Molecular consequences of cellular UDP-glucose deficiency

A Chinese hamster fibroblast mutant cell line, deficient in UDP-glucose (UDPG), denoted as Qc, and its counterpart cell line with normalized levels of UDPG (G3), were used to determine some molecular consequences of chronic UDPG deficiency. The UDPG deficient cells lacked glycogen, had elevated rates of glucose uptake and a decreased capacity to synthesize glycogen after 120h of culture. In Qc cells glycogen synthase (GS), which synthesizes glycogen using UDPG as the glucosyl donor, did not bind UDPG. This was associated with almost complete inactivation of the enzyme (= 5% of G3 activity). The amount of GS protein in Qc cells was 50% of the amount in G3 cells and this decrease was associated with lower GS mRNA levels in the UDPG deficient cells.

Comparable results for GS were obtained when G3 cells were cultured in glucose free medium. Thus, in addition to serving as a glucosyl donor, UDPG can regulate GS activity by non-substrate dependent mechanisms. Qc cells also exhibited increased levels of seven stress proteins: one mitochondrial chaperone (GRP75) and six chaperones of the endoplasmic reticulum (GRP58, ERP72, GRP78, GRP94, GRP170 and calreticulin). These proteins are also upregulated in cells cultured under hypoxia or glucose starvation. Since upregulation of the ER chaperones is a feature of ER stress, two of the major ER stress responses, the Unfolded Protein Response (UPR) and the ER Overload Response (EOR) were analysed. In mammalian cells, triggering the UPR leads to activation of the transcription factors ATF6 and XBP-1/TREB5, thereby inducing expression of ER chaperones and the transcription factor CHOP/GADD 153. However, a decrease in the cellular levels of UDPG did not induce the expression of CHOP/GADD 153, nor the activation of ATF6 or XBP-1/TREB5. Thus, the upregulation of the selective set of ER chaperones in Qc cells is elicited via a signalling pathway that is molecularly distinct from the classical UPR.

The classical EOR is characterized by accumulation of membrane proteins in the ER. This leads to a Ca" and Reactive Oxygen Intermediates (ROls) mediated nuclear factor kappaB (NFkappaB) activation. To investigate whether Qc cells trigger an EOR, constitutive NF-kappaB activation in these cells was investigated. Qc cells exhibited increased levels of Ca2+, ROIs and a higher NF-kappaB activation than G3 cells. Treatment of Qc cells with Ca2+ antagonists or inhibitors of ROls abrogated the NF-kappaB activation. Hence, NF-kappaB is activated in Qc cells via Ca2+ and ROIs. Moreover, NFkappaB activation in the Qc cells occurred via the phosphoinositide 3-kinase (PI3K) /Akt pathway. This activation might function as a survival strategy for Qc cells since inhibition of NF-kappaB activity resulted in apoptosis of these cells.

In conclusion, this work revealed the role of UDPG as a non-substrate-dependent regulator of glycogen synthase activity. Furthermore, it provides evidence supporting that a cellular UDPG deficiency induces the upregulation of a set of stress proteins and triggers a non classical EOR when is important for cell survival.

List of scientific papers

I. Higuita JC, Alape-Giron A, Thelestam M, Katz A (2003). A point mutation in the UDP-glucose pyrophosphorylase gene results in decreases of UDP-glucose and inactivation of glycogen synthase. Biochem J. 370(Pt 3): 995-1001.
https://pubmed.ncbi.nlm.nih.gov/12460121

II. Higuita JC, Thelestam M, Katz A (2004). Glucose starvation results in UDP-glucose deficiency and inactivation of glycogen synthase. Arch Biochem Biophysics. 425(2): 242-8.
https://pubmed.ncbi.nlm.nih.gov/15111133

III. Flores-Diaz M, Higuita JC, Florin I, Okada T, Pollesello P, Bergman T, Thelestam M, Mori K, Alape-Giron A (2004). A Cellular UDP-glucose Deficiency Causes Overexpression of Glucose/Oxygen-regulated Proteins Independent of the Endoplasmic Reticulum Stress Elements. J Biol Chem. 279(21): 21724-31. Epub 2004 Mar 12
https://pubmed.ncbi.nlm.nih.gov/15020602

IV. Higuita JC, Frisan T, Thelestam M (2004). Constitutive activation of NF-kappaB - a strategy for survival in UDP-glucose deficient cells. [Manuscript]