The Journal of Experimental Biology 208, 2819-2830 2819

Published by The Company of Biologists 2005

doi:10.1242/jeb.01730

Commentary
Organic osmolytes as compatible, metabolic and counteracting cytoprotectants
in high osmolarity and other stresses
Paul H. Yancey
Biology Department, Whitman College, Walla Walla, WA 99362, USA
e-mail: yancey@whitman.edu

Accepted 1 June 2005

Summary
Organic osmolytes are small solutes used by cells of counteract perturbations by urea (e.g. in elasmobranchs
numerous water-stressed organisms and tissues to and mammalian kidney), inorganic ions, and hydrostatic
maintain cell volume. Similar compounds are accumulated pressure in deep-sea animals. Trehalose and proline in
by some organisms in anhydrobiotic, thermal and possibly overwintering insects stabilize membranes at subzero
pressure stresses. These solutes are amino acids and temperatures. Trehalose in insects and yeast, and anionic
derivatives, polyols and sugars, methylamines, polyols in microorganisms around hydrothermal vents,
methylsulfonium compounds and urea. Except for urea, can protect proteins from denaturation by high
they are often called ‘compatible solutes’, a term temperatures. Third, stabilizing solutes appear to be used
indicating lack of perturbing effects on cellular in nature only to counteract perturbants of
macromolecules and implying interchangeability. macromolecules, perhaps because stabilization is
However, these features may not always exist, for three detrimental in the absence of perturbation. Some of these
reasons. First, some of these solutes may have unique solutes have applications in biotechnology, agriculture and
protective metabolic roles, such as acting as antioxidants medicine, including in vitro rescue of the misfolded protein
(e.g. polyols, taurine, hypotaurine), providing redox of cystic fibrosis. However, caution is warranted if high
balance (e.g. glycerol) and detoxifying sulfide (hypotaurine levels cause overstabilization of proteins.
in animals at hydrothermal vents and seeps). Second,
some of these solutes stabilize macromolecules and
counteract perturbants in non-interchangeable ways. Key words: osmolyte, antioxidant, pressure, urea, trimethylamine
Methylamines [e.g. trimethylamine N-oxide (TMAO)] can oxide, hypotaurine, temperature, compatible solute, counteracting,
enhance protein folding and ligand binding and compensatory.

Introduction: osmolytes in osmoconformers and

osmoregulators
Water is widely regarded as the most important molecule of Osmoconformers are most commonly found in the oceans
life, and an organism’s ability to cope with changes in its and include most types of life other than most vertebrates and
internal water content is essential for survival. In particular, some arthropods. The salts (mainly NaCl) of ocean water
loss of internal water is a common threat, arising from yield an average osmotic concentration of
evaporation into air, during the excretion of wastes or from ~1000·milliosmoles per liter (1000·mOsm), well above the
osmosis into concentrated aqueous surroundings. The latter ~300–400·mOsm created by the basic solutes found in most
may occur in an external saline environment, by extracellular cells (K+, metabolites, proteins, etc.). To prevent osmotic
freezing, or from diseases that cause osmotic imbalances (e.g. shrinkage, internal fluids of marine osmoconformers have
diabetes and its associated hyperglycemia). Traditionally, about the same osmotic pressure as their environment (e.g.
organisms have been divided into two broad categories in 1000·mOsm). However, while extracellular fluids in
terms of adaptations to water stress: osmoconformers, which multicellular organisms are typically dominated by NaCl, the
usually use organic osmolytes to keep cellular osmotic pressure major osmotic components inside cells (which raise osmotic
equal to that of the external fluid environment, and pressure above the basal level of 300–400·mOsm) are usually
osmoregulators, which use ion transport to homeostatically organic osmolytes (Fig.·1). These osmolytes can be up- or
regulate internal osmotic pressures. downregulated in many species to prevent osmotic shrinkage

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2820 P. H. Yancey

Alpha-AAs GPC

1000 Taurine Sorbitol + inositols

Hypotaurine Urea
Cell solute concentrations (mmol l–1, estimated)

Thiotaurine Ser-P-Eth-X
800
TMAO β-Alanine
Betaine Unknown
600

400

200

0
Shark Worm Snail Clam Worm Snail Snail Clam Clam Clam Riftia Riftia Mammal
(shallow) (shallow) 2.9 km 2.9 km vent seep seep seep vest. troph. renal
0.5 km 4 km 6 km

Fig.·1. Patterns of osmolyte distributions in marine animals and mammalian kidneys, shown as estimates of intracellular concentrations. Panels
from left to right: (1) sharks and other elasmobranchs are dominated by urea and TMAO (data for Squalus acanthias); (2) shallow-water
invertebrates, such as the polychaete worm Glycera, snail Mitrella carinata and clam Saxidomus giganteus, are typically dominated by taurine,
betaine and α-amino acids (AAs) such as glycine; (3) invertebrates from 2.9·km depth, such as the polychaete worm Glycera and snail Neptunea
lyrata, have less taurine and other amino acids and more scyllo-inositol, GPC, and unknowns, while a snail (Depressigyra globulus) from
hydrothermal vents at 1.5·km depth has high levels of hypotaurine and thiotaurine; (4) vesicomyid clams (Calyptogena spp.) from sulfide seeps
have hypotaurine and thiotaurine and show a depth-related increase in an unsolved serine–phosphoethanolamine solute (Ser-P-Eth-X) and an
unknown methylamine; (5) vestimentiferan tubeworms (Riftia pachyptila) from hydrothermal vents at 2.6·km depth have high amounts of
hypotaurine and an unknown methylamine, both in vestiment tissue (Vest.) and trophosome (Troph., location of sulfide-oxidizing microbial
symbionts), which also has high levels of thiotaurine; (6) mammalian renal cells (inner medulla) have varying levels of sorbitol, myo-inositol,
GPC, betaine and taurine (along with urea). Data from Peterson et al. (1992); Yin et al. (2000); Yancey et al. (2002); Fiess et al. (2002);
Rosenberg et al. (2003).

