Water efflux in response to hyperosmotic stress causes cells to shrink, protein and DNA damage, cell cycle arrest and death. Cells must therefore adapt to changes in external osmolality; one conserved mechanism is to increase intracellular osmolytes to maintain osmotic homeostasis. New research from Possik et al., (2015) has identified a role for FLCN-1, mediated by AMPK activity, in the maintenance of glycogen stores required for rapid production of the osmolyte glycerol in C.elegans.
AMPK, a key regulator of cellular energy homeostasis, is negatively regulated by FLCN. It was previously reported that flcn-1 loss in C. elegans (flcn-1(ok975) mutants) resulted in AMPK-dependent, increases in glycolysis and autophagy enhancing resistance to metabolic stresses (Possik et al., 2014). To determine if flcn-1 loss also confers resistance to hyperosmotic stress Possik et al. (2015) assessed survival on high concentration NaCl plates. The flcn-1(ok975) worms showed increased survival, reduced shrinkage and a faster recovery from paralysis in comparison to wild type worms. Re-expression of flcn-1 reversed the phenotype confirming its role in increased hyperosmotic stress resistance.
In C. elegans hyperosmotic stress triggers increased glycerol-3-phosphate dehydrogenase-1 (gpdh-1) transcription and production of glycerol from glycogen (Lamitina et al., 2004). Electron microscope imaging of flcn-1(ok975) worms identified a significant increase in the size and number of glycogen deposits in comparison to wild type worms, which were depleted after NaCl treatment (Possik et al., 2015). Glycogen synthesis and degradation are mediated by glycogen synthase (gsy-1) and glycogen phosphorylase (pygl-1) respectively. Inhibition of either enzyme strongly reduces survival following NaCl treatment supporting key roles for glycogen accumulation and breakdown in hyperosmotic stress resistance.
As repression of the C.elegans AMPK-α subunits aak-1 and aak-2 abolished the increased survival, enhanced hyperosmotic stress resistance in flcn-1(ok975) worms must be AMPK-dependent. Loss of aak-1 and aak-2 also reduced glycogen accumulation in both wild type flcn-1 and flcn-1(ok975) worms. This suggests that the increased glycogen accumulation seen in flcn-1(ok975) worms is a result of the chronic AMPK activation due to FLCN-1 loss, but also that AMPK signalling plays a role in normal maintenance of glycogen stores.
Degradation of glycogen produces glucose-6-phosphate, a metabolite that can be converted to glycerol by gpdh-1 or gpdh-2. The flcn-1(ok975) worms showed a two-fold increase in gpdh-1 expression under non-stressed conditions and a higher basal level of glycerol. Additionally a marked increase in both gpdh-1 and gpdh-2 expression was reported after two hours treatment with NaCl. Simultaneous inhibition of gpdh-1 and gpdh-2 was required to strongly reduce the increase in survival in flcn-1(ok975) worms. These results support a key role for glycogen store metabolism by gpdh enzymes in resistance to hyperosmotic stress.
FLCN loss is associated with the development of renal tumours in BHD patients and model systems. Analysis of Flcn-null mouse kidneys and patient tumour samples showed increased glycogen accumulation in comparison to healthy tissue. Glycogen biosynthesis and degradation gene expression levels in human non-BHD renal cell carcinoma samples were also upregulated and expression of 46% of these genes was negatively correlated with FLCN expression.
The breakdown of glycogen stores can also produce the metabolites required for glycolysis, a process that protects cells from multiple metabolic stresses. Therefore increased glycogen stores could play dual roles in tumourigenesis as a source of energy and oncolytes: cells able to survive hyperosmotic or metabolic stresses can accumulate more DNA damage thereby increasing the risk of neoplastic transformation. Greater understanding of how tumour cells resist normal cell death programmes can help in the development of new targeted treatments.
- Lamitina ST, Morrison R, Moeckel GW, Strange K (2004). Adaptation of the nematode Caenorhabditis elegans to extreme osmotic stress. Am J Physiol Cell Physiol. Apr;286(4):C785-91. PMID: 14644776.
- Possik E, Jalali Z, Nouët Y, Yan M, Gingras MC, Schmeisser K, Panaite L, Dupuy F, Kharitidi D, Chotard L, Jones RG, Hall DH, Pause A (2014). Folliculin regulates ampk-dependent autophagy and metabolic stress survival. PLoS Genet. Apr 24;10(4):e1004273. PMID: 24763318.
- Possik E, Ajisebutu A, Manteghi S, Gingras MC, Vijayaraghavan T, Flamand M, Coull B, Schmeisser K, Duchaine T, van Steensel M, Hall DH, & Pause A (2015). FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores. PLoS genetics, 11 (10) PMID: 26439621.