Glucose regulates protein catabolism in ras-transformed fibroblasts through a lysosomal-dependent proteolytic pathway.

Transformed cells are exposed to heterogeneous microenvironments, including low D-glucose (Glc) concentrations inside tumours. The regulation of protein turnover is commonly impaired in many types of transformed cells, but the role of Glc in this regulation is unknown. In the present study we demonstrate that Glc controls protein turnover in ras-transformed fibroblasts (KBALB). The regulation by Glc of protein breakdown was correlated with modifications in the levels of lysosomal cathepsins B, L and D, while autophagic sequestration and non-lysosomal proteolytic systems (m- and mu-calpains and the zeta-subunit of the proteasome) remained unaffected. Lactacystin, a selective inhibitor of the proteasome, depressed proteolysis, but did not prevent its regulation by Glc. The sole inhibition of the cysteine endopeptidases (cathepsins B and L, and calpains) by E-64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] was also not sufficient to alter the effect of Glc on proteolysis. The Glc-dependent increase in proteolysis was, however, prevented after optimal inhibition of lysosomal cysteine and aspartic endopeptidases by methylamine. We conclude that, in transformed cells, Glc plays a critical role in the regulation of protein turnover and that the lysosomal proteolytic capacity is mainly responsible for the control of intracellular proteolysis by Glc.

Glucose controls cathepsin expression in Ras-transformed fibroblasts.

Overexpression and altered trafficking of cathepsins have been associated with the malignant properties of tumors and transformed cells. A characteristic phenotype of transformed cells is also a profound deviation in their metabolism (aerobic glycolysis, glutaminolysis) which enables them to adapt to extreme nutritional conditions. However, whether the altered metabolism may change the expression of proteinases involved in malignancy has not been determined. Herein we present evidences in Kirsten-virus-transformed 3T3 fibroblasts (KBALB) that D-glucose selectively increases active forms of cathepsins L, B, and D, without altering other lysosomal nonproteolytic hydrolases (beta-D-glucosaminidase, acid phosphatase, beta-D-glucuronidase, and beta-D-galactosidase). D-Glucose did not modify mRNA levels for cathepsin B or L and did not affect secretion of pro-cathepsin L. However, D-glucose enhanced strongly the amount of the mature forms of cathepsins B and L, without altering their preferential distribution to light endosomal fractions. Induction by d-glucose of intracellular mature cathepsins B and L required a high growth density of KBALB cells and was reproduced in BALB/3T3 fibroblasts stably transfected with a constitutively activated form of Ras. d-Glucose induction of active cathepsins however was not observed in nontransformed BALB/3T3. D-Mannose, in contrast to nonmetabolized sugars (D-galactose, or L-glucose), caused a similar increase in lysosomal cathepsin activities in dense KBALB cells. The D-glucose analogue, 3-O-methyl-D-glucose, which is transported but not further metabolized, did not reproduce the d-glucose effects. Our findings indicate that, dependent on the nutrient supply and as a consequence of their altered metabolism, transformed cells may modulate the production of active proteinases implicated in malignant progression.