or swelling if the osmotic concentration of the environment also accumulated by some organisms in thermal and
changes. anhydrobiotic stresses, and possibly under high hydrostatic
Osmoregulators, which in the oceans include vertebrates pressure. These solutes are typically (and sometimes
other than hagfish, coelacanths and elasmobranchs (sharks, misleadingly) called ‘compatible’ solutes, based on the concept
skates, etc.), are quite different. Such animals typically have that they do not perturb cellular macromolecules even when
regulatory organs (e.g. gills, kidneys) that work to keep the solutes are at high concentrations (Brown and Simpson,
internal body fluids at ~400·mOsm or less in marine species, 1972). However, as will be discussed, many of these solutes
obviating the need for organic osmolytes. This is the pattern have cytoprotective properties, such as antioxidation and
inherited by terrestrial vertebrates, which typically have stabilization of proteins, that go beyond simple compatibility
~300·mOsm body fluids. (The brine shrimp Artemia in desert and that vary from solute to solute.
salt lakes is another example of a strong osmoregulator.)
However, there are major exceptions to this generalized
pattern, with some osmoregulators utilizing organic osmolytes Types of organic osmolytes
in certain situations. For example, in mammals (considered to Many different small molecules are known to serve as
be exemplary osmoregulators), cells of the kidney medulla organic osmolytes and other compatible solutes. As has been
osmoconform to the high osmotic concentrations in that extensively reviewed previously (Yancey et al., 1982; Yancey,
organ’s extracellular fluid. And, as will be discussed later, 2001), these solutes fall into a few major chemical categories
osmoregulating fishes in the deep sea have very high levels of (Fig.·2): small carbohydrates including sugars (e.g. trehalose),
an organic solute that is a major osmolyte in some polyols (glycerol, inositols, sorbitol, etc.) and derivatives (such
osmoconformers. as o-methyl-inositol); amino acids (glycine, proline, taurine,
Organic solutes similar or identical to organic osmolytes are etc.) and derivatives (e.g. ectoine); methylamines [such as N-

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 Compatible and counteracting osmolytes 2821
trimethylamine oxide (TMAO) and glycine betaine] and hyperosmotic culture medium uses primarily sorbitol as an
methylsulfonium solutes including dimethylsulfonopropionate osmolyte. When production of sorbitol is inhibited, cell growth
(DMSP); and urea. Except for urea (used only by relatively few decreases in parallel with declining cell sorbitol content
types of animals), these categories are widespread in (inhibition had no effect in normal medium, in which these
occurrence; for example, glycine betaine is found in every cells use little sorbitol; Yancey et al., 1990a). However,
kingdom of life, and taurine is widespread among marine addition of glycine betaine (normally absent) to the medium
animals and some mammalian organs. Carbohydrate osmolytes largely restores viability (Moriyama et al., 1991). (3) With
occur in archaea, fungi, algae, plants and mammalian
kidneys, and possibly deep-sea invertebrates. Sugars
and polyols are usually the dominant solutes Carbohydrates
accumulated in organisms that tolerate or avoid POLYOLS CYCLITOLS ANIONIC POLYOLS
Glycerol Sorbitol myo-Inositol Diglycerol phosphate
freezing, such as terrestrial plants, insects, H2-C-OH
amphibians and some polar fishes. Also, many H2-C-OH H2C-OH |
organisms use mixtures of osmolyte categories; e.g. | | H-C-OH
H-C-OH H-C-OH OH OH |
the mammalian kidney uses the polyols myo-inositol | | | | H-C-H
and sorbitol, the methylamines H2-C-OH HO-C-H |
OH
glycerophosphorylcholine (GPC) and glycine | | O
H-C-OH | |
betaine, and the amino acid taurine (the organ also | HO OH O=P-O –
has high urea as both a waste product and an osmotic H-C-OH | |
| |
agent that helps concentrate the urine) (Fig.·1, OH O
H2C-OH |
mammal renal bar). What selective forces have H-C-H
resulted in this widespread use of organic osmolytes, |
H-C-OH
with the (metabolically less costly) inorganic ions |
usually not used? H2-C-OH

Amino acids and derivatives

The basic compatibility hypothesis α-TYPES CYCLIC TYPES β-TYPES
As noted earlier, organic osmolytes are typically Glycine Proline Ectoine Taurine Hypotaurine
called compatible solutes based on the hypothesis N+H3 N+H3 N+H3
N+H2 N+H2
that these solutes (other than urea) do not interact |
O–
| |
H-C-CH3 O– H-C-H H-C-H
with macromolecules in detrimental ways; thus, they | H 2 C CH-C H3C-C CH-C
| |
O O
can be safely up- and downregulated with little C N CH2 H-C-H H-C-H
/ \\ H2C CH2 | |
impact on cellular functions (Brown and Simpson,
O– O CH2 O=S=O S=O
1972; Yancey et al., 1982). This is in stark contrast | |
to inorganic ions, which at high concentrations O– O–
typically bind to and destabilize proteins and nucleic
acids. Indeed, exposure of some cells to high NaCl Methylammonium and methylsulfonium solutes
medium can produce breaks in DNA (Kültz and TMAO Glycine GPC Choline-O- β-DMSP
Chakravarty, 2001). betaine sulphate
CH3
In concert with the compatibility hypothesis, most |
osmolytes are neutral (either zwitterionic or lacking CH3 CH3 H3C-N+-CH3 CH3 H3C CH3
| | |
charges) at physiological pH, although some H3C-N+-CH3 |
H3C-N+-CH3 CH2 H3C-N+-CH3 S+
bacterial and archaeal osmolytes are anionic (e.g. | | | |
O– CH2 |
CH2 CH2
diglycerol phosphate; Fig.·2) and are paired with K+ | CH2
| |
C |
to achieve neutrality (Martin et al., 1999). In its O CH2 CH2
/ \\ |
simplest form, the compatibility hypothesis also O– O | |
O=P-O– O C
suggests that organic osmolytes are interchangeable, | | / \\
i.e. that a cell can be osmotically protected with a O=S=O O– O
O |
variety of compatible osmolytes whether it normally | O–
CH2
uses them or not. There is evidence to support this, |
as illustrated by the following examples. (1) Growth H-C-OH
|
of Escherichia coli in saline growth media can be H2C-OH
improved with a variety of osmolytes, some not used
naturally by the bacterium, added to the medium Fig.·2. Examples of organic osmolytes in three of the four major categories; the
(Hanson et al., 1991). (2) One cultured line of fourth category, urea, is not shown. TMAO, trimethylamine N-oxide; GPC,
mammalian kidney medullary cells (PAP-HT25) in glycerophosphorylcholine; DMSP, dimethylsulfonoproprionate.

