PI: David Thomson

Evaluation of Academic Objectives

The incidence of obesity and the health consequences that accompany it have increased rapidly over the past decade. Understanding the molecular pathways involved in its development will be crucial in developing novel treatment strategies for obesity and associated disorders. Low-grade inflammation likely plays a major role in many of the complications of obesity. We proposed to study the role of liver kinase B1 (LKB1) in the development of inflammation and insulin resistance in obese mice and saturated fatty acid-induced insulin-resistant cells.

For the most part, the research went well. As only $13,300 of the requested $20,000 was funded, we focused on Specific Aim 2 of the proposal, where we assessed the effect of high-fat diet induced obesity in skeletal muscle-specific LKB1 knockout (LKB1-KO) mice and littermate wild-type (WT) mice. We also had some major challenges. In particular, the move from the WIDB to the LSB in 2014 resulted in our having to reduce our mouse colony down, and then a prolonged “burning out” process before moving them into the new pathogen free animal facility. This delayed the project until well into 2015 as we built the mouse colony back up. Nonetheless, we have obtained valuable data (outlined in results section below) that has provided great learning opportunities to graduate and undergraduate students in the lab.

Evaluation of Mentoring Environment

The mentoring environment continued to be essentially what I aim for it to be. Although my availability to be in the lab with students ebbs and flows, I felt good about the amount of time spent with students. I was appreciative of the efforts of my graduate and more experienced undergraduates, who also helped in teaching lab skills to the other students. Lab meetings are going very well- the students seem more and more comfortable with scientific content and we have good discussions of not only our research but other literature in the field. I am hoping to improve the social interaction with my lab. I’ve normally had a lot of fun interaction outside of the lab with students (playing sports, hiking, parties, etc.) and this has slipped a bit, and I hope to reverse that trend. Otherwise I think the environment is working well for helping the students learn how to do the lab work and how to think properly about the results.

Student Participants & Academic Deliverables

Ting Chen

• Graduate Student
• Involved in most aspects of the study.
• Dissertation and publication (expected submission in April 2017) resulting from this funding is forthcoming.
• Abstract for Experimental Biology 2017 (April, 2017) is pending (will submit at end of month).

Marc Matsumura:

• Undergraduate Student
• First author on abstract and poster presentation at the Integrative Biology of Exercise meeting (international meeting) in Phoenix, AZ, October 2016:

High-fat diet drives an LKB1-dependent attenuation of insulin-induced skeletal muscle p70S6k phosphorylation.

Insulin is an important anabolic hormone. Its signaling through Akt promotes maintenance or increases in muscle mass by activating mTOR, the classic master-regulator of protein synthesis and by decreasing proteolysis. High-fat diet-induced obesity impairs insulin-sensitivity and decreases insulin-stimulated mTOR signaling to its target p70S6k. AMP-activated protein kinase (AMPK) blocks mTOR activity, protein synthesis and muscle growth, and AMPK is activated by high-fat diet feeding, suggesting that it may play a role in high-fat diet-induced resistance of mTOR to insulin stimulation. Therefore, we hypothesized that a loss of AMPK activity through the loss of skeletal muscle LKB1 (the primary AMPK activator in skeletal muscle), would prevent the attenuation of insulin-induced mTOR signaling with high-fat diet-induced obesity. To test this, skeletal muscle specific LKB1 knockout (KO) and wild-type littermate (WT) mice were fed a standard chow diet (SD) or a high-fat diet (HFD) for 14 weeks. Extensor digitorum longus (EDL) muscles were then removed and incubated in the presence or absence of insulin for 30 minutes. Phosphorylation of AMPK, Akt substrates, p70S6k and the p70S6k target rpS6 were measured by western blotting. We found that AMPK phosphorylation was increased while phosphorylation of Akt substrates, p70S6k and rpS6 were decreased by HFD in WT but not KO muscles. Insulin stimulation of Akt substrate, p70S6k and rpS6 phosphorylation was likewise blunted by HF diet in WT but not KO muscles. We conclude, then, that high-fat diet attenuation of basal and insulin-stimulated anabolic signaling via Akt and mTOR is dependent on LKB1.

Michael Harding:

• Undergraduate Student
• Involved in mitochondrial studies

Eric Cheung

• Undergraduate Student
• First author on abstract and poster presentation at the Integrative Biology of Exercise meeting (international meeting) in Phoenix, AZ, October 2016, but not on data from project funded by this grant.

Conrad Ashby

• Undergraduate Student

JJ Tsukamoto

• Undergraduate Student
• Planning on abstract for Experimental Biology 2017 (April, 2017) on gene expression from this project (will hopefully submit at end of month).

Major Results

1. Both control and obese LKB1-KO mice tend to have better glucose tolerance than wild-type animals.
2. LKB1-KO protects mice somewhat from obesity-induced Insulin resistance as measured by intraperitoneal insulin tolerance testing. This is contrary to our hypothesis.
3. The typical decline in Akt phosphorylation of its substrates in muscles from obese mice is eliminated by LKB1-KO. This is consistent with the insulin sensitivity data.
4. Diet-induced obesity decreases anabolic signaling downstream of the mechanistic target of rapamycin, and this decrease is prevented by LKB1-KO (see Mark Matsumura’s abstract above).
5. A few inflammation-associated genes (IER3, Bcl3) were increased by high-fat diet feeding and this was blocked by LKB1-KO. Most inflammatory genes, however, were not affected by the diet.
6. Mitochondrial respiration is decreased by LKB1-KO regardless of diet status.
7. The high-fat diet induced increase in complex III of the electron transport chain was not affected by LKB1-KO.
8. The mitochondrial master-regulator, PGC1, increased in skeletal muscle with high-fat feeding, and this was prevented by LKB1-KO.

We are continuing to explore potential mechanisms for the improved insulin action in the LKB1-KO mice.

Budget Expenditures