Characterizing the role of Characterizing the role of IGF-axis on the growth IGF-axis on the growth restriction during short restriction during short photoperiod in Siberian photoperiod in Siberian

hamster (hamster (Phodopus Phodopus sungorussungorus) )

Ei K. SweEi K. Swe

Bio-466HBio-466H

Biological Science DepartmentBiological Science Department

IntroductionIntroduction

• Photoperiod is an important factor in Photoperiod is an important factor in physiology of temperate animals physiology of temperate animals

• can regulate behavior, reproduction, and can regulate behavior, reproduction, and growthgrowth

• It has been known that in SD (short-It has been known that in SD (short-day)photoperiod, animals undergo much less day)photoperiod, animals undergo much less growthgrowth

• we don’t know what the mechanism behind itwe don’t know what the mechanism behind it• ““IGF (Insulin-like Growth Factor) -axis” may IGF (Insulin-like Growth Factor) -axis” may

be regulating suppressed growth during SD. be regulating suppressed growth during SD.

BackgroundBackground• IGF-axisIGF-axis

– IGF/IGFBP/IGF-receptor axis directly regulates IGF/IGFBP/IGF-receptor axis directly regulates somatic growth in vertebratessomatic growth in vertebrates

– IGF-I is most important of all IGFs for somatic IGF-I is most important of all IGFs for somatic growthgrowth

– In IGFBP family, we will look at IGFBP-1 and In IGFBP family, we will look at IGFBP-1 and IGFBP-3IGFBP-3

– IGFBP-1 has inhibitory effect on binding of IGFBP-1 has inhibitory effect on binding of IGF-I to its receptorIGF-I to its receptor

– IGFBP-3 has stimultory effect.IGFBP-3 has stimultory effect.

Background (Cont’d)Background (Cont’d)

• CortisolCortisol– well-known as stress hormonewell-known as stress hormone– associated with long-term stressed associated with long-term stressed

conditioncondition

AnimalsAnimals• Use Siberian hamster Use Siberian hamster

((Phodopus sungorusPhodopus sungorus) ) as a as a temperate mammal modeltemperate mammal model

• they undergo a lot of they undergo a lot of changes in different changes in different photoperiodsphotoperiods

• AdvantageAdvantage– small- easy to handlesmall- easy to handle– abundantabundant– easy to apply to higher mammalseasy to apply to higher mammals– available from other project on available from other project on

campuscampus

HypothesisHypothesis

• If hamsters are housed in winter-like SD If hamsters are housed in winter-like SD (short-day) photoperiod, IGF-I and (short-day) photoperiod, IGF-I and IGFBP-3 level will be decreased, and IGFBP-3 level will be decreased, and cortisol and IGFBP-1 level will be cortisol and IGFBP-1 level will be increased.increased.

• Also, mRNA levels of IGF-I and IGFBP-3 Also, mRNA levels of IGF-I and IGFBP-3 in liver and muscle will be in liver and muscle will be suppressed ,and that of IGFBP-1 will be suppressed ,and that of IGFBP-1 will be elevated. elevated.

Specific AimsSpecific Aims

• To compare IGF-I and cortisol levels in different To compare IGF-I and cortisol levels in different photoperiodsphotoperiods

• To compare IGFBPs (IGFBP-1 and IGFBP-3) in To compare IGFBPs (IGFBP-1 and IGFBP-3) in different photoperiodsdifferent photoperiods

• To see tissue-specific mRNA for IGF-I, IGFBP-1 To see tissue-specific mRNA for IGF-I, IGFBP-1 and IGFBP-3 in different tissue types: liver and and IGFBP-3 in different tissue types: liver and musclemuscle

Those findings will characterize the mechanism Those findings will characterize the mechanism of IGF-axis in the relationship between short-of IGF-axis in the relationship between short-day photoperiod and somatic growth change.day photoperiod and somatic growth change.