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2822 P. H. Yancey
Table 1. Summary of protective properties of osmolytes through metabolic reactions
Cytoprotective property Compatible solutes in nature
Antioxidation Hypotaurine; DMSP; polyols (e.g. water-stressed plant)
Redox balancing, hypoxia Proline, β-alanine betaine, glycerol (e.g. Dunaliella alga)
Sulphide detoxification/storage Hypotaurine (e.g. hydrothermal vent tubeworm, Riftia)
Sulphate detoxification Choline-O-sulphate (e.g. mangrove plant)
Energy reserve Glucose, trehalose, etc. (e.g. frozen wood frog)
Predator repellent DMSP, trans-hydroxyprolinebetaine (e.g. diatom)
Ca2+ modulation Taurine? (e.g. mammalian neuron)

normal cells of rat renal medulla, in both primary cultures in ways that may not be related to osmotic balance. Neonatal
(Rohr et al., 1999) and in vivo (Yancey et al., 1990b), inhibition cats, for example, become blind if raised without taurine due
of sorbitol synthesis triggered a compensating increase in to improper retinal development. However, it is not certain
glycine betaine, such that there were no short-term osmotic how taurine exerts its developmental effects. The compound is
imbalances or apparent damage. These are but a few of many said to be cytoprotective by acting as an antioxidant, a calcium
examples. modulator, a synaptic neuromodulator and a membrane
These experiments suggest that some osmolytes (even from stabilizer (Schaffer et al., 2003). Most of these effects appear
different chemical categories) are functionally to be indirect (e.g. by taurine affecting the actions of other
interchangeable. Thus, perhaps the reason osmolytes vary compounds) rather than direct actions of the taurine molecule
among organisms is simply due to different diets or itself. Brain taurine contents decline with age in mammals,
metabolisms that are unrelated to water stress. This may often with other solutes such as glutamate becoming important as
be the case; for example, the widespread use of (non- osmolytes (Trachtman et al., 1995; Miller et al., 2000). This
nitrogenous) carbohydrate and sulfonium osmolytes in decline, which is not understood, appears to be the basis for
photosynthesizers may arise from nitrogen limitation. including taurine in most of the new so-called ‘energy’ drinks.
However, long-term effects of interchanging osmolytes have There is still much not understood about this solute, something
not been adequately tested. Also, the compatibility concept that drinkers of the taurine-rich energy drinks should consider.
does not readily explain why there is such an enormous variety The metabolic effects of taurine and other osmolytes are
of organic osmolytes, found in all kingdoms of life; Fig.·2 summarized in Table·1, and metabolic roles of other osmolytes
shows only a few examples of the dozens of different known that are better understood are discussed in more detail below.
organic osmolytes. Nor does it explain why many organisms
employ a complex mixture of osmolytes. As will become Antioxidation
apparent, much remains to be learned about the reasons for this In some cases, osmolytes may be compatible (i.e. they do
variety, but it may result from unique properties of some not perturb protein structures), while simultaneously being
osmolytes, properties that may be helpful only with certain active cytoprotectants by serving as antioxidants. For example,
stresses. These cytoprotective properties fall into two broad it has been found that cyclitols (cyclic polyols; Fig.·2) and
categories: (1) protective metabolic reactions and (2) polyols such as mannitol, which are used by many plants for
counteraction of destabilizing forces on macromolecules. water retention, may also scavenge free radicals generated
during drought and cold and other stresses; proline and betaine
(also common osmolytes in plants) were not effective (Orthen
Metabolic protection et al., 1994; Shen et al., 1999). Taurine has already been
It is becoming clear that some osmolytes and related solutes mentioned; it cannot scavenge reactive oxygen species (ROS)
are not metabolically inert but rather engage in unique but rather seems to enhance other cellular antioxidant
reactions that can protect cells in various ways other than functions. However, taurine can directly bind HOCl (a reactive
osmotically. Taurine (Fig.·2) is perhaps the most intensely molecule produced by mammalian leukocytes) to form N-
studied, and most mysterious, compatible solute in this regard. chlorotaurine (Schaffer et al., 2003). Glycine betaine has also
This sulfur-based, non-protein amino acid is a major, often the been implicated in reducing lipid peroxidation in plants
dominant, osmolyte in many marine invertebrates such as (reviewed in Cushman, 2001). Finally, DMSP (Fig.·2), a major
bivalves in shallow waters (Fig.·1, clam shallow bar). It is not osmolyte of marine algae, also has antioxidant properties
clear why this is, nor why taurine contents in at least some (Sunda et al., 2002).
marine invertebrates decrease with depth in the oceans (Fig.·1, Of all solutes accumulated at relatively high concentrations
shallow, 2.9·km and seep bars; Pruski et al., 2000a; Fiess et al., in some situations, hypotaurine, with its reactive sulfur atom
2002). Taurine is also relatively high in mammalian heart and (Fig.·2), is one of the strongest antioxidants, able to scavenge
brain cells, where it can serve as a major osmolyte in severe OH radicals (which bond to the sulfur atom, converting
dehydration (reviewed in Miller et al., 2000; Olson et al., hypotaurine into taurine) as well as HOCl (Aruoma et al.,
2003). It is also essential for mammalian neural development 1988). Hypotaurine is known to occur at osmotically

