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Metabolic Coupling Determines the Activity: Comparison of 11β-Hydroxysteroid Dehydrogenase 1 and Its Coupling between Liver Parenchymal Cells and Testicular Leydig Cells.

Li X, Hu G, Li X, Wang YY, Hu YY, Zhou H, Latif SA, Morris DJ, Chu Y, Zheng Z, Ge RS - PLoS ONE (2015)

Bottom Line: S3483, a G6P transporter inhibitor, reversed the G6P-mediated increases of 11β-HSD1 reductase activity.The depletion of Leydig cells eliminated Hsd11b1 (encoding 11β-HSD1) expression but did not affect the expression of H6pd (encoding H6PDH) and Slc37a4 (encoding G6P transporter).In conclusion, the availability of H6PDH determines the different direction of 11β-HSD1 in liver and Leydig cells.

View Article: PubMed Central - PubMed

Affiliation: The Second Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, ZJ 325000, PR China.

ABSTRACT

Background: 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) interconverts active 11β-hydroxyl glucocorticoids and inactive 11keto forms. However, its directionality is determined by availability of NADP+/NADPH. In liver cells, 11β-HSD1 behaves as a primary reductase, while in Leydig cells it acts as a primary oxidase. However, the exact mechanism is not clear. The direction of 11β-HSD1 has been proposed to be regulated by hexose-6-phosphate dehydrogenase (H6PDH), which catalyzes glucose-6-phosphate (G6P) to generate NADPH that drives 11β-HSD1 towards reduction.

Methodology: To examine the coupling between 11β-HSD1 and H6PDH, we added G6P to rat and human liver and testis or Leydig cell microsomes, and 11β-HSD1 activity was measured by radiometry.

Results and conclusions: G6P stimulated 11β-HSD1 reductase activity in rat (3 fold) or human liver (1.5 fold), but not at all in testis. S3483, a G6P transporter inhibitor, reversed the G6P-mediated increases of 11β-HSD1 reductase activity. We compared the extent to which 11β-HSD1 in rat Leydig and liver cells might be coupled to H6PDH. In order to clarify the location of H6PDH within the testis, we used the Leydig cell toxicant ethane dimethanesulfonate (EDS) to selectively deplete Leydig cells. The depletion of Leydig cells eliminated Hsd11b1 (encoding 11β-HSD1) expression but did not affect the expression of H6pd (encoding H6PDH) and Slc37a4 (encoding G6P transporter). H6pd mRNA level and H6PDH activity were barely detectable in purified rat Leydig cells. In conclusion, the availability of H6PDH determines the different direction of 11β-HSD1 in liver and Leydig cells.

No MeSH data available.


Related in: MedlinePlus

Effects of G6P transporter inhibitor S3483 on intact rat liver and Leydig cell 11β-HSD1 direction.0.015 × 106 liver or 0.025 × 106 Leydig cells were used to measure 11β-HSD1 oxidase (CORT→11DHC) or 0.015 × 106 liver or 0.05 × 106 Leydig cells for reductase (11DHC→CORT) activities when the substrates were incubated with the cells for 30–120 min. S3483 was added into cells with concentration of 0.01–100 μM 10 min before addition of substrates. Mean ± SEM, n = 3~7. The asterisks designate significant differences compared to control (CON, no S3483) at *P < 0.05, **P < 0.01, and *** P < 0.001.
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pone.0141767.g001: Effects of G6P transporter inhibitor S3483 on intact rat liver and Leydig cell 11β-HSD1 direction.0.015 × 106 liver or 0.025 × 106 Leydig cells were used to measure 11β-HSD1 oxidase (CORT→11DHC) or 0.015 × 106 liver or 0.05 × 106 Leydig cells for reductase (11DHC→CORT) activities when the substrates were incubated with the cells for 30–120 min. S3483 was added into cells with concentration of 0.01–100 μM 10 min before addition of substrates. Mean ± SEM, n = 3~7. The asterisks designate significant differences compared to control (CON, no S3483) at *P < 0.05, **P < 0.01, and *** P < 0.001.

