Taken together, the data suggest that bacteria suffer damage from glycation in a manner similar to that of eukaryotic cells. As shown here, some amount of glycation was detectable in cells growing under standard laboratory conditions in batch culture (Fig. ), most likely due to the action of reactive electrophiles that are generated as part of normal metabolism. When the culture was supplemented with glucose, the amount of glycation increased significantly (Fig. ). Although E. coli
has evolved various protective mechanisms against toxic electrophiles (12
), we speculate that glycation may nevertheless contribute to cell death and negatively affect the long-term survival of these and other bacterial populations during long-term stationary phase (14
Carnosine is a naturally occurring dipeptide with the ability to act as an antioxidant and/or antiglycation agent in cells (19
). Synthesized endogenously by carnosine-anserine synthetase in many eukaryotes, it is found in long-lived mammalian tissues (such as neurons and muscle tissue) at concentrations up to 20 mM (37
). It has been shown that protein cross-links induced in vitro
by methyglyoxal are eliminated in the presence of carnosine (24
). Carnosine may react directly with MG and sequester it; its amino group and imidazole ring may bind to reactive dicarbonyl groups (3
), although the structure of MG-carnosine adducts has yet to be determined (37
). Alternatively, Hipkiss and Brownson (22
) have proposed that proteins may become “carnosinylated” at carbonyl groups and that this may protect them from degradation and/or cross-linking. Our findings provide further evidence for carnosine's ability to suppress the formation of AGEs in vivo
(Fig. ). Carnosine protected cells from lethal concentrations of glucose (Fig. ) and methylglyoxal (Fig. ). Furthermore, it protected E. coli
even when added after 3 days of exposure to toxic concentrations of glucose (Fig. ), and more carnosine was needed to protect gloA
mutants, which are less capable of degrading intracellular methylglyoxal, than to protect wild-type cells (Fig. ). The slight increase in sensitivity of gloB
single mutants was likely due to the fact that the functional gloA
gene product can remove MG, resulting in accumulation of a less toxic intermediate (29
). A proposed model of protection against AGE formation is shown in Fig. .
FIG. 6. Proposed model of carnosine protection against glycation. Carnosine most likely interacts with reactive carbonyl intermediate compounds (e.g., glyoxal and methylglyoxal) to inhibit the formation of advanced glycated end products (e.g., CML and carboxyethyl (more ...)
Other chemical agents with possible antiglycating ability were investigated using our bacterial model system. Folic acid (folate/vitamin B9
) protected E. coli
from toxic concentrations of both glucose and methylglyoxal (Table ). Although the exact mechanism of action for this effect has not been determined, it has been shown that folate can modulate cellular glutathione levels, which may act as a defense against oxidant and alkylating agent damage, since glutathione is an essential component of the glyoxylase I/II system(s) used to detoxify methylglyoxal (13
). In one study, rats fed a high-folate diet were found to have greater hepatic glutathione concentrations than were those on standard or folate-deficient diets (5
). Other investigators found that methylglyoxal-resistant E. coli
mutants have increased activity in their glutathione-forming enzyme system (34
). Therefore, we postulate that the protective ability of folate under glycation-prone conditions may be due to its influence on glutathione. Further studies using glyoxylase mutants should help to clarify the role(s) of folate and/or glutathione in protection against glycating agents.
Aminoguanidine (AG) has previously been shown to reduce the severity of AGE-influenced pathologies in mammalian systems (27
), probably by reaction with glycation intermediates and/or toxic dicarbonyl compounds via its free amino groups (4
). We demonstrated that AG can also display protective effects in a bacterial system, allowing E. coli
to survive when grown in medium containing lethal concentrations of glucose or methylglyoxal (Table ). It may be useful to determine if AG acts synergistically with carnosine or any of the other antiglycation agents previously discussed.
We have developed a novel prokaryotic model system to study the effects of compounds that may protect against glycation. This study focused on carnosine, an agent that may be one of several compounds that Gallant and colleagues (18
) refer to as “geroprotectors,” substances that can prevent some of the deterioration associated with the aging process. Additionally, folate and aminoguanidine displayed protective effects in the presence of agents known to induce glycation (Table ). Other chemicals, such as grape seed extract and aspirin, may also have protective benefits (E. D. Pepper and S. E. Finkel, unpublished observation). Further studies using this bioassay may shed light on the biological relevance of these compounds in long-term survival of bacteria and clinically relevant eukaryotic systems.