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 Compatible and counteracting osmolytes 2823
significant levels in two situations: mammalian reproductive (vestimentiferan tubeworms, vesicomyid clams) that house
fluids (where it appears to act as an osmolyte and may protect sulfide-oxidizing microbial symbionts. Pruski et al. (2000a)
sperm and eggs from oxygen radicals; Setchell et al., 1993) and hypothesized that the solutes either protect from sulfide
marine animals living in sulfide-laden waters (see Sulfide radicals and/or store and transport sulfide (for future use by the
detoxification, below). Why it is not used extensively symbionts) nontoxically, as follows:
elsewhere is not clear, but it is possible that using a strong
(hypotaurine) +NH3-CH2-CH2-SO2– + HS˙ →
antioxidant in the absence of radicals could lead to cell damage +
NH3-CH2-CH2-SO2–-SH (thiotaurine)·.
in some way. Thus, it may be accumulated primarily for its
antioxidation properties in specific situations, with an osmotic As evidence for the storage function, hypotaurine is high in
role being a secondary one. all tissues in these animals, but thiotaurine has been found only
in non-trace amounts in symbiont-bearing tissues: gills in
Redox balance and hypoxia protection vesicomyid clams and trophosome in vestimentiferans. This
Some osmolytes are not actively protective in themselves, led to a proposal that thiotaurine is a marker of symbiosis
but their synthesis may be. Glycerol, the archetypical (Pruski et al., 2000b). Studies in our laboratory suggest the
compatible solute (Brown and Simpson, 1972), accumulates in hypotaurine–thiotaurine reaction has a greater, body-wide
certain water-stressed yeasts and algae to high levels (up to cytoprotective role against sulfide in some species: we found
several molar in species in salt ponds and lakes). Glycerol has that two species of vent gastropods without endosymbionts
been shown to be largely compatible with protein function, but have both hypotaurine and thiotaurine as major osmolytes
its synthesis also requires the use of NADH. This may be (Fig.·1, snail vent bar) and that the ratio of thiotaurine to
essential for maintaining cellular redox balance (by hypotaurine decreases in animals held in the laboratory without
regeneration of NAD+) during anaerobic metabolism; indeed, sulfide (Rosenberg et al, 2003).
mutant yeasts unable to make glycerol are not only highly A different form of sulfur detoxification may be involved in
sensitive to osmotic stress but also accumulate excessive some mangrove plants (angiosperms). Species of Aegialitis
NADH and thus cannot grow (Ansell et al., 1997). Glycerol mangroves use choline-O-sulfate as their primary osmolyte. It
may also help reduce radical oxygen production (Shen et al., has been proposed that the synthesis of this solute serves to
1999). Proline accumulation as an osmolyte in water-stressed detoxify sulfate, a major anion in seawater that can be
plants may also be primarily for maintaining redox states, inhibitory at high concentrations (Hanson et al., 1994). Plants
rather than for (or in addition to) compatibility or stabilizing are more vulnerable than animals to inhibitory ion
properties (reviewed in Cushman, 2001). accumulation since most do not have excretory tissues. The
Other osmolytes may protect cells during hypoxia by other methylsulfonium osmolyte DMSP (Fig.·2) may serve a similar
mechanisms. β-alanine betaine, a major osmolyte in several role in marine algae.
species of salt-marsh plants, appears to replace glycine betaine
(found in related plants). Unlike glycine betaine, β-alanine Other metabolic roles, and compatibility revisited
betaine requires no direct use of oxygen to produce it, possibly Other important metabolic and protective functions have been
favoring its use in hypoxic muds of salt marshes (Hanson et attributed to some osmolytes. Other possible functions for
al., 1994). Recently, high cellular levels of trehalose in fruit taurine have already been mentioned (see Table·1). Certainly,
flies and transfected mammalian cells have been found to carbohydrate osmolytes such as glucose, sorbitol and trehalose
confer enhanced resistance to hypoxia. However, this effect (commonly accumulated with temperature stress such as
has attributed to protein stabilization (Chen and Haddad, freezing) can serve as immediate sources of energy after an
2004), the second cytoprotective category that will be organism emerges from a stress-induced dormancy. Defense
discussed later. against predators is another possible function of some osmolytes.
DMSP (Fig.·2), widespread in marine microalgae, can be broken
Sulfide/sulfate detoxification down into a gas, DMS (dimethylsulfide), and acrylate, which
Large concentrations of two taurine derivatives – may serve to repel grazers such as copepods (Wolfe, 2000; Van
hypotaurine (Fig.·2) and thiotaurine – have been reported as Alstyne and Houser, 2003). In some terrestrial plants,
major organic components of marine invertebrates living at hydroxyprolinebetaine is accumulated in water stress; an isomer
hydrothermal vents and cold seeps (reviewed in Pruski et al., of this (trans-4-hydroxy-L-prolinebetaine) is a strong inhibitor
2000a). We have shown that these solutes are osmolytes in the of animal acetylcholine esterase and therefore may deter
sense that they create much of the osmotic pressure of cells, herbivores (Hanson and Burnet, 1994).
and because they effectively replace the common osmolytes The active metabolic roles of osmolytes that have been
(e.g. taurine, glycine) of non-vent and non-seep invertebrates presented here indicate that many compatible solutes are not
(Fig.·1, clam seep and Riftia bars; Yin et al., 2000; Fiess et al., interchangeable, which has significant implications for
2002). But they may have another role. Vents and seeps emit practical applications (more will be said on this later).
high quantities of H2S, a gas that is toxic to animals but that However, the basic compatibility concept is still probably
is a primary energy source for some microorganisms. Initially, correct in the sense that most or all of these compounds should
the two taurine derivatives were found in animals not bind to and perturb most macromolecules.

