Expression of Kv1.3 Channels and Insulin Receptor in Hypothalamic Neurons

Alex Palmer1, Carl Vitzthum2, Megan Doczi1

1Norwich University, 2Colby College

Introduction

The hypothalamus is a region of the brain that is responsible for maintaining the homeostatic balance between food intake and energy expenditure. Voltage gated potassium (Kv) channels help govern neuronal excitability and resting membrane potential, in many types of neurons. Evidence indicates that circulating metabolic signals such as insulin and glucose can modulate the function of Kv1.3.1-3 Circulating levels of insulin in the blood can interact with various circumventricular organs of the brain, including hypothalamic nuclei, where insulin receptors (IR) have been detected. 4, 5 As a result, insulin signaling has been shown to affect the hypothalamic regulation of food intake and body weight.6 We hypothesize that metabolic factors such as insulin and glucose can affect neuronal function through interactions with the Kv1.3 ion channel. Understanding the effects of this mechanism may lead to possibilities of new obesity treatment and diet awareness, impacting human health.

Kv1.3 channel function is directly modulated by IR tyrosine kinase activity suggesting a link between insulin stimulation and Kv1.3 channel activity.1 To identify Kv1.3 and the Insulin Receptor as potential candidates for mediating the neuronal excitability of avian hypothalamic neurons, we analyzed mRNA expression of these proteins. In previous experiments, RT-PCR analysis was used to detect fragment sizes for the Kv1.3 channel and -actin housekeeping gene present in hypothalamic tissue microdissected from E8 and E12 embryonic time points (Figure 1). These data indicate that Kv1.3 mRNA is expressed in the hypothalamus during avian embryonic development, and identify the Kv1.3 channel as a candidate for mediating the neuronal excitability of hypothalamic neurons. This project focuses on the comparison of gene expression in intact tissue and dissociated hypothalamic cultures. Our hypothesis is that dissociated hypothalamic cultures will have the same gene expression of Kv1.3 and Insulin Receptor as the intact tissue, thus enabling us to use these cultures as a model system. By using RT-PCR primer sets for Kv1.3 from previous work, we can detect whether expression is found in the hypothalamic cultures. By using previously published RT-PCR primer sets for IR7, we can detect and determine whether IR is present and whether it effects the neuronal firing of the Kv1.3 channel in both the intact and dissociated hypothalamic tissues.

Figure 1: Kv1 Channel mRNA in the Intact E12 Avian Hypothalamus. RT-PCR analysis of fragment sizes for the Kv1.2 (448 bp), Kv1.3 channel (474 bp), Kv1.5 (347 bp), and B-actin housekeeping gene (247 bp) are present in hypothalamic tissue microdissected from E12 embryonic time point (Carl Vitzthum). –RT indicates no reverse transcriptase control.

The long-term goal of this project is to analyze whether peripheral metabolic signals can modulate the developmental regulation of Kv1.3 channels in avian hypothalamic neurons, thus critically affecting neuronal excitability during the early patterning of feeding circuits. We are the first laboratory to study the function of Kv1.3 channels in the embryonic hypothalamus. This work will have a significant impact on the field of hypothalamic development and provide valuable insight into the role of ion channels in the regulation of homeostatic feeding circuits.

MATERIALS AND METHODS

Chicken Embryos

Fertile white leghorn chicken eggs were obtained from Sunrise Farms, Catskill, NY and stored at 6 °C. The eggs were transferred and maintained in a forced draft incubator at 37.5 °C with 55% relative humidity until embryonic day 12 (E12). Embryos were sacrificed by decapitation and the hypothalamus was microdissected and dissociated for use in culture and for experimentation.

Hypothalamic Dissociation

Hypothalamic tissue was isolated from embryonic day 12 (E12) chickens by microdissection. Tissues were harvested on ice in sterile filtered Dulbecco’s Phosphate Buffered Saline (DPBS) supplemented with 0.1% glucose and 1.0% penicillin/streptomycin. Hypothalamic dissections were mechanically dispersed by gentle trituration with a flame-polished Pasteur. Cells were resuspended in Neurobasal medium supplemented with b-27, GlutaMAX, and 1.0% penicillin/streptomycin, and subsequently plated on poly-L-lysine coated cover slips.

Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)

RNA was isolated from the avian dissociated hypothalamic cultures using RNAeasy mini kit from Qiagen. Equal amounts of RNA were reverse transcribed and equal amounts of cDNA were amplified. Table 1 identifies the primer sets that were used to detect the Kv1.3 channel, β-actin housekeeping gene, and Insulin Receptor.

Table 1. Kv1.3 channel and β-actin PCR primer setsmRNA Primer Sequences Product SizeKv1.3 Fwd: 5’- GGG CGC ATC CGA CGA CCG TCA AT-3’

Rev: 5’- ACA GGC AAA GAA GCG CAC CAA CA-3’474 bp

β-actin Fwd: 5’- TGG ATG ATG ATA TTG CTG CG-3’Rev: 5’- CTC CAT ATC ATC CCA GTG G-3’

247 bp

IR Fwd: 5’- CGC TGA GAA TAA CCC TGG TC-3’Rev: 5’- GCT GCC ATC TGG ATC ATT TC- 3’

58 bp

RESULTS & DISCUSSION

Figure 2. Kv1 mRNA in E12 Avian Hypothalamic Cultures. RT-PCR analysis of fragment sizes for Kv1.2 (448 bp), Kv1.3 (474 bp), Kv1.5 (347 bp), and B-actin housekeeping gene (247 bp) present in dissociated hypothalamic culture from E12 embryo. –RT indicates no reverse transcriptase control.

Figure 3. Insulin Receptor mRNA in E12 Avian Cultures and Intact Tissue. RT-PCR analysis of fragment size for the Insulin Receptor (58 bp) present in both dissociated hypothalamic culture from embryonic day 12 (E12) and E12 intact hypothalamic tissue.

Dissociated hypothalamic cultures of E12 embryos express the same mRNA of Kv1.3 and IR as intact hypothalamic tissue. This indicates that our dissociated cultures are growing the same neurons as intact tissue and express the same genes. This is important because we can use the hypothalamic cultures as a representative model system. These results are the first to confirm hypothalamic insulin receptor expression in the avian embryo. The expression of IR at such an early developmental period indicates that these genes are active and may participate in the patterning of hypothalamic circuits.

FUTURE DIRECTIONS

More data will need to be gathered to confirm that hypothalamic dissociated cultures express the same protein for Kv1.3 and IR as intact tissue at embryonic time period E12. We will gather these data by immunofluorescence and western blot. Immunofluorescence will allow us to visualize the specific proteins we are looking for in the culture and western blotting will allow us to detect the specific proteins in the samples of tissue. By combining these data, it will give us a strong confirmation that the protein for Kv1.3 and IR is expressed the same in dissociated hypothalamic cultures as intact tissue.

REFERENCES

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