Jan.

Inhibition of Adenosine Monophosphate-Activated Protein Kinase-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Signaling Leads to Hypercholesterolemia and Promotes Hepatic Steatosis and Insulin Resistance

Kim Loh  1 , Shanna Tam  1 , Lisa Murray-Segal  1 , Kevin Huynh  2 , Peter J Meikle  2 , John W Scott  1   3   4 , Bryce van Denderen  1 , Zhiping Chen  1 , Rohan Steel  1 , Nicholas D LeBlond  5 , Leah A Burkovsky  5 , Conor O'Dwyer  5 , Julia R C Nunes  5 , Gregory R Steinberg  6 , Morgan D Fullerton  5 , Sandra Galic  1 , Bruce E Kemp  1   3
Affiliations

Inhibition of Adenosine Monophosphate-Activated Protein Kinase-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Signaling Leads to Hypercholesterolemia and Promotes Hepatic Steatosis and Insulin Resistance

Kim Loh et al. Hepatol Commun. .

Abstract

Adenosine monophosphate-activated protein kinase (AMPK) regulates multiple signaling pathways involved in glucose and lipid metabolism in response to changes in hormonal and nutrient status. Cell culture studies have shown that AMPK phosphorylation and inhibition of the rate-limiting enzyme in the mevalonate pathway 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase (HMGCR) at serine-871 (Ser871; human HMGCR Ser872) suppresses cholesterol synthesis. In order to evaluate the role of AMPK-HMGCR signaling in vivo, we generated mice with a Ser871-alanine (Ala) knock-in mutation (HMGCR KI). Cholesterol synthesis was significantly suppressed in wild-type (WT) but not in HMGCR KI hepatocytes in response to AMPK activators. Liver cholesterol synthesis and cholesterol levels were significantly up-regulated in HMGCR KI mice. When fed a high-carbohydrate diet, HMGCR KI mice had enhanced triglyceride synthesis and liver steatosis, resulting in impaired glucose homeostasis. Conclusion: AMPK-HMGCR signaling alone is sufficient to regulate both cholesterol and triglyceride synthesis under conditions of a high-carbohydrate diet. Our findings highlight the tight coupling between the mevalonate and fatty acid synthesis pathways as well as revealing a role of AMPK in suppressing the deleterious effects of a high-carbohydrate diet.

