Characterizing the role of IGF- axis on the growth restriction during short photoperiod in Siberian hamster (Phodopus sungorus) Ei K. Swe Bio-466H Biological Science Department

Introduction Photoperiod is an important factor in physiology of temperate animals Photoperiod is an important factor in physiology of temperate animals can regulate behavior, reproduction, and growth can regulate behavior, reproduction, and growth It has been known that in SD (short- day)photoperiod, animals undergo much less growth It has been known that in SD (short- day)photoperiod, animals undergo much less growth we don’t know what the mechanism behind it we don’t know what the mechanism behind it “IGF (Insulin-like Growth Factor) -axis” may be regulating suppressed growth during SD. “IGF (Insulin-like Growth Factor) -axis” may be regulating suppressed growth during SD.

Background IGF-axis IGF-axis –IGF/IGFBP/IGF-receptor axis directly regulates somatic growth in vertebrates –IGF-I is most important of all IGFs for somatic growth –In IGFBP family, we will look at IGFBP-1 and IGFBP-3 –IGFBP-1 has inhibitory effect on binding of IGF-I to its receptor –IGFBP-3 has stimultory effect.

Background (Cont’d) Cortisol Cortisol –well-known as stress hormone –associated with long-term stressed condition

Animals Use Siberian hamster ( Phodopus sungorus) as a temperate mammal model Use Siberian hamster ( Phodopus sungorus) as a temperate mammal model they undergo a lot of changes in different photoperiods they undergo a lot of changes in different photoperiods Advantage Advantage –small- easy to handle –abundant –easy to apply to higher mammals –available from other project on campus

Hypothesis If hamsters are housed in winter-like SD (short-day) photoperiod, IGF-I and IGFBP- 3 level will be decreased, and cortisol and IGFBP-1 level will be increased. If hamsters are housed in winter-like SD (short-day) photoperiod, IGF-I and IGFBP- 3 level will be decreased, and cortisol and IGFBP-1 level will be increased. Also, mRNA levels of IGF-I and IGFBP-3 in liver and muscle will be suppressed,and that of IGFBP-1 will be elevated. Also, mRNA levels of IGF-I and IGFBP-3 in liver and muscle will be suppressed,and that of IGFBP-1 will be elevated.

Specific Aims To compare IGF-I and cortisol levels in different photoperiods To compare IGF-I and cortisol levels in different photoperiods To compare IGFBPs (IGFBP-1 and IGFBP-3) in different photoperiods To compare IGFBPs (IGFBP-1 and IGFBP-3) in different photoperiods To see tissue-specific mRNA for IGF-I, IGFBP-1 and IGFBP-3 in different tissue types: liver and muscle To see tissue-specific mRNA for IGF-I, IGFBP-1 and IGFBP-3 in different tissue types: liver and muscle  Those findings will characterize the mechanism of IGF-axis in the relationship between short-day photoperiod and somatic growth change.

Experimental Design Animals Animals –use 20 LD and 40 SD female hamsters –SD will be artificially set for 8hr in light and 16hr in dark –LD will be artificially set for 16hr in light and 8hr in dark –tissue and blood sampled every three weeks: week- 3,6,9 & 12

Experimental Design (Cont’d) Tissue storage Tissue storage –blood -put in 4°C (in ice) first, then centrifuged and stored the serum in -80 °C –tissue (liver and muscle) -put in RNA later first, then stored in -80 °C

Experimental Design (Cont’d) Techniques Techniques 1) Radioimmunoassay (RIA) for serum level of IGF-I and cortisol 2) Western immunoblot for IGFBPs (specific): IGFBP-1 and IGFBP-3, using rabbit anti-rat IGFBP-1 antiserum and rabbit anti-rat IGFBP-3 antiserum for specific protein 3) RNA-isolation - RNA from liver and muscle tissue by single step method of RNA isolation by acid guanidinuum thiocyanate-phenol chloroform extraction. 4) Northern Blot for mRNA level determination of IGF-I, IGFBP-1 and IGFBP-3 in liver and muscle

Experimental Design (Cont’d) Statistical Analysis Statistical Analysis –Data from all measurements will be analyzed using one-way ANOVA with p<0.05 as the level of significance

Expected Results Fig-1: The expected serum level of IGF-I and cortisol in LD and SD, measured by RIA. Significant difference between SD and LD (p<0.05). n=40 for SD and n=20 for LD

Expected Results (Cont’d) Fig-2: The expected serum level of IGFBP-1 and IGFBP-3 in LD and SD, measured by Western Immunoblot assay. Significant difference between two groups (p<0.05). n=40 for SD and n=20 for LD.

Expected Results (Cont’d) Fig-3: mRNA level of IGF-I, IGFBP-1 and IGFBP-3 in liver and muscle of LD and SD. There is no significant difference between two tissue types in both groups (LD and SD), but there is significant difference between the groups in the mRNA levels of each protein (p<0.05).n=40 for SD and n=20 for LD.

Discussion During winter-like short-day photoperiod, animals undergo physiological changes to save energy During winter-like short-day photoperiod, animals undergo physiological changes to save energy some energy expensive processes (reproduction, growth, etc) are shut down physiologically to save energy some energy expensive processes (reproduction, growth, etc) are shut down physiologically to save energy From this study, we can determine the mechanisms in IGF-axis on regulation of growth in mammals in different photoperiods. From this study, we can determine the mechanisms in IGF-axis on regulation of growth in mammals in different photoperiods. The mechanisms controlling growth are likely to be applicable in most mammalian species, including humans. The mechanisms controlling growth are likely to be applicable in most mammalian species, including humans.

Acknowledgement Dr. Kevin Kelley Dr. Kevin Kelley HHMI honor program HHMI honor program Dr. A. Z. Mason Dr. A. Z. Mason Dr. Kelly Young Dr. Kelly Young