Participation of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase Isoform 3 and Pyruvate Dehydrogenase Complex in the Regulation of Lactate Production By FSH and bFGF in Rat Sertoli Cells

Presentation Number: THR-125
Date of Presentation: March 5th, 2015

María Fernanda Riera*, Mariana Regueira, María Noel Galardo, Eliana Herminia Pellizzari, Selva Beatriz Cigorraga and Silvina Beatriz Meroni
Centro de Investigaciones Endocrinológicas, Dr César Bergadá-CONICET-División de Endocrinología-FEI, Ciudad Autónoma de Buenos Aires, Argentina

Abstract

Sertoli cells (SC) actively metabolize glucose and the majority of it is converted to lactate. Spermatocytes and spermatids are unable to use glucose and they do prefer lactate as an energy source. In this context, it is widely accepted that one of the most important SC nurse functions is to provide lactate for germ cells and that the mechanisms that regulate its production may be relevant to the maintenance of spermatogenesis.

The availability of pyruvate, which is converted into lactate, might be regulated by FSH and bFGF in SC to ensure lactate supply to germ cells. The aim of this study was to analyze two possible mechanisms which may be involved in pyruvate availability. Firstly, regulation of the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase isoform 3 (PFKFB3) responsible for fructose-2-6-biphosphate (Fru2,6P2) production. Fru2,6P2 is an allosteric regulator of phosphofructokinase 1, the rate limiting step in glycolysis, which increases glycolytic flux. Secondly, negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation, which will increase pyruvate availability as a consequence of a lower conversion of pyruvate to acetyl-CoA.

Cultures of SC obtained from 20-day-old rats were incubated for 48 h without (basal) or with FSH (100 ng/ml) or bFGF (30 ng/ml). Lactate levels were determined in conditioned media. Cell extracts were utilized to evaluate phosphorylated PDC (P-PDC) levels by Western blot and mRNA levels by RQPCR. Results are expressed as mean±SD (n=3).

Analysis of PFKFB3 expression showed that FSH increased PFKFB3 mRNA levels (2.1±0.32*; *p<0.05 vs. basal) while bFGF did not modify it. The physiological relevance of this mechanism was revealed by the fact that FSH-stimulated lactate production was inhibited in the presence of the specific PFKFB3 inhibitor 3-PO (10μM) (basal: 12.3±0.7; FSH: 17.6±1.8**; FSH+3-PO: 12.7±1.1# μg/μgDNA, **p<0.01 vs. basal; #p<0.01 vs. FSH).

As for the regulation of PDC, expression of pyruvate dehydrogenase kinases (PDKs) isoforms and levels of P-PDC were analyzed. We observed that FSH increased PDK3 and decreased PDK4 mRNA levels (PDK3: 1.8±0.4*; PDK4: 0.5±0.1*; *p<0.05 vs. basal) and that these variations were not accompanied by changes in P-PDC levels. On the other hand, bFGF increased the expression of all isoforms (PDK1: 1.9±0.2*; PDK2:1.7±0.4*; PDK3: 1.7±0.3*; PDK4: 1.6±0.2*; *p<0.05 vs. basal) and increased P-PDC levels. The physiological relevance of PDC regulation was evidenced by the fact that bFGF-stimulated lactate production was inhibited in the presence of the PDK inhibitor dichloroacetate (DCA, 10μM) (basal: 7.5±1.5; bFGF: 14.3±1.7*; bFGF+DCA: 10.3±0.7# μg/μgDNA, *p<0.05 vs. basal; #p<0.5 vs. bFGF). 

Altogether, these results suggest that regulation of pyruvate availability may be a mechanism involved in FSH- and bFGF-regulation of lactate production in SC.

 

Nothing to Disclose: MFR, MR, MNG, EHP, SBC, SBM