● Cells were scraped and spun down for protein extraction.

● Western Blot was performed and pAKT and pGSK-3β primary antibodies were used to probe the membranes. Constitutively expressed heat shock protein (HSP) was used to normalize protein concentration.

● Densitometry was performed with Image J software and protein expression was calculated.

● This experiment was done in duplicates to have an n=4.

Experiments were performed using an adult mouse astrocytic cell line, C8-D1A, hereby referred to in this poster as astrocytes. The GLP-1 receptor (GLP1-R) agonist Liraglutide was used to treat the cells.

Experiment 1● Determine if there is a functional GLP1-R receptor on the

astrocytes● Astrocytes were treated with 100 nM liraglutide or 100 nM

forskolin for the following times: 5, 7.5, 10, 15, 30 and 60 minutes.

● Levels of cAMP were measured using cAMP-Glo™ Max Assay (Promega, V1681) following the manufacturer's protocol to determine if GLP-1 was acting on GLP1-R, a G protein-coupled receptor.

Experiment 2● Examine the role of GLP-1 on insulin action in the

astrocytes● The effects on insulin action were determined by studying

the phosphorylation of AKT and GSK-3β.

● The astrocytes were divided into five six-well plates. ● The cells were either treated with GLP-1, insulin, or a

combination of both. ● There were five treatment groups total: Vehicle, Insulin

alone, Insulin + GLP-1 added 18 hr. prior, Insulin + GLP-1 added 30 min. prior, and GLP-1 alone.

● 100 nM GLP-1 was given 18 hours before harvest to one treatment group and 30 minutes before harvest to another.

● Insulin was added 30 minutes before harvest in various concentrations of 1 nM, 10 nM, and 100 nM.

● It appears that astrocytes express a functional GLP-1R, suggesting the possibility that previously studied neuroprotective effects of GLP-1 might utilize an astrocytic mechanism

● GLP-1 was shown to act on astrocytes and mimic insulin signaling at baseline conditions, supporting my hypothesis

● GLP-1 was shown to enhance astrocytic insulin signaling, supporting my hypothesis. This should ultimately increase glycogen synthesis

● GLP-1 effects on astrocytes suggest a potential role in attenuating insulin resistance and decreased glycogen storage seen in stress-induced astrocytes

● Conduct the experiments in the presence of palmitate to determine if GLP-1 can reclaim insulin signaling and glycogen storage in stress-induced astrocytes

● Calculate actual glycogen stores in astrocytes treated with GLP-1 to determine if GLP-1 action enhances glycogen storage

GLP-1 acts on astrocytes to enhance insulin signaling, providing a mechanism by which GLP-1 analogs may offer neuroprotection to patients with diabetes-associated neurodegenerative diseases such as Alzheimer’s.

Experiment 1: Do astrocytes express a functional receptor for GLP-1?● Western blot analysis indicated that our astrocyte cell

line expresses the receptor for GLP-1 and the expression of the receptor was not altered by any of our experimental cell treatments (data not shown).

• There is growing evidence linking type 2 diabetes (T2D) with dementia and neurodegenerative diseases such as Alzheimer’s disease (AD)

• The incretin hormone glucagon-like peptide 1 (GLP-1), utilized for its insulinotropic properties in the treatment of T2D, is also synthesized in the CNS as a neuropeptide and has been demonstrated to have neuroprotective effects

• Astrocytes are a type of glial cell with a central role in neuronal health through involvement with CNS metabolism, glycogen storage and structural support. They are the only type of brain cells that store glycogen, which is utilized in learning and memory

● Our lab has demonstrated in an astrocyte cell line that glycogen synthesis is responsive to insulin. At higher doses of insulin, these astrocytes respond with increased glycogen storage

• To this, we have seen that palmitate (used to simulate an obesogenic physiology) causes insulin resistance, reduced activation via phosphorylation of protein kinase B (AKT, a marker of insulin signaling) and lower inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK-3β, a negative regulator of glycogen synthesis), and thus decreased glycogen storage in astrocytes

• This suggests that obesity or diabetes-induced disruption of astrocytic functions may be implicated in the pathogenesis of neurodegenerative diseases

• Little data is available about the effects of GLP-1 on glial cell function

• OBJECTIVES: To determine if astrocytes express a functional GLP-1 receptor and to examine whether GLP-1 action mimics and/or enhances insulin action on astrocytes

Methods

Background

Chris Ha, University of North Texas Health Science CenterKevin Niswender, MD, PhD and the Niswender Laboratory

Vanderbilt University School of Medicine, Nashville, TN

Conclusion

Implications of GLP-1 for neuroprotection

Figures 3-5. GLP-1 is shown to enhance the phosphorylation of GSK-3B. This is observed when astrocytes are treated acutely both at 30 min. as well as at 18 hours. The enhancement of insulin signaling by GLP-1 is most apparent at 10nM insulin, inducing a near maximal response.

Figures 6-8. Similar to its effects on GSK-3B, GLP-1 is also observed to enhance the phosphorylation of AKT.

Figure 2. Liraglutide (GLP-1) treatment of astrocytes shows a time dependent increase in cAMP levels, indicating there is a functional GLP-1R receptor. Forskolin was used as a positive control as it stimulates adenylyl cyclase to increase cAMP levels independent of the GLP-1 receptor

Results

Figure 1. Diagram of insulin signaling pathway

Experiment 2: What are the effects of GLP-1 on insulin action in astrocytes?

Summary

Future Directions

HypothesisGLP-1 will act on astrocytes and mimic insulin action at baseline conditions. In the presence of insulin, GLP-1 should enhance insulin action, exhibiting a mechanism by which astrocytic glycogen storage can ultimately be augmented.

Phosphorylation of Glycogen Synthase Kinase