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2824 P. H. Yancey
Table 2. Summary of protective roles of counteracting solutes through stabilization of macromolecules and membranes
Stabilising property Counteracting solutes in nature
Counteract urea inhibition Methylamines, especially TMAO (e.g. shark)
Increase thermostability Trehalose, anionic polyols (e.g. vent archaeon)
Protect membranes in freezing Trehalose; proline (e.g. frozen insect)
Preserve in dry state Carbohydrates, especially trehalose (e.g. dried yeast)
Counteract inorganic ion inhibition Methylamines (e.g. salt marsh plant)
Counteract hydrostatic pressure TMAO; other solutes? (e.g. deep-sea fish)

Stabilization and counteraction appear to accumulate significant amounts of counteracting

The basic compatibility concept is misleading in another osmolytes (Withers and Guppy, 1996). Inhibition by urea may
way. Numerous studies, often unrelated to research on natural actually be useful during estivation. However, at least some of
compatible solutes, have shown that these types of solutes can their enzymes are more resistant to urea than are enzymes of
stabilize macromolecular structures (proteins, membranes) in other species (Grundy and Storey, 1994; Fuery et al., 1997).
a variety of conditions. However, not all compatible solutes are Thus, as originally noted for elasmobranch proteins (Yancey
equal in this regard (although nearly all are stabilizers at high et al., 1982), there may be more than one way to adapt to high
concentrations). Moreover, this property is not necessarily a urea. Perhaps the metabolic cost of making counteracting
benefit in and of itself (as will be discussed later). In nature, osmolytes is disfavored in estivation.
stabilizing ability seems to be used only when there are stresses Methylamines can also offset some perturbing effects of
that directly destabilize macromolecules and membranes. salts. Methylated derivatives of glycine (sarcosine,
These stresses include perturbing solutes, anhydrobiosis, high dimethylglycine and glycine betaine) can counteract NaCl
temperature, freezing and high hydrostatic pressure (Table·2). inhibition of a plant enzyme’s activity, with protection
increasing with degree of methylation (Pollard and Wyn Jones,
Perturbing solutes: urea and salts 1979). Many other studies show counteraction of salt inhibition
Some organic osmolytes are able to offset, or ‘counteract’, by methylamines, including complex cellular systems
effects of solutes that also build up in osmotic stress and that (reviewed by Yancey, 2001).
perturb macromolecules. Urea is such a perturbant. It is a
highly concentrated waste produce in mammalian kidneys and Anhydrobiosis
urine, and it is the major organic osmolyte in marine Disaccharides, most notably trehalose, commonly build up
elasmobranch fishes (ureosmotic animals) (Fig.·1, shark and in anhydrobiotic dormant organisms (e.g. baker’s yeast,
mammal renal bars). At concentrations in these fishes and resurrection plants, tardigrades). However, these sugars do not
mammalian kidneys (e.g. several hundred millimolar), urea exhibit non-interactive compatibility and are not osmolytes
destabilizes many macromolecular structures and inhibits since the organisms lose most of their water. Rather, these
functions such as ligand binding. However, these animals have solutes appear to bind to macromolecules and membranes, in
other osmolytes, mainly methylamines such as TMAO and essence replacing water molecules and maintaining the basic
GPC (Figs·1,·2). These solutes do not exhibit simple structure of these large biomolecules. Moreover, trehalose
compatibility, but rather show strong enhancement of protein forms a glass-like state (i.e. it vitrifies) in the dry state, which
activity and stability at physiological concentrations. For also helps preserve cellular structures. Trehalose appears to be
TMAO, this property is additive with urea’s effects such that better than other biological sugars in forming a protective
they counteract, most effectively at about a 2:1 urea:TMAO vitrified state (Crowe et al., 1998). Trehalose also is a non-
ratio (Fig.·3A), which is similar to physiological levels reducing sugar, which, unlike glucose and some other
(roughly 400:200·mmol·l–1 in shallow-water elasmobranch monosaccharides, does not engage in ‘browning’ (Maillard)
fishes; Fig.·1, shark bar). Counteraction of urea has been reactions that can damage proteins during drying (reviewed by
extensively confirmed in a variety of protein systems (reviewed Tunnacliffe and Lapinski, 2003).
by Yancey, 2001) and has also been recently demonstrated for Although in vitro experiments have clearly established the
nucleic acids in the form of bacterial tRNA (Fig.·3B; Gluick efficacy of trehalose, recent studies are questioning its role in
and Yadav, 2003). TMAO is usually a better stabilizer than anhydrobiosis in nature. In particular, bdelloid rotifers have
other osmolytes, including glycine betaine and glycerol (Ortiz- been found to undergo reversible anhydrobiosis without
Costa et al., 2002; Russo et al., 2002; Yancey et al., 2004; accumulating trehalose or similar solute (Tunnacliffe and
Kumar et al., 2005), perhaps explaining why TMAO is Lapinski, 2003). The issue raised by these observations
preferred in ureosmotic fishes. Like basic compatibility, remains unresolved.
counteraction occurs whether a protein is from a urea-
accumulating tissue or not (e.g. bacterial tRNA noted above). Freezing
Accumulation of high levels of urea also occur in some Freezing is another stress faced by many ectotherms in
amphibians, especially estivating frogs. However, they do not which specific small solutes play a role. Strategies to survive

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 Compatible and counteracting osmolytes 2825
Control TMAO addition, certain amino acids such as proline also accumulate
Urea Urea + TMAO in some freeze-tolerant animals (Storey and Storey, 1996;
Neufeld and Leader, 1998), but not to levels that would suggest
50 4 an antifreeze function. In fact, there is some evidence that
A B cryoprotectants fall into two categories with distinct roles.
% Reactivation (after 30 min)

First, the carbohydrates such as glycerol act as both colligative

–ΔG Unfolding (kcal mol–1)