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Figures

Figure 1
Figure 1
The Ser871Ala mutation renders HMGCR insensitive to AMPK phosphorylation and inhibition. (A) Genotyping using PCR for determining WT, HET, and homozygous HMGCR KI mice. (B,C) The catalytic domains of WT and Ser871Ala KI mouse HMGCR were expressed as GST‐fusion proteins and incubated with or without bacterial‐expressed CaMKK‐activated AMPK before (B) mass spectrometry analysis or (C) assay for HMGCR activity using a radioisotope‐based 14C‐HMG CoA reduction assay. Results are means ± SEM, n = 3 per group (*P < 0.05, when comparing no AMPK versus +AMPK). (D) Relative Hmgcs1 and Hmgcr mRNA expression determined in the liver by quantitative RT‐PCR using Rpl32 as a housekeeping gene. Results are means ± SEM, n = 12 WT, n = 11 HMGCR KI. (E) Liver from WT and HMGCR KI mice were subjected to immunoblotting using antibodies against HMGCR and actin and quantified using ImageJ software. Results are means ± SEM, n = 3 per genotype. Abbreviations: HET, heterozygous; Rpl32, ribosomal protein L32.
Figure 2
Figure 2
Elevated serum and hepatic cholesterol levels in high‐carbohydrate‐fed HMGCR KI mice. (A) Cholesterol synthesis in WT and HMGCR KI hepatocytes in response to AMPK activators A769662 and AICAR. Results are means ± SEM, n = 3 independent experiments (each experiment contains at least three replicates; *P < 0.05, when comparing WT versus HMGCR KI; ## P < 0.01, ### P < 0.001, when comparing between different treatments). Liver and serum samples were collected from (B,C) chow‐fed WT and HMGCR KI mice or (D,E) high‐carbohydrate‐fed WT and HMGCR KI mice. Cholesterol levels, including total cholesterol, free cholesterol, and cholesteryl ester, were measured. (F) Serum LDL/VLDL cholesterol from high‐carbohydrate‐fed WT and HMGCR KI mice. (B‐F) Results are means ± SEM, n = 7‐10 per genotype (*P < 0.05, ***P < 0.001, when comparing WT versus HMGCR KI).
Figure 3
Figure 3
Enhanced hepatic cholesterol synthesis in vivo in HMGCR KI mice. (A) Rates of cholesterol synthesis in the liver were measured in WT and HMGCR KI mice fed a high‐carbohydrate diet for 10 weeks. Mice were injected with 3H2O, and livers and plasma were collected 1 hour later. Livers were saponified, sterol‐containing lipid was extracted, and the incorporated tracer was measured by scintillation counting. (B) In vivo hepatic cholesterol synthesis in livers from high‐carbohydrate‐fed WT and HMGCR KI mice. (C) Quantitative RT‐PCR was performed to measure expression of genes for cholesterol synthesis pathway enzymes, including Fdps, Sqle, and Dhcr7, from the livers of high‐carbohydrate‐fed WT and HMGCR KI mice. (B,C) Results are means ± SEM, n = 6‐8 per genotype (*P < 0.05, when comparing WT versus HMGCR KI). Abbreviations: Dhcr7, 7‐dehydrocholesterol reductase; Fdps, farnesyl diphosphate synthase; Sqle, squalene epoxidase.
Figure 4
Figure 4
Increased adiposity in high‐carbohydrate‐fed HMGCR KI mice. WT and HMGCR KI mice were fed a high‐carbohydrate diet for 10 weeks. (A,B) Absolute and incremental weekly body weights of WT and HMGCR KI mice. (C,D) Absolute and normalized epididymal fat mass in WT and HMGCR KI mice. Results are means ± SEM, n = 7‐10 per genotype (*P < 0.05, **P < 0.01, when comparing WT versus HMGCR KI).
Figure 5
Figure 5
Increased adiposity and hepatic lipid accumulation in high‐carbohydrate‐fed HMGCR KI mice. WT and HMGCR KI mice were fed a high‐carbohydrate diet for 10 weeks. (A) Liver weights of WT and HMGCR KI mice. (B) Oil Red O stain of liver tissue from high‐carbohydrate‐fed WT and HMGCR KI mice. (C,D) Hepatic and serum triglyceride levels in WT and HMGCR KI mice. (E) Masson’s trichrome stain of liver tissue from high‐carbohydrate‐fed WT and HMGCR KI mice. (F) F4/80 immunohistochemistry of liver tissue from high‐carbohydrate‐fed WT and HMGCR KI mice. Relative hepatic Il6, Il1b, and Tnfa mRNA expression determined in the liver by quantitative RT‐PCR. Results are means ± SEM, n = 6‐10 per genotype (*P < 0.05, when comparing WT versus HMGCR KI). Abbreviation: Tnfa, tumor necrosis factor alpha.
Figure 6
Figure 6
Enhanced capacity for de novo triglyceride synthesis in HMGCR KI liver. (A) Triglyceride synthesis in WT and HMGCR KI primary hepatocytes in response to AMPK activators A769662 and AICAR. Results are means ± SEM, n = 3 independent experiments (each experiment contains at least three replicates; *P < 0.05, when comparing WT versus HMGCR KI). (B,C) mRNA expression of lipogenic genes, including Srebf1, Fasn, Acaca, and Acacb, from livers of high‐carbohydrate‐fed WT and HMGCR KI mice. Results are means ± SEM, n = 7‐10 per genotype (*P < 0.05, when comparing WT versus HMGCR KI).
Figure 7
Figure 7
Impaired glucose homeostasis in high‐carbohydrate‐fed HMGCR KI mice. WT and HMGCR KI mice were fed a high‐carbohydrate diet for 10 weeks. (A) Ad libitum fed and overnight‐fasted blood glucose levels from WT and HMGCR KI mice. (B) WT and HMGCR KI mice were fasted for 6 hours and injected intraperitoneally with 1 mg/g of glucose. Blood glucose was measured, and the area under the curve was calculated. (C) WT and HMGCR KI mice were fasted for 4 hours and injected intraperitoneally with 0.5 mU/g of insulin; blood glucose was measured, and the area under the curve was calculated. (D) Soleus and EDL muscles were isolated from WT and HMGCR KI mice, and 2‐deoxy‐D‐glucose uptake was assessed under basal conditions and in response to insulin. (E,F) Liver and muscle from high‐carbohydrate‐fed WT and HMGCR KI mice were isolated and processed for immunoblotting using antibodies specific for phosphorylated Akt (Ser473) and actin. Results are means ± SEM; (A‐C,E,F) n = 7‐10 per genotype, (D) n = 5‐6 per genotype (*P < 0.05, when comparing WT versus HMGCR KI). Abbreviations: 2DG, 2‐deoxy‐D‐glucose; pAkt, phosphorylated Akt.
Figure 8
Figure 8
Increased HGP in high‐carbohydrate‐fed HMGCR KI mice. (A‐F) Hyperinsulinemic‐euglycemic clamp studies in high‐carbohydrate‐fed WT and HMGCR KI mice. (A) Blood glucose levels, (B) glucose infusion rate, and (C) whole‐body glucose disappearance rate during the clamp. (D,E) Hepatic glucose output during basal and insulin‐stimulated conditions and expressed as percent suppression. (F) Quantitative RT‐PCR was performed to measure expression of genes for gluconeogenic genes, including Pck1 and G6pc, from the livers of high‐carbohydrate‐fed WT and HMGCR KI mice after the clamp. Results are means ± SEM, n = 4 per genotype (**P < 0.01, ***P < 0.001, when comparing WT versus HMGCR KI). Abbreviations: G6pc, glucose 6‐phosphatase; Pck1, phosphoenolpyruvate carboxykinase 1.
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