Drugs are chemical substances of natural or synthetic origin used for diagnosis, prevention, treatment, and control of disorders. Most drugs are specific to control and cure diseases and, although their curing effects outweigh their side effects, most are associated with side effects. One example is aspirin, which is a salicylate drug, prescribed as an analgesic, antipyretic, and anti-inflammatory medication; it has been reported to cause Reye syndrome (sudden brain damage and liver problems) when given to children suffering from flu and chicken pox (Belay et al., 1997). The other side effects of aspirin are gastrointestinal ulcers and stomach bleeding (Sorensen et al., 2000).
C. elegans has a well conserved IGF-1/insulin-like signaling pathways. daf-2 encodes an insulin-like receptor in C. elegans, which is a orthologue of the mammalian insulin/IGF receptor family (Kimura et al. 1997). In daf-2(e1370) animals, the decrease of insulin-like signaling results in increased dauer formation. Quantification of daf-2(e1370) dauer formation in C. elegans can be used as an index to measure the level of IGF-1/insulin signaling: enhancement of IGF-1 signaling causes increased dauer formation while decrease in IGF-1 signaling causes cessation in number of dauers formed.
We examined the effect of the drugs silymarin, Godex, aspirin, and pravastatin in modulating IGF-1/insulin signaling. Effects of these drugs on IGF-1/insulin signaling have not been explored. Silymarin is a lipophilic flavonolignan (flavanone derivative) extracted from the seeds and fruits of Silybum marianum (milk thistle). Godex is composed of carnitine orotate, which is the major chemical constituent, it also contains seventeen amino acids as well as vitamin B2, B6, and B12. Both silymarin and Godex are known hepatoprotectant drugs and are mainly used in the clinical treatment of liver cirrhosis, acute liver intoxication, and chronic hepatitis (Al Anati et al., 2009; Park et al., 2001). Aspirin is a salicylate drug used as an analgesic, antipyretic, and anti-inflammatory medication. Pravastatin, a statin, is issued for lowering cholesterol and preventing cardiovascular disease, and is generally well tolerated (Pfeffer et al., 2002).
To test the effect of the above mentioned drugs on IGF-1/insulin signaling, we used daf-2(e1370) animals. Synchronized eggs of daf-2(e1370) were seeded on drug treated (100 µM) and control plates at 22ºC. After 4 days of incubation the number of dauers present on the respective plates were counted manually and further confirmation of dauer was done by SDS treatment. The mean percentage ± S.D of dauer formation on control plates was 41.8±6.1 whereas the drug treated plates showed respective percentage of 1.8±0.7 (p<0.0001) for silymarin, 4.9±1.2 (p<0.0001) for aspirin, and 23.6±2. (p<0.0001) for Godex. All the three drugs showed a statistically significant effect on suppressing dauer formation. The mean percentage of dauer formation on pravastatin treated plate was 78.8 ±11.9, which was also statistically significant (p<0.0001) as compared to control (41.8±6.1).
Our results show that both the hepatoprotective drugs silymarin and Godex, as well as the well-known analgesic drug aspirin, suppress the IGF-1/insulin signaling pathway, while the cholesterol-lowering drug pravastatin enhances this pathway. Our results thus suggest that daf-2(e1370) animals can be used to examine the effect of pharmacological drugs on IGF-1/insulin signaling. This strategy might be useful to examine the effect of drugs in relation to IGF-1 signaling and can be further extrapolated to study the effect of different drugs in relation to insulin signaling in human subjects.
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References
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Belay ED, Bresee JS, Holman RC, Khan AS, Shahriari A, and Schonberger LB. (1999). Reye’s syndrome in the United States from 1981 through 1997. N. Engl. J. Med. 340, 1377–1382.
Kimura KD, Tissenbaum HA, Liu Y, and Ruvkun G. (1997). daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942-946.
Park MS, Kang JS, Jeon JY, Baek SW, Lim GS, and Kwak MJ. (2001). Effectiveness of Godex capsule on chronic liver disease patients. J. Korean Soc. Clin. Pharmacol. Ther. 9, 151–162.
Pfeffer MA, Keech A, Sacks FM, Cobbe SM, Tonkin A, Byington RP, Davis BR, Friedman CP, and Braunwald E. (2002). Safety and tolerability of pravastatin in long term clinical trials: prospective Pravastatin Pooling (PPP) Project. Circulation 105, 2341-2346.
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Alzheimer’s disease and gender differences in C. elegans
The prevalence and severity of Alzheimer’s disease (AD) are influenced by gender such that two thirds of AD patients are women. However, the biological mechanisms underlying these sex differences are not fully understood. AD begins with slight memory loss and confusion, and eventually leads to severe mental impairment (Mc Khann et al., 1984). It has been previously shown by Guillozet et al. that the formation of amyloid plaques and neurofibrillary tangles in the brain are responsible for memory and other cognitive functions (Guillozet et al., 2003). A recent human trial of AD treatments has proven to be largely unsuccessful. A probable reason is that multiple factors such as food, social relationships, physical activity, gender, climate and stress all play a role in its pathology. One of the most important factors is gender (Viña and Lloret, 2010). Lack of biological gender model systems for AD limits our understanding of the role that gender play in AD pathogenesis. Elucidating the role of gender in an AD model is crucial to prevention, diagnosis, and treatment of AD in different sexes.
In order to examine the role of gender on the etiology of AD, a C. elegans model was used to emulate the pathological processes of AD. The worm apl-1 gene encodes two identical isoforms, orthologous to the human amyloid precursor protein (APP) involved in AD (Wentzell and Kretzschmar, 2010). Recently, a transgenic animal (CL4176) expressing Aβ42 in muscle tissues (Drake et al., 2003) has been established as a model. Interestingly, the Aβ protein expressed in C. elegans self-aggregates like Aβ1–42, and forms fibrillar structures (Link, 1995).
We used male and hermaphrodite CL4176 transgenic animals and performed a paralysis assay (Drake et al., 2003). Synchronized eggs were maintained at 16 ºC and males and hermaphrodites were sorted at late L4 stage. Sorted worms were subjected to 25 ºC to induce the transgene expression and the paralysis was scored successively at 2 hour intervals. The hermaphrodite animals paralyzed earlier in comparison to the males. The mean time duration at which 50% worms were paralyzed was 34.6 hours and 27.3 hours, for males and hermaphrodites, respectively (p< 0.001) (Figure 1A, 1B). The data shows that the male CL4176 takes significantly more time to undergo paralysis compared to the hermaphrodites.
In this paper, we provide a way to study the mechanism and treatment strategy of AD disease based on gender. The observed phenotype is consistent with human beings (Viña and Lloret, 2010). Further studies will allow us to investigate the molecular pathways involved in AD in relation to sex and significantly impact our understanding to identify novel therapeutics in the future.
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References
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Guillozet AL, Weintraub S, Mash DC, and Mesulam MM. (2003). Neurofibrillary tangles, amyloid, and memory in aging and mild cognitive impairment. Arch. Neurol. 60, 729-736.
Link, CD. (1995). Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 92, 9368–9372.
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Viña J, and Lloret A. (2010). Why women have more Alzheimer’s disease than men: gender and mitochondrial toxicity of amyloid-beta peptide. J. Alzheimers Dis. S527- 533.
Wentzell J, and Kretzschmar D. (2010). Alzheimer’s disease and tauopathy studies in flies and worms. Neurobiol. Dis. 40, 21–28.