Mentions: The directionality of 11β-HSD1 in intact rat liver parenchymal cells and Leydig cells was determined using endogenous cofactor (Fig 1). In our experiments, we first performed the time-course reaction in order to determine the linear range of 11β-HSD1 oxidase and reductase. We found that the reductase activity was in linear reaction during 0–1 h and the oxidase activity was in linear reaction during 0–4 h. In liver cells, 11β-HSD1 reductase activity was 3 fold higher than oxidase activity (Fig 1A). The percentage of 11β-HSD1 reductase activity in 0.015 × 106 liver cells after 0.5 h incubation was 20.1%, while that of 11β-HSD1 oxidase activity in 0.015 × 106 liver cells after 2 h incubation was 14.6%. However, in intact Leydig cells, the percentage of 11β-HSD1 oxidase activity in 0.025 × 106 Leydig cells after 30-min incubation was 16.29%, with a velocity of 313.0 pmol/106 cells.hr, while that of 11β-HSD1 reductase activity in 0.05 × 106 cells after 2 h incubation was 41.79% with a velocity of 98.76 pmol/106 cells.hr. This showed that 11β-HSD1 oxidase was significantly higher than reductase activity (Fig 1C and 1D). To further test whether H6PDH rendered this difference, G6P transporter inhibitor S3483 was used. Our rationale was that if the reductase activity of 11β-HSD1 was metabolically coupled to H6PDH, then availability of the substrate for H6PDH, G6P, would also be important. Addition of S3483 significantly increased the oxidation and decreased the reduction of 11β-HSD1 in intact liver cells (Fig 1A and 1B). This is what would be expected because the metabolic coupling between H6PDH and 11β-HSD1 has previously been shown [9, 10]. In contrast, addition of S3483 (up to 100 μM) to intact Leydig cells had no effect on either oxidation or reduction by 11β-HSD1 (Fig 1C and 1D). When 100 μM S3483 was used, it had a little bit cytotoxicity (data not shown), while other concentrations of S3483 did not affect both liver and Leydig cell viability (data not shown).


Metabolic Coupling Determines the Activity: Comparison of 11β-Hydroxysteroid Dehydrogenase 1 and Its Coupling between Liver Parenchymal Cells and Testicular Leydig Cells.

Li X, Hu G, Li X, Wang YY, Hu YY, Zhou H, Latif SA, Morris DJ, Chu Y, Zheng Z, Ge RS - PLoS ONE (2015)

Effects of G6P transporter inhibitor S3483 on intact rat liver and Leydig cell 11β-HSD1 direction.0.015 × 106 liver or 0.025 × 106 Leydig cells were used to measure 11β-HSD1 oxidase (CORT→11DHC) or 0.015 × 106 liver or 0.05 × 106 Leydig cells for reductase (11DHC→CORT) activities when the substrates were incubated with the cells for 30–120 min. S3483 was added into cells with concentration of 0.01–100 μM 10 min before addition of substrates. Mean ± SEM, n = 3~7. The asterisks designate significant differences compared to control (CON, no S3483) at *P < 0.05, **P < 0.01, and *** P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4631333&req=5

