1. The ability to adapt to changing osmotic conditions is critical for cellular and organismal
survival. Though many of the pathways and mechanisms that define these induced
effects are unknown, conserved determinants can be observed. Prior studies in the
budding yeast, Saccharomyces cerevisiae, have shown that inducing hyperosmotic
conditions through addition of sorbitol to the growth medium is sufficient to increase
both chronological and replicative lifespans. Our group has found that the addition of
either peptone or sorbitol to the growth medium of the nematode, Caenorhabditis
elegans can induce a similar longevity effect, resulting in an increase in lifespan of
approximately 35% in both wild type (N2) and several mutant backgrounds. We
hypothesize that this effect on lifespan results from an adaptive response to the
hyperosmotic conditions. One conserved response to hyperosmolarity is increased
biosynthesis of intracellular glycerol. To determine whether glycerol synthesis is
required for lifespan extension, we examined the effect of sorbitol addition on lifespan
in gpdh-1(ok1558); gpdh-2(kb33) double-mutant animals. These genes encode the
enzyme glycerol 3-phosphate dehydrogenase and are required for elevation of glycerol
levels under hyperosmotic conditions. We found that addition of sorbitol was no longer
able to extend the lifespan of gpdh-1(ok1558); gpdh-2(kb33) animals, supporting the
hypothesis that glycerol accumulation plays a necessary role in the longevity response
to hyperosmotic stress in C. elegans. Next, we plan to determine whether increased
glycerol biosynthesis through overexpression of glycerol 3-phosphate dehydrogenase is
sufficient to extend lifespan in the absence of osmotic stress. These studies will provide
further understanding of the mechanisms that control aging and the interaction
between longevity and osmotic changes in the environment.
Abstract
Effects of mutation on osmotic stress response
pathway in sorbitol conditions
Conclusions and Future Directions
Acknowledgements
Both 10x peptone and 5% sorbitol conditions
are shown to increase lifespan in wild type (N2)
gpdh-1(ok1558);gpdh-2(kb33) double-knock-out-
mutant show no signs of increased lifespan
in 5% sorbitol conditions
Methods
Worms are grown on NGM
solid medium seeded with
Na22, well fed until eggs
are highly amplified
Bleach prep
when adults
are sufficient
After 2 days, transfer
stage L4 larval worms
to condition plates,
35 worms/plate
5% Sorbitol
200mM (4x) NaCl
20℃
2. Measurement of Glycerol Levels in C. elegans
Well-fed worms were rinsed off plates with M9, spun down, and then rinsed with fresh
M9 twice and re-suspended in 500μl M9. 500μl of the pellet was dropped by transfer
pipette into liquid nitrogen. Frozen worms were ground to a fine powder with
homogenizer cooled in nitrogen. The power was neutralized with 1N perchloric acid
(PCA) to extract organic solutes and precipitate proteins. After centrifugation, the acid
supernatant was neutralized with 10N KOH. Glycerol levels were measured with a
commercially available UV-based glycerol assay kit (R-Biopharm Inc, Washington, MO
USA). PCA precipitated pellets were solubilized with 0.1N NaOH, and protein content
was measured with BCA assay Kit (Thermo Fisher Scientific Inc, Rockford, IL USA) and
used for the normalization of glycerol levels.
gpdh-1::GFP
Actin
NGM 10x Peptone 5% Sorbitol
***
***
***
***
**
Both 10x peptone and 5% sorbitol conditions show increase
induced gpdh-1 expression to hyperosmotic conditions
Dosage dependent effects were recorded for differing hyperosmotic conditions in regards to lifespan.
200mM NaCl and 5% Sorbitol selected for further experimentation hypersosmotic conditions.
0
2
4
6
8
10
12
14
16
NGM 5% Sorbitol 4x Salt
Proportionofwormglycerol
(normalized to the
protein concentration)
 Increasing the osmolarity of the growth medium with sorbitol increases nematode lifespan
and stress resistance
 Previous studies in our lab found that sorbitol promotes longevity by a mechanism distinct
from insulin-like signaling, sir-2.1, AMP kinase, and hypoxic response
 Additionally, our lab concluded that Dietary Restriction (defined as the limitation of food
without malnutrition) fails to further extend lifespan in combination with 5% sorbitol
 Induction of osmotic stress response pathway is necessary for lifespan extension from
sorbitol
 gpdh-1(ok1558);gpdh-2(kb33) double-mutant animals did not have increased lifespans
under hyperosmotic stress
 Glycerol accumulation via enzyme glycerol 3-phosphate dehydrogenase plays an important
role in increasing C. elegans lifespan under hyperosmotic conditions
 Future plans: determine whether increased glycerol biosynthesis through overexpression
of glycerol 3-phosphate dehydrogenase is sufficient to extend lifespan in the absence of
osmotic stress.
We’d like to thank the Kaeberlein laboratory, Devon Chandler-Brown, Erica D. Smith, the
Lamitina laboratory, and our mentors Haeri Choi, PhD and Matt Kaeberlein, PhD.
Mutations in these four genes, known to promote osmotic stress resistance in
C. elegans, resulted in non-long-lived lifespans in osr-1(rm-1) and osm-11(n1604) knock-
out mutants and significant life extension in mutant animals of osm-5(p813) and osm-
7(n1515). Sorbitol conditions induced increased lifespan in all four mutant lifespans,
indicating the induction of the osmotic stress response pathway is necessary for lifespan
extension from sorbitol and sufficient in extending lifespan in these conditions.
Glycerol accumulation via enzyme glycerol 3-phosphate dehydrogenase induced by sorbitol conditions shown with
wild-type (N2) worms (left), but absent in identical conditions with gpdh-1;gpdh-2 mutant worms
The role of glycerol biosynthesis in lifespan extension
from hyperosmotic conditions in Caenorhabditis elegans
Shirley Park, Vikram Akam, Haeri Choi and Matt Kaeberlein*
Department of Pathology, University of Washington
Control
10x Peptone
1. Measuring C. elegans lifespan
-100
-80
-60
-40
-20
0
1x Salt 2x Salt 4x Salt 6x Salt 10x Salt
%DifferenceinLifespan
Background
Dr. Todd Lamitina discovered expression of gpdh-1 and gpdh-2
mediate the accumulation of the organic osmolyte glycerol,
essential for the survival of the C.elegans in conditions of osmotic
stress.
(Proc Natl Acad Sci USA. 2006 Aug 8;103(32):12173-8
Benefits of Using C. elegans
 non-parasitic nematode
 consume bacteria
 easy to grow
 short lifespan