Excretion and Conservation of Glycerol

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Excretion and Conservation of Glycerol During
Cold Acclimation in Copes Gray Treefrog Hyla
chrysoscelis
J. West, D. Goldstein, K. Rivera, J.
Frisbee, S.L. Zimmerman, and C.M. Krane
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Background and Objectives

• Freeze Tolerance
• Aquaporin/Aquaglyceroporin
• In vivo mechanism of freeze-tolerance in Copes
  gray treefrog, Hyla chyrsoscelis
• Cryopreservation

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• http//www.pbs.org/wgbh/nova/sciencenow/3209/05.ht
  ml

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H. Chrysoscelis Habitat

• Wooded areas near water in eastern and central US
• Congregate at ponds for breeding in March-July
• Sister species H. veriscolor is tetraploid, with
  larger RBC and slower trill

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Strategies for Survival at Low Temperatures

• 1. Freeze Tolerance
• Ice forms in ECF
• Must be slow minimize cell damage and osmotic
  stress
• Minimize cell volume loss
• Maintenance of homeostasis under ischemic
  conditions

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Strategies for Survival at Low Temperatures

• 2. Freeze Avoidance
• inhibition of ice nucleation
• colligative depression of freezing point using
  antifreeze proteins
• supercooling

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Cryoprotectants and Water Flow

• When a frog freezes, ice forms only in ECF.
• Second, ice is relatively pure and is formed of
  water but no solutes.
• When the solution outside the cells gradually
  freezes, the salts and other solutes there become
  quite concentrated. This condition creates an
  osmotic imbalance across the water-permeable cell
  membrane.
• The pressure differential is relieved when water
  inside the cell flows outside.

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• However, if too much water leaves, the cell
  shrinks excessively and the plasma membrane may
  fail.
• This dynamicnot mechanical damage from jagged
  ice crystalsis thought to be a primary mechanism
  of freezing injury to tissues.

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Cryoprotectants

• Cryoprotectants (glucose and glycerol) help cells
  retain water by
• 1) colligatively reducing the quantity of ice
  that forms and
• 2) increasing the intracellular solute
  concentration and thereby limiting the osmotic
  pressure differential.

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Aquaporin/Aquaglyceroporin Function More than
Water Channels

• Osmosensors
• Cell migration
• Fat metabolism
• Glycerol metabolism, gluconeogenesis
• Freeze Tolerance

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Aquaporins--Aquaglyceroporins
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Transmembrane Structure of The Aquaporin Family
Of Water Channels
repeat-2
repeat-1
C
A
E
Extracellular
Intracellular
D
B
NH2
C
N
P
A
COOH
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A. thaliana AQPs
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Hypothesis

• Copes gray treefrogs accumulate glycerol as part
  of their strategy for cold acclimation
• Adjustments of renal function will help to
  conserve glycerol either a drop in GFP or
  enhanced glycerol reabsorption
• Transmembrane movement of glycerol and of water
  is associated with Aquaporin/Aquaglyceroporin
  expression

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Methods

• 14C-Inulin clearance as an estimate of GFR
• GFR UV/P
• FR (filtered excreted)/filtered x 100
• Amount filtered GFR X plamsa inulin
• Amount excreted UFR x urine inulin
• Degenerate RT-PCR, subcloning
• RT-PCR of cDNA panel

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Plasma and Urine Glucose and Glycerol
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Reduction in GFR in Cold-Acclimated and
Dehydrated Frogs
GFR was significantly different in each of the
three groups (p lt 0.001).
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Reduction in Urine Flow Rate in Cold-Acclimated
and Dehydrated Frogs
Urine flow rate was significantly reduced in the
latter two groups, which did not differ
significantly from each other ( p lt 0.001)
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Urine Excretion Rates of Glucose and Glycerol
For glycerol, all three groups differed
significantly (p lt 0.001),whereas the cold and
dehydrated groups differed from warm-acclimated
animals but not from each other for glucose (p lt
0.001)
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Mechanism?

• FR (filtered excreted)/filtered x 100
• Fractional reabsorption of glycerol
• 64 in warm acclimated
• 82 in cold acclimated
• Role for aquaporins?

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HC-1 272 a.a.
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HC-2 280 a.a.
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HC-3 292 a.a.
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AQP/GLP Functional Residues
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Thermal- and Tissue-Specific Expression of HC-1,
HC-2, and HC-3
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What Weve Learned

• Cold acclimation reduces GFR and UFR, with an
  increase in circulating and tissue glycerol
• Fractional glycerol reabsorption is increased
• Three AQP/GLP cloned from gray tree frogs
• Thermal- and tissue-specific regulation

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Cryopreservation
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