Kate Erickson and Dr. Benjamin Bikman, Department of Physiology and Developmental Biology

The purpose of this project was to determine the cellular mechanisms that mediate AMPK-induced reductions in lipotoxicity. We predicted that AMPK activation inhibits lipotoxic ceramide biosynthesis via targeted reduction in transcription of a critical, rate-limiting enzyme involved in de novo ceramide synthesis.

This study aimed to provide greater understanding of the mechanisms that mediate obesity-induced morbidity, with particular attention on a well-known agent of lipotoxicity—ceramide.¹ Ceramide accumulates in tissues during obesity and mediates a host of deleterious metabolic effects and may even exacerbate obesity by reducing metabolic rate.² Our studies reveal a novel therapeutic target, viz. ceramide metabolism, for an existing set of drugs that could treat and even prevent diverse complications associated with obesity.

AMP-activated protein kinase (AMPK), is a cellular energy sensor, responding to work and energetic need by reducing anabolic reaction (e.g. fatty acid synthesis) and increasing certain catabolic processes (e.g. fatty acid oxidation). AMPK has been a topic of popular discussion and several current therapeutics exist that are known to activate AMPK. Because AMPK activation results in fatty acid oxidation, one would expect an universal reduction in all cellular lipids. However, based on our preliminary data, AMPK activation with the drug AICAR elicits a selectively robust reduction in ceramides, with only modest reductions in other, less deleterious, lipids [e.g. triacyglycerol (TAG) and diacylglycerol (DAG)]. Further evidence of a ceramide-specific effect for AMPK is our preliminary observation that AMPK activation inhibits transcription of a key rate-limiting enzyme in de novo ceramide synthesis—serine palmitoyltransferase (SPT). Specifically, we found that AMPK activation with AICAR resulted in a robust reduction in SPT transcript levels.

Our initial research plan was to increase sample sizes and statistical power for cell culture experiments. This involved extensive use of murine muscle cell cultures, involving various laboratory techniques and skills (e.g. culture maintenance, culture expansion, cell differentiation, treatment, and harvesting). For protein and transcript measurements, we utilized Western blotting and quantitative real-time polymerase chain reaction. For analysis of lipids, we extracted lipids from samples by an isolation/concentration protocol partly developed by Dr. Bikman. However, actual lipid analysis (e.g. ceramide, TAG, DAG) was performed in collaboration with Dr. John Prince (Chemistry Dept.).

In addition to cell culture experiments, we attempted to confirm our cellautonomous findings with an in vivo model. Due to the often exaggerated and finely controlled responses of cell cultures to experimental treatments, we tried to determine whether our cell culture conclusions apply to the entire organism. Specifically, male Wistar rats were divided into three treatment groups—standard diet (SD), high-fat diet (HFD) and high-fat diet plus daily AICAR administration, which activates muscle AMPK. Diet-induced obesity in rodents through HFD has been shown to induce muscle ceramide accumulation⁴ and, similar to cell culture,³ we saw that AICAR treatment in HFD-fed rats selectively reduced ceramide levels via reduced SPT transcription. At the conclusion of the treatment period, animals were sacrificed and tissues harvested for analysis. The same outcomes and techniques used in cell culture experiments (see above) were used with tissue analysis.

Our experiments and predictions about their outcomes were fairly straightforward to execute and analyze. The techniques selected were performed successfully by myself, Dr. Bikman, and a graduate student member of our lab, and results strongly reinforce our hypotheses. Our only obstacle in obtaining critical data was difficulty in creating efficient correspondence with Dr. Prince’s lab for lipid analysis. Another consideration of the results of our study is the less robust decrease in SPT transcription in HFD rats treated with AICAR. We received rat tissue from animals that were also being used evaluate effects of a diet high in unsaturated fats. Palmitate, one of the components of de novo ceramide biosynthesis, is a saturated fatty acid. While the treatment conditions of the rats were still controlled within the parameters of our
experimental design (i.e., was still a high-fat diet), we had not considered this detail in planning our treatment. This variation in diet-type may account for the slightly less convincing data seen in the in vivo model.

The results of this study were presented in poster format at the American Physiological Society Intersociety Meeting: The Integrative Biology of Exercise VI in Denver, Colorado in October 2012. Additionally, a manuscript based on our findings was recently accepted for publication in the professional, peer-reviewed journal, Diabetology and Metabolic Syndrome.

References

1. Summers SA. Ceramides in insulin resistance and lipotoxicity. Prog Lipid Res. 2006;45:42-72
2. Bikman BT, Summers SA. Ceramides as modulators of cellular and whole-body metabolism. Journal of clinical investigation. 2011;121:doi: 10.1172/JCI57144
3. Bikman BT, Zheng D, Reed MA, Hickner RC, Houmard JA, Dohm GL. Lipidinduced insulin resistance is prevented in lean and obese myotubes by aicar treatment. American journal of physiology. Regulatory, integrative and comparative physiology. 2010;298:R1692-1699
4. Holland WL, Bikman BT, Wang LP, Yuguang G, Sargent KM, Bulchand S, Knotts TA, Shui G, Clegg DJ, Wenk MR, Pagliassotti MJ, Scherer PE, Summers SA. Lipid-induced insulin resistance mediated by the proinflammatory receptor tlr4 requires saturated fatty acid-induced ceramide biosynthesis in mice. J Clin Invest. 2011;121:1858-1870