40
3 antifreezes, and, in freeze tolerance, as osmolytes (i.e. they
reduce loss of cellular water), while at the same time being
30
compatible with macromolecules. Non-carbohydrate solutes
2 might be substituted for this role, but carbohydrates may be
20 preferred as the easiest to both synthesize and transport across
1 membranes rapidly. They also form a ready energy source for
10 use upon emergence from freezing.
By contrast, a second group of cryoprotectants may have
0 0 stabilizing functions that other solutes do not. In particular,
Shark LDH Bacterial tRNA proline and trehalose appear to bind to head groups of
membrane phospholipids, in effect replacing water molecules.
Fig.·3. Old and new examples of counteraction between urea and Thus, they can stabilize membranes during cell shrinkage
trimethylamine N-oxide (TMAO). (A) Extent of refolding of
(Rudolph and Crowe, 1985; Storey and Storey, 1996).
denatured great white shark A4-lactate dehydrogenase (LDH) in
physiological buffer with no osmolytes (control), 400·mmol·l–1 urea,
High temperature
200·mmol·l–1 TMAO or combined urea:TMAO (2:1) (data from
Yancey and Somero, 1979). (B) Free energy (ΔG) of unfolding of E. Almost all natural osmolytes and other compatible solutes
coli tRNAfmet in physiological buffer with no osmolytes (control), can increase protein thermal stability in vitro; although for
2·mol·l–1 urea, 1·mol·l–1 TMAO or combined urea:TMAO (2:1) (data most osmolytes, this occurs only at non-physiologically high
from Gluick and Yadav, 2003). concentrations. However, certain carbohydrate solutes may be
used in living organisms to counteract temperature disruption
of proteins. For example, heat stress induces accumulation of
body temperatures below freezing fall into two categories: trehalose in yeast, in which the disaccharide can protect
freeze avoidance and freeze tolerance. Avoiders (whose body enzymes from thermal denaturation (Singer and Lindquist,
fluids do not freeze) use a variety of mechanisms such as non- 1998).
colligative antifreeze proteins, reduced nucleation sites and Hyperthermophilic archaea from marine hydrothermal vents
supercooling. Many avoiders also accumulate (in all body accumulate β-mannosylglycerate, di-myo-inositol phosphate
fluids) high levels of colligative antifreezes, or cryoprotectants, and K+ at high temperatures and salinities. One species has
which are typically compatible carbohydrates such as glycerol. high levels of diglycerol phosphate at high temperatures
Well-studied model animals that use glycerol include gall moth (Fig.·2; Martin et al., 1999). Both trehalose and anionic
(Epiblema scudderiana) caterpillars (Storey and Storey, 1996) osmolytes such as these sugar phosphates (paired with K+) can
and rainbow smelt, which, unlike most teleost fish, is nearly an stabilize proteins at high temperatures (even boiling in some
osmoconformer due to the accumulation of glycerol as an cases), while other osmolytes are much less effective. One
antifreeze (Raymond, 1992). study showed this type of counteraction to be effective on
Freeze tolerators, by contrast, let their extracellular fluids proteins of archaea, yeast and mammals, suggesting a universal
freeze with the aid of ice nucleators; however, intracellular ability (Santos and da Costa, 2002).
fluids typically do not freeze due to the presence of, once again,
colligative cryoprotectants such as glycerol, trehalose and Hydrostatic pressure in the deep sea
sorbitol. In this situation, cells shrink somewhat due to The most recent example of counteraction has been found
increasing extracellular concentrations caused by ice in the deep sea, where high hydrostatic pressure destabilizes
formation. However, shrinkage is limited by the compatible protein structure and ligand binding. Although some proteins
solutes serving as osmolytes as well as antifreezes. Model appear to have evolved pressure resistance, many have not or
animals include gall fly (Eurosta solidaginis) larvae and have done so incompletely (Siebenaller and Somero, 1989).
intertidal barnacles, which use glycerol, wood frogs (Rana Our recent studies suggest that some osmolytes can help with
sylvatica), which use glucose, and New Zealand alpine wetas pressure. In shallow marine animals, TMAO (Fig.·2) is either
(Hemideina maori), which use trehalose (Baust and Lee, 1982; absent or found at less than 100·mmol·kg–1 wet mass (except
Storey and Storey, 1996; Neufeld and Leader, 1998). in ureosmotic fish such as sharks). However, deep-sea teleost
Carbohydrates are also found as cryoprotectants in many fishes (osmoregulators usually thought to have low organic
plants. osmolyte levels), as well as certain crustaceans, skates and
Thus, small carbohydrates have been selected as colligative other osmoconforming animals, have up to 300·mmol·kg–1
antifreezes independently in different taxa and strategies. In TMAO, increasing with depth (Fig.·4A). Initially, we found the

THE JOURNAL OF EXPERIMENTAL BIOLOGY

2826 P. H. Yancey
increase in TMAO down to 3·km depth (Gillett et al., 1997; species and within the same species (Fig.·4A; Yancey et al.,
Kelly and Yancey, 1999); recently, we have found that the 2004). In osmoconformers, high levels of TMAO essentially
pattern extends linearly down to 4.8·km both among different replace the common osmolytes of shallow relatives, e.g.
glycine in shrimp, urea in skates, which, in a species from 3·km
depth, had a 1:2 urea:TMAO ratio rather than the typical 2:1
–䊊– Gadid + –䉫– Shrimp
ratio of shallow elasmobranchs (Rajids, Fig.·4A). A similar
macrourids
–䊉– Rajids (TMAO) pattern has been confirmed for some sharks (Treberg and
–䉭– Scorpaenids
--䊏-- Rajids (urea) Driedzic, 2002).
Since hydrostatic pressure is the only environmental factor
400 that is linear with depth, we hypothesized that TMAO might
A
Osmolyte content (mmol kg–1 wet mass)

counteract pressure effects. Indeed, TMAO (but not other

common osmolytes) in vitro was able to offset pressure
300 inhibition of (1) stability of several homologues of lactate
dehydrogenase, (2) polymerisation of actin, (3) enzyme-
substrate binding for two enzymes and (4) growth of living
yeast cells (Yancey and Siebenaller, 1999; Yancey et al., 2002,
200
2004). One example is shown in Fig.·4B. Other hypotheses to
explain the high TMAO in deep-sea animals, such as diet,
buoyancy, energy savings (Kelly and Yancey, 1999) and
100 byproduct of lipid storage (Seibel and Walsh, 2002), do not
readily explain the highly linear pattern. Thus, TMAO may not
be serving primarily as an osmolyte but rather as a pressure
0 counteractant.
0 1000 2000 3000 4000 5000
Other researchers have found that some sugars and polyols
Depth of capture (m) can counteract pressure destabilization of bacterial enzymes
(Saad-Nehme, 2001), a concern for the food industry, which is
0.1 MPa increasingly using pressure for sterilization. These findings
140 25 MPa * raise the possibility that other osmolytes might help counteract
B* * pressure in nature.
*,†
Km of NADH (% of control)