pone.0141767.g001: Effects of G6P transporter inhibitor S3483 on intact rat liver and Leydig cell 11β-HSD1 direction.0.015 × 106 liver or 0.025 × 106 Leydig cells were used to measure 11β-HSD1 oxidase (CORT→11DHC) or 0.015 × 106 liver or 0.05 × 106 Leydig cells for reductase (11DHC→CORT) activities when the substrates were incubated with the cells for 30–120 min. S3483 was added into cells with concentration of 0.01–100 μM 10 min before addition of substrates. Mean ± SEM, n = 3~7. The asterisks designate significant differences compared to control (CON, no S3483) at *P < 0.05, **P < 0.01, and *** P < 0.001.
Mentions: The directionality of 11β-HSD1 in intact rat liver parenchymal cells and Leydig cells was determined using endogenous cofactor (Fig 1). In our experiments, we first performed the time-course reaction in order to determine the linear range of 11β-HSD1 oxidase and reductase. We found that the reductase activity was in linear reaction during 0–1 h and the oxidase activity was in linear reaction during 0–4 h. In liver cells, 11β-HSD1 reductase activity was 3 fold higher than oxidase activity (Fig 1A). The percentage of 11β-HSD1 reductase activity in 0.015 × 106 liver cells after 0.5 h incubation was 20.1%, while that of 11β-HSD1 oxidase activity in 0.015 × 106 liver cells after 2 h incubation was 14.6%. However, in intact Leydig cells, the percentage of 11β-HSD1 oxidase activity in 0.025 × 106 Leydig cells after 30-min incubation was 16.29%, with a velocity of 313.0 pmol/106 cells.hr, while that of 11β-HSD1 reductase activity in 0.05 × 106 cells after 2 h incubation was 41.79% with a velocity of 98.76 pmol/106 cells.hr. This showed that 11β-HSD1 oxidase was significantly higher than reductase activity (Fig 1C and 1D). To further test whether H6PDH rendered this difference, G6P transporter inhibitor S3483 was used. Our rationale was that if the reductase activity of 11β-HSD1 was metabolically coupled to H6PDH, then availability of the substrate for H6PDH, G6P, would also be important. Addition of S3483 significantly increased the oxidation and decreased the reduction of 11β-HSD1 in intact liver cells (Fig 1A and 1B). This is what would be expected because the metabolic coupling between H6PDH and 11β-HSD1 has previously been shown [9, 10]. In contrast, addition of S3483 (up to 100 μM) to intact Leydig cells had no effect on either oxidation or reduction by 11β-HSD1 (Fig 1C and 1D). When 100 μM S3483 was used, it had a little bit cytotoxicity (data not shown), while other concentrations of S3483 did not affect both liver and Leydig cell viability (data not shown).

Bottom Line: S3483, a G6P transporter inhibitor, reversed the G6P-mediated increases of 11β-HSD1 reductase activity.The depletion of Leydig cells eliminated Hsd11b1 (encoding 11β-HSD1) expression but did not affect the expression of H6pd (encoding H6PDH) and Slc37a4 (encoding G6P transporter).In conclusion, the availability of H6PDH determines the different direction of 11β-HSD1 in liver and Leydig cells.

View Article: PubMed Central - PubMed

Affiliation: The Second Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, ZJ 325000, PR China.

ABSTRACT

Background: 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) interconverts active 11β-hydroxyl glucocorticoids and inactive 11keto forms. However, its directionality is determined by availability of NADP+/NADPH. In liver cells, 11β-HSD1 behaves as a primary reductase, while in Leydig cells it acts as a primary oxidase. However, the exact mechanism is not clear. The direction of 11β-HSD1 has been proposed to be regulated by hexose-6-phosphate dehydrogenase (H6PDH), which catalyzes glucose-6-phosphate (G6P) to generate NADPH that drives 11β-HSD1 towards reduction.

Methodology: To examine the coupling between 11β-HSD1 and H6PDH, we added G6P to rat and human liver and testis or Leydig cell microsomes, and 11β-HSD1 activity was measured by radiometry.

Results and conclusions: G6P stimulated 11β-HSD1 reductase activity in rat (3 fold) or human liver (1.5 fold), but not at all in testis. S3483, a G6P transporter inhibitor, reversed the G6P-mediated increases of 11β-HSD1 reductase activity. We compared the extent to which 11β-HSD1 in rat Leydig and liver cells might be coupled to H6PDH. In order to clarify the location of H6PDH within the testis, we used the Leydig cell toxicant ethane dimethanesulfonate (EDS) to selectively deplete Leydig cells. The depletion of Leydig cells eliminated Hsd11b1 (encoding 11β-HSD1) expression but did not affect the expression of H6pd (encoding H6PDH) and Slc37a4 (encoding G6P transporter). H6pd mRNA level and H6PDH activity were barely detectable in purified rat Leydig cells. In conclusion, the availability of H6PDH determines the different direction of 11β-HSD1 in liver and Leydig cells.

No MeSH data available.


Related in: MedlinePlus