c. elegans: therapeutic targets in metabolic syndrome. · abstract. environmental factors that...

Abstract

Environmental factors that contribute to metabolicsyndrome and type 2 diabetes include increased caloricintake with fat accumulation in muscle and liver. Asmitochondrial dysfunction is implicated in thesepathologies, we examined the effects of chronic stearicand oleic fatty acid supplemented diets on mitochondrialfunction and stress pathways in the nematode C.elegans. Both the bacteria on which the nematodes fedand the nematodes accumulated oleic acid whencultured on oleic-supplemented media. However, whencultured on stearic acid, only bacteria accumulated thesaturated fat. To investigate mitochondrial and cellularstress, dietary effects on stress proteins HSP-6, HSP-60and HSP-4 were measured using transcriptional greenfluorescent protein constructs. Mitochondrial hsp-6::gfplevels were elevated on stearic acid diets, but not onoleic acid. Nematode respiration was unchanged andATP levels were increased on both diets. In addition, diet-induced oxidative stress was not observed. Chronicdietary saturated fat induced a mitochondrial stresspathway without bioenergetic dysfunction in C. elegans,suggesting that the HSP-6 unfolded protein responsemitigated stress associated with exposure to andmetabolism of saturated fat. These data argue forinvestigation of targets in mitochondrial stress pathwaysfor treating syndromes associated with exposure tosaturated fat.

IntroductionHuman populations have been shifting towards a

more sedentary lifestyle accompanied with an increasein nutrient rich diets. These trends have led toincreased obesity which is associated with the onset ofmetabolic diseases such as type 2 diabetes (T2D).1,2 Ithas been suggested that mitochondrial dysfunctionmay contribute to the onset of T2D.3 In this study, wetested the hypothesis that increased dietary fat leads tomitochondrial dysfunction and oxidative stress using C.elegans as a model organism. C. elegans is a small (1mm), semitransparent nematode whose genome iscompletely sequenced.4 The worms are easily culturedand have a relatively short lifespan (2-3 weeks)allowing for observations of dietary effects from birth(hatching) to adulthood. In addition, a mutant strainwas available, nnt-1, that is susceptible to oxidativestress due to a deleted gene that has been linked withglucose intolerance and T2D.5-7

We tested the effects of saturated and unsat-urated fatty acids, stearic and oleic acid, which areboth 18-carbon fatty acids found in the human diet. C.elegans strains were cultured on an OP50 E. colibacterial lawn grown on nematode growth media(NGM) in agar. Stearic (18:0) or oleic (18:1Δ9) fattyacids were mixed with a detergent (tergitol or igepal)and added to NGM to a final concentration of 0.25 to1.00 mM. OP50 were grown on fat supplementedmedia for two days, then nematode eggs weretransferred to the bacterial lawn, grown for 3 days, andthe 1-day adult animals were collected for analyses.

Conclusions

Dose-dependent increases in stearic and oleic acid wereobserved in the bacterial food of C. elegans when grownon fat-supplemented media (Fig 1A). Similar fatty acidcomposition was observed in wild-type N2 and nnt-1mutant animals on each diet (Fig 1B and 1C).

Although stearic acid composition was increased in thebacterial food, it was not increased in either worm strain,suggesting that worms did not store but metabolizedstearic acid. Oleic acid composition increased in wormsin a dose-dependent manner, however, indicating thatwas stored.

Worms raised on saturated fat had increased expression of the mitochondrial hsp-6::gfp but not the ER chaperone (Fig 2), indicating that a mitochondrial unfolded protein response, UPRmt, was induced in response to saturated fatty acid metabolism. Increased saturated fat in the mitochondrial matrix may disrupt hydrophobic peptide folding, possibly leading to unequal subunit availability of protein complexes that assemble in the mitochondrial matrix, leading to lipid-induced stress.

Stress responses to chronic dietary fat did not appear toinvolve oxidative stress as supplemented fat did notelevate H2O2 (Fig 3) or lipid peroxidation (Fig 4) in worms.

Chronic fatty acid dietary stress upregulated an HSP-6UPRmt in C. elegans without adversely effecting lifespan(data not shown) or mitochondrial indices of respiration (Fig5) and ATP levels (Fig 6), suggesting that the UPRmt

mitigated any dietary stress.

These data argue for investigation of targets in mito-chondrial stress pathways to treat syndromes associatedwith chronic exposure to saturated fat.

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common, serious and costly. Ogden, C. et al., Prevalence of Obesity in the United States, 2009-2010. NCHS Data Brief No. 82, January 2012. Web. http://www.cdc.gov/obesity/data/adult.html.

2) Steyn, N. P., J. Mann, P. H. Bennett, N. Temple, P. Zimmet et al. (2004) Diet, nutrition and the prevention of type 2 diabetes. Public Health Nutr. 7: 147-165.

3) Lowell, B; Shulman, G. (2005). Mitochondrial dysfunction and type 2 diabetes. Science 307: 384-387.

4) Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics. 77: 71–94. 5) Arkblad et al. (2005) A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide

transhydrogenase displays increased sensitivity to oxidative stress. Free Radic Biol Med 38: 1518-1525.

6) Freeman, H., Shimomura, K., Cox, R., Ashcroft, F. (2006). Nicotinamide nucleotide transhydrogenase: a link between insulin secretion, glucose metabolism and oxidative stress. Biochem Soc Trans 34: 806-810.

7) Freeman, H. C., A. Hugill, N. T. Dear, F. M. Ashcroft, and R. D. Cox. (2006) Deletion of nicotinamide nucleotide transhydrogenase: a new quantitative trait locus accounting for glucose intolerance in C57BL/6J mice. Diabetes 55: 2153-2156.

Carin Thomas, Maya Magana, John Carter, Jennifer Watts‡ and Lucinda Carnell Departments of Chemistry# and Biological Sciences†, Central Washington University, Ellensburg, WA, and School of Molecular Biosciences‡, Washington State University, Pullman WA, USA

Chronic dietary saturated fat induces a mitochondrial unfolded protein response in C. elegans: therapeutic targets in metabolic syndrome.

AcknowledgmentsThis work was supported by the NIDDK (NIH 1R15DK089443-01), the C. elegans Genetic Center (Minneapolis, MN) and theDepartments of Biological Sciences and Chemistry at CentralWashington University.

Results

Respiration

ATP

Heat Shock Proteins

Fatty Acid Composition