120 † We have recently found that some deep-sea animals

(echinoderms, some mollusks, polychaetes, vestimentiferans,
100
etc.) do not have TMAO, probably because their taxa lack the
80 biosynthesis pathways. However, all have high levels of
potentially stabilizing (and often unusual) osmolytes, including
60
the polyol scyllo-inositol, and other methylamines, including
40 glycine betaine, GPC and several unsolved methylamines
(Fig.·1, 2.9·km bars) (Fiess et al., 2002; Yancey et al., 2002).
20 Also, vesicomyid clams from 2–6.4·km depth contain an
unsolved serine-phosphate-ethanolamine compound that
0
increases linearly with depth, forming over 60% of the
l
AO

ne
ro

ito
in

osmolyte pool of the deepest species (Fig.·1, clam 4·km and

ci
nt

ta

os
TM

ly
Co

Be

In

6·km bars; Fiess et al., 2002). Since organic phosphates (e.g.

-
yo
m

diglycerol phosphate, GPC) have been found to be stabilizers

of proteins in other situations, perhaps this compound is also
Fig.·4. Trimethylamine N-oxide (TMAO) as a possible pressure a stabilizer.
counteractant in deep-sea animals (see also Fig.·1 for other deep-sea Deep-sea bacteria have been found to accumulate the
osmolytes). (A) Contents of TMAO (and urea in rajids, as shown) in osmolyte β-hydroxybutyrate in correlation with exposure to
muscles as a function of depth in shrimp, rajids (skates) and teleost hydrostatic pressure as well as to osmotic pressure (Martin et
fishes: gadid (cod) and related macrourids (grenadiers), plus al., 2002). The investigators proposed the term ‘piezolyte’ for
scorpaenids (rockfish) (data from Kelly and Yancey, 1999; Yancey et
solutes that are accumulated at high pressure. (This suggests
al., 2004). (B) Effect of 250·mmol·l–1 osmolytes on NADH Km of A4-
lactate dehydrogenase (LDH) from deep-sea grenadier
that parallel terms such as ‘thermolyte’, ‘cryolyte’ and
(Coryphaenoides armatus). Measurements were made at atmospheric ‘anhydrolyte’ might be considered!) Whether the serine-
pressure (0.1·MPa) and 250·atmos (25·MPa), showing that TMAO phosphate or β-hydroxybutyrate can offset the effects of
counteracts pressure better than other common solutes. *Significant pressure is unknown. However, a recent study on marine
increase compared to 0.1·MPa water control; †significant decrease bacteria has shown that adaptation to salinity synergistically
compared to 25·MPa water control (modified from Yancey et al., 2004). enhances survival at high pressure, suggesting that some

THE JOURNAL OF EXPERIMENTAL BIOLOGY

 Compatible and counteracting osmolytes 2827
osmolytes may protect against both stresses in these organization through stronger hydrogen bonding among water
microorganisms (Kaye and Baross, 2004). molecules. (By contrast, urea weakens water–water hydrogen
bonding.) Possibly, the peptide bond of proteins is less able to
Mechanisms of stabilization interact with (i.e. be hydrated by) the organized water around
The compatible and counteracting hypotheses predict that TMAO than with bulk water.
solute–macromolecule effects are universal, i.e. stabilization Other stabilizers may work through more direct interactions,
should occur with proteins or membranes from any organism as discussed earlier for membrane interactions of trehalose and
regardless of whether it uses osmolytes or not (Wyn Jones et other solutes used in anhydrobiosis and freezing. Taurine has
al., 1977; Yancey et al., 1982). How can these effects be also been reported to bind to membranes through ionic
universal, given the great diversity in macromolecular interactions (Schaffer et al., 2003). The charged osmolytes of
structures? The mechanisms are not fully known, but universal hyperthermophiles (mannosylglycerate, diglycerol phosphate;
water–solute–macromolecule interactions are involved for Fig.·2) appear to enhance native protein conformations through
many osmolytes and related solutes. Destabilizers such as electrostatic interactions, in addition to preferential exclusion
some salt ions and urea generally bind to proteins, causing (Faria et al., 2004).
them to unfold because this exposes more groups that undergo
thermodynamically favorable binding with the destabilizer
(Fig.·5C). By contrast, many stabilizing solutes do not bind to The ‘yin and yang’ of cytoprotection
proteins; indeed, they are excluded from a protein’s hydration Are stabilization and counteraction simply another aspect of
layer (the water molecules adjacent to a protein’s surface) compatibility, as it is often portrayed? Not necessarily. At its
(Timasheff, 1992). Recently termed the ‘osmophobic’ effect inception, the ‘counteracting-osmolytes’ hypothesis proposed
by Bolen and Baskakov (2001), exclusion arises from an that a mixture of urea and methylamine is more beneficial than
apparent repulsion between stabilizers and the peptide either solute alone, since a methylamine such as TMAO might
backbone, explaining how this effect can be universal. Because ‘overstabilize’ proteins, e.g. making them too rigid for optimal
of this repulsion, proteins will tend to fold up more compactly, function or causing them to precipitate (Yancey et al., 1982).
since this will reduce exposure of the peptide-bond backbone This concept has not received much attention, but there is
to thermodynamically unfavorable interactions with the evidence supporting it, as follows.
stabilizing solute (Fig.·5A,B). (1) Strong stabilizers such as TMAO and trehalose appear
Why are stabilizing solutes repelled by the protein to be high in organisms only when there is a perturbant present
backbone? New studies by Bennion and Daggett (2004) show (e.g. urea, pressure, high temperature). The pattern of
that TMAO enhances water structure (Fig.·5), causing greater increasing TMAO with depth in marine animals (Fig.·4A)