Experimental DesignExperimental Design• AnimalsAnimals

– use 20 LD and 40 SD female use 20 LD and 40 SD female hamstershamsters

– SD will be artificially set for SD will be artificially set for 8hr in light and 16hr in dark8hr in light and 16hr in dark

– LD will be artificially set for LD will be artificially set for 16hr in light and 8hr in dark16hr in light and 8hr in dark

– tissue and blood sampled tissue and blood sampled every three weeks: week-every three weeks: week-3,6,9 & 123,6,9 & 12

Experimental Design Experimental Design (Cont’d)(Cont’d)

• Tissue storageTissue storage– blood -put in 4°C (in ice) first, then blood -put in 4°C (in ice) first, then

centrifuged and stored the serum in -80 centrifuged and stored the serum in -80 °C °C

– tissue (liver and muscle) -put in RNA tissue (liver and muscle) -put in RNA later first, then stored in -80 °C later first, then stored in -80 °C

Experimental Design Experimental Design (Cont’d)(Cont’d)

• TechniquesTechniques1) 1) RadioimmunoassayRadioimmunoassay (RIA) for serum level of IGF-I and (RIA) for serum level of IGF-I and cortisolcortisol

2) 2) Western immunoblotWestern immunoblot for IGFBPs (specific): IGFBP-1 and for IGFBPs (specific): IGFBP-1 and IGFBP-3, using rabbit anti-rat IGFBP-1 antiserum and rabbit IGFBP-3, using rabbit anti-rat IGFBP-1 antiserum and rabbit anti-rat IGFBP-3 antiserum for specific proteinanti-rat IGFBP-3 antiserum for specific protein

3) 3) RNA-isolationRNA-isolation - RNA from liver and muscle tissue by single - RNA from liver and muscle tissue by single step method of RNA isolation by acid guanidinuum step method of RNA isolation by acid guanidinuum thiocyanate-phenol chloroform extraction.thiocyanate-phenol chloroform extraction.

4) 4) Northern BlotNorthern Blot for mRNA level determination of IGF-I, IGFBP- for mRNA level determination of IGF-I, IGFBP-1 and IGFBP-3 in liver and muscle1 and IGFBP-3 in liver and muscle

Experimental Design Experimental Design (Cont’d)(Cont’d)

• Statistical AnalysisStatistical Analysis– Data from all measurements will be Data from all measurements will be

analyzed using one-way ANOVA with analyzed using one-way ANOVA with p<0.05 as the level of significancep<0.05 as the level of significance

Expected ResultsExpected 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

0100200300400500600700800

LD SD

ng/ml IGF-I

Cortisol

Expected Results (Cont’d)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.

0

50

100

150

200

250

300

350

400

450

500

LD SD

ng/ml IGFBP-1

IGFBP-3

Expected Results (Cont’d)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.

muscle liver muscle liver

LD LD SD SD

IGF-I

IGFBP-1

IGFBP-3

DiscussionDiscussion• During winter-like short-day photoperiod, animals During winter-like short-day photoperiod, animals

undergo physiological changes to save energyundergo physiological changes to save energy

• some energy expensive processes (reproduction, some energy expensive processes (reproduction, growth, etc) are shut down physiologically to save growth, etc) are shut down physiologically to save energyenergy

• From this study, we can determine the From this study, we can determine the mechanisms in IGF-axis on regulation of growth in mechanisms in IGF-axis on regulation of growth in mammals in different photoperiods.mammals in different photoperiods.

• The mechanisms controlling growth are likely to be The mechanisms controlling growth are likely to be applicable in most mammalian species, including applicable in most mammalian species, including humans. humans.

AcknowledgementAcknowledgement

• Dr. Kevin KelleyDr. Kevin Kelley

• HHMI honor programHHMI honor program

• Dr. A. Z. MasonDr. A. Z. Mason

• Dr. Kelly YoungDr. Kelly Young