FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores

Abstract

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

Fig 1. Loss of flcn-1 confers resistance to hyperosmotic stress.

(A-C, F) Percent survival (A, B, F) and mean survival (C) of indicated worm strains exposed to 400mM and 500mM NaCl. (D) Percent recovery from paralysis of wt and flcn-1(ok975) animals after 2 hours from exposure to 400mM NaCl. Data represent mean ± SEM, n≥ 3. (E) Representative images of wt and flcn-1(ok975) animals treated with 400mM NaCl for 48 hours.

Fig 2. Loss of flcn-1 increases glycogen content, which mediates resistance to hyperosmotic stress.

(A) Representative electron micrographs from longitudinal sections of the hypodermis in indicated nematodes strains exposed or not to 400mM NaCl for 16 hours. Arrows represent glycogen stores. Scale bars: 2μm. (B, C) Iodine staining (B) and quantification of staining intensities (C) of indicated worm strains treated or not with 400mM NaCl for 16 hours. Data represent mean ± SEM, n≥ 3. (D, E) Percent survival to 400mM NaCl of indicated worm strains treated with indicated RNAi. (F) Relative mRNA levels of indicated target genes in indicated strains with or without 400mM NaCl treatment for 2 hours. Data represent the mean ± SEM, n≥ 3.

Fig 3. The increased survival to hyperosmotic stress and the accumulation of glycogen in flcn-1 mutant worms require AMPK.

(A-D) Percent survival of indicated worm strains exposed to 400mM NaCl. (A) aak-2(ok524), (B) aak-2(gt33), (C) aak-1(tm1944), (D) aak-1(tm1944); aak-2(ok524). (E, F) Iodine staining (E) and quantification of staining intensities (F) of indicated worm strains. Scale bar:100μm. Data represent the mean ± SEM of at least 3 independent experiments.

Fig 4. Glycogen degradation heightens glycerol levels and protects animals from hyperosmotic stress.

(A) Representative scheme of glycogen metabolism and osmolyte production in worms. (B-D) Relative mRNA levels of gpdh-1 and gpdh-2 (B, C) and glycerol content (D) in wt and flcn-1(ok975) L4/young adult animals treated with or without 400mM NaCl for 2 hours. Data represent mean ± SEM, n ≥3. (E) Percent survival of indicated worm strains exposed to 400mM NaCl.

Fig 5. The FLCN-dependent glycogen accumulation is conserved from C. elegans to humans.

(A-B) PAS staining of kidney sections from wt and Flcn kidney-specific KO mice (A) and human BHD kidney tumor in comparison with an adjacent region from the same individual (B). Scale bars:100μm. (C) Table indicating the upregulated glycogen metabolism genes in kidney tumors (KIRC, KIRP, and KICH) as compared to normal kidney. The sign (+) indicates genes that are upregulated in these tumors. The values are indicated in S5 Table. (D) Heat map indicating correlation of expression between glycogen metabolism genes and FLCN in KIRC, KIRP, and KICH tumors. Green and red colors indicate genes that are negatively or positively correlated with FLCN expression, respectively.

Fig 6. Graphical representation of FLCN-1/AMPK hyperosmotic stress resistance pathway.

Loss of flcn-1 chronically activates AMPK and leads to glycogen accumulation under normal conditions. Upon exposure to hyperosmotic stress, glycogen is rapidly degraded leading to the production of glycerol and animal survival.