A B C

Bulk H2O
U
U
U
+ΔV
U
Pressure Urea
U
S Normal TMAO
U
folding/
TMAO U

U
T T
T S

Fig.·5. Model of trimethylamine N-oxide (TMAO), urea and pressure effects on protein folding, based on basic pressure effects (Siebenaller
and Somero, 1989), counteracting effects (Yancey et al., 1982) and osmolyte physicochemical studies of Timasheff (1992), Bolen and Baskakov
(2000) and Bennion and Daggett (2004). Small spheres represent water molecules. (A) An unfolded protein and/or substrate (S) with hydration
layers at a higher density than that of bulk water. (B) Thus, upon folding and/or ligand binding, there is a net expansion (+ΔV) as water molecules
are released into bulk water during folding. If this is the case, hydrostatic pressure will inhibit folding and/or binding (A). (C) Addition of urea
(U) enhances unfolding since that maximizes favorable binding interactions. In B, TMAO (T) is surrounded by its own structured water layer,
which disfavors exposure of the protein’s peptide backbone and of the substrate to bulk water. TMAO thus favors folding and binding, reducing
the total order (higher in A and C).

THE JOURNAL OF EXPERIMENTAL BIOLOGY

2828 P. H. Yancey
1.8 (5) Hypotaurine is one of the most reactive antioxidants of
*
all known compatible solutes, and yet its use in nature at high
1.5 concentrations is rare. Perhaps it is too reactive for ordinary
antioxidant needs.
Gm (nS pF–1)
1.2
(6) Some cryoprotectants such as dimethylsulfoxide and
0.9 ethylene glycol, which can protect protein structure in
freeze–thaw cycles, will denature proteins at higher
0.6 temperatures. This may be due to the fact that hydrophobic
interactions increase with temperature, such that these solutes
0.3
* may be excluded from proteins at low but not high
0 temperatures (Arakawa et al., 1990).
nt e

In ed
Ta tol

TM e
So O

l
ito
U typ

in
A
at

i
ur
os

rb
re
ild

Practical applications of osmolytes

W

ΔF508 As has been reviewed elsewhere (Cushman, 2001; Yancey,

2001), properties of osmolytes are becoming increasingly
Fig.·6. Specific ion conductance (Gm) of the wild-type and ΔF508 useful in molecular biology, agriculture and biotechnology.
mutant cystic fibrosis transmembrane-conductance regulator (CFTR)
For example, Welch and colleagues have suggested that
in forskolin-stimulated transfected mouse fibroblast cells. The
untreated mutant cells have very low conductance compared with the
stabilizing osmolytes, which they call ‘chemical chaperones’,
wild type, while cells exposed to 300·mmol·l–1 of the indicated might rescue misfolded proteins in human diseases (Welch and
osmolytes had conductance rates as great or greater than the wild type Brown, 1996). Recently, we found that addition of various
(modified from Howard et al., 2003). *Significant difference mammalian osmolytes and TMAO can indeed restore function
compared to wild type. of one form of cystic fibrosis mutant protein (Fig.·6; Howard
et al., 2003). TMAO can also prevent misfolding of prion
proteins (Bennion et al., 2004). Crop plants are also being
illustrates this: if high TMAO is beneficial to deep-sea animals, engineered to accumulate a variety of so-called compatible
why isn’t it used more extensively by (non-ureosmotic) solutes for stress conditions (Cushman, 2001). Some of these
shallow animals? As another example, the mammalian renal solutes, especially taurine and sometimes inositol and glycine
medulla appears to regulate one of its methylamine osmolytes, betaine, are major ingredients of a number of energy or sports
GPC, to maintain a constant ratio to urea, rather than for drinks. However, caution is warranted in all these usages. If,
osmotic stress alone (Peterson et al., 1992). Again, if this in fact, many of these solutes have unique metabolic reactions
methylamine is a simple compatible solute, why not use it at and/or stabilizing properties, then they could cause harmful
high levels under all water-stress conditions? Perhaps the costs reactions or protein aggregates if used where their non-osmotic
of synthesis and retention create a tradeoff in the use of some properties are not needed.
osmolytes, but perhaps the compounds are harmful in the
absence of a perturbant.
(2) Methylamines at high concentrations can be detrimental Unanswered questions and conclusions
to protein function in the absence of a perturbant, at least in Much remains to be learned about the evolution of osmolyte
vitro. For example, TMAO inhibits some enzymes (Yancey et systems. Water–solute–protein interactions are still
al., 1982), and it can enhance formation of non-functional incompletely understood, for example. Regardless of the
protein aggregates (Devlin et al., 2001), including β-amyloid mechanisms of osmolyte function, it seems clear that their
formation (Yancey, 2001). universal properties should speed up adaptation to water stress
(3) Using cultured renal cells, we found that adding high conditions relative to the alternative, i.e. evolving
urea or glycine betaine alone at high concentrations to the macromolecular structures to preserve function in a
medium greatly reduced cell growth. However, adding both concentrated ion solution (Yancey et al., 1982). However, it
partly or fully together restored normal growth (Yancey and should be noted that the possible co-evolution of protein
Burg, 1990). structures with cellular osmolyte compositions has received
(4) In yeast, high trehalose concentration, induced by little study. Also, non-osmotic protective roles for osmolytes
temperature stress, protects enzymes at high temperatures, but have been well documented in some instances; but in other
rather strikingly inhibits them at normal temperatures. Yeasts cases the selective rationales for osmolyte patterns and types
that cannot eliminate their trehalose suffer when they return to in many organisms remain speculative or are not known.
normal temperatures. This has been termed ‘the yin and yang Further studies on unique properties of osmolytes need to be
of trehalose’ (Singer and Lindquist, 1998). This phrase nicely conducted.
captures the important conclusion arising from these In conclusion, a variety of other stresses (oxidative, protein-
observations, namely that some ‘compatible’ solutes may be perturbing, etc.) can co-occur with water stress, and many
harmful in the absence of a perturbant. osmolytes probably have unique properties that protect cells

THE JOURNAL OF EXPERIMENTAL BIOLOGY

 Compatible and counteracting osmolytes 2829
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Gluick, T. C. and Yadav, S. (2003). Trimethylamine N-oxide stabilises RNA
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