Glycogen regulates cellular proliferation in the context of aging, tumorigenesis, and hepatic regeneration

Abstract

Glycogen is a branched polysaccharide that serves as an intracellular store of glucose that can be mobilized to maintain homeostasis or to fuel cellular processes. Glycogen is synthesized by glycogen synthase, which is present in two different isoforms: liver glycogen synthase (LGS) is mainly expressed in the liver, while muscle glycogen synthase (MGS) is expressed everywhere else. Recent studies are starting to uncover new roles for glycogen besides just being a glucose depot. Importantly, glycogen metabolism has been implicated in the normal aging process in species ranging from Saccharomyces cerevisiae to humans. However, the implication of glycogen in senescence, a hallmark of aging, is less well understood. Senescence is a tumor suppressive response that results in an irreversible cell cycle arrest, and can be induced by a variety of cellular stressors. Glycogen has previously been shown to accumulate in the context of senescence, however the significance of this remains unclear. Taking this into account, we aim to elucidate the role of glycogen in proliferation specifically in the context of aging, tumorigenesis and hepatic regeneration.

To achieve this, we used two knock-out (KO) models where glycogen synthesis is disrupted: 1) mouse embryonic fibroblasts (MEFs) isolated from MGS KO embryos and, 2) LGS KO mice which lack hepatic glycogen. These two models allowed us to test what occurs in proliferative contexts when glycogen is absent both in vitro and in vivo.

First, we subjected WT MEFs to replicative senescence (RS), where MEFs were passaged every time the plate reached confluence, until cells entered a growth arrested state (senescence). We determined that glycogen accumulates, and further observed that MGS is activated during senescence.

To test whether the presence of glycogen affects the senescent response, we subjected glycogen-free MEFs to RS, and observed that they exhibit various markers of senescence: flattened cell morphology, positive senescence- associated B-gal staining, and an increased expression of senescence protein markers. Interestingly, MGS KO MEF overcome the senescent phase faster than WTs by becoming immortalized at an earlier time point. After transcriptomic analysis, we determined that MGS WT MEFs show an enrichment of the TGF-b pathway during senescence, while MGS KO MEFs are depleted. Furthermore, we found that the transcriptional signatures of senescent MGS KO MEFs are transcriptionally more similar to actively proliferating cells than to senescent WT MEFs. These results suggest that in the absence of glycogen, MEFs enter a pseudo-senescent state allowing them to immortalize faster than wild type counterparts.

Once immortalized, MGS KO MEFs continue exhibiting a proliferative advantage over wild types, in addition to increased migratory and clonogenic capacities. Furthermore, we tested the metabolic consequence of removing glycogen by performing live cell analysis, which reveals a metabolic shift towards glycolysis. These results bring the Warburg effect to mind, suggesting that glycogen-free cells reprogram their metabolism to satisfy their energetic needs.

Lastly, we question whether glycogen is important in proliferative contexts in vivo. For this, we used LGS KO mice and subjected them to two hepatic proliferative challenges: hepatic regeneration through partial hepatectomy (Phx) and hepatocellular carcinoma (HCC) induction.

After Phx, we showed that there is a higher proportion of LGS KO hepatocytes in the S phase of the cell cycle which suggests that glycogen is an important regulator of hepatocytic proliferation.

In the pathological proliferative context of N-nitrosodiethylamine (DEN), we showed that LGS KO mice present higher mortality and tumor burden than controls. Therefore, our results suggest that glycogen is playing a protective role in animals exposed to DEN.

In conclusion, our results indicate that glycogen is an important modulator in the context of cellular proliferation and aging, and positions this polysaccharide as a novel target for therapeutic interventions to combat aging and possibly, cancer.