Limits...
Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase.

Rizzo MA, Piston DW - J. Cell Biol. (2003)

Bottom Line: Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO).Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK.GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 735 Light Hall, Nashville, TN 37232, USA.

ABSTRACT
Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic beta cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic beta cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

Show MeSH
Regulation of GK by insulin requires NO. (A) βTC3 cells were starved for 4 h before loading with DAF-FM and analysis using confocal microscopy. The change in DAF intensity is represented as the change in fluorescence/initial fluorescence (dF/F0) from the average of at least 10 cells treated with 100 nM insulin (indicated by the arrow, ▪) or left untreated (○). (B) FRAP measurements were taken in cells expressing GK-YFP (yellow) and proinsulin-CFP (cyan) by selectively photobleaching YFP fluorescence in a small region (white box) of the cell containing several granules. (C) Fluorescence recovery of GK-YFP to CFP-labeled insulin granules was measured after photobleaching GK-YFP in starved cells (○) and after insulin treatment (100 nM, 5 min, ▪). The bleaching period is indicated by the arrow and broken lines. (D) FRAP measurements show fluorescence intensity of granule-associated GK-YFP immediately after photobleaching (white bars) and 2 s after photobleaching (black bars) and expressed as the percentage of prebleached fluorescence intensity. Cells were starved previously in BMHH for 3 h before insulin treatment (5 min), pretreatment with L-NAME (5 mM, 10 min) before insulin treatment (100 nM, 5 min), and after treatment with SNAP (100 μM, 1 min). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (E) Cells expressing CFP-GK-YFP were examined for FRET by fluorescence microscopy as indicated. Statistical significance (P < 0.05 by ANOVA or t test as appropriate) is denoted by an asterisk. Cells were treated under the same conditions as in D. (F) Nitrosylated proteins were precipitated with neutravidin-agarose after biotinylation of S-nitrosylated proteins from cell lysates. Cells were treated as above where indicated. GK was detected in unreacted lysates and precipitated fractions by Western blot using an antibody to GK (Jetton and Magnuson, 1992).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172922&req=5

fig1: Regulation of GK by insulin requires NO. (A) βTC3 cells were starved for 4 h before loading with DAF-FM and analysis using confocal microscopy. The change in DAF intensity is represented as the change in fluorescence/initial fluorescence (dF/F0) from the average of at least 10 cells treated with 100 nM insulin (indicated by the arrow, ▪) or left untreated (○). (B) FRAP measurements were taken in cells expressing GK-YFP (yellow) and proinsulin-CFP (cyan) by selectively photobleaching YFP fluorescence in a small region (white box) of the cell containing several granules. (C) Fluorescence recovery of GK-YFP to CFP-labeled insulin granules was measured after photobleaching GK-YFP in starved cells (○) and after insulin treatment (100 nM, 5 min, ▪). The bleaching period is indicated by the arrow and broken lines. (D) FRAP measurements show fluorescence intensity of granule-associated GK-YFP immediately after photobleaching (white bars) and 2 s after photobleaching (black bars) and expressed as the percentage of prebleached fluorescence intensity. Cells were starved previously in BMHH for 3 h before insulin treatment (5 min), pretreatment with L-NAME (5 mM, 10 min) before insulin treatment (100 nM, 5 min), and after treatment with SNAP (100 μM, 1 min). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (E) Cells expressing CFP-GK-YFP were examined for FRET by fluorescence microscopy as indicated. Statistical significance (P < 0.05 by ANOVA or t test as appropriate) is denoted by an asterisk. Cells were treated under the same conditions as in D. (F) Nitrosylated proteins were precipitated with neutravidin-agarose after biotinylation of S-nitrosylated proteins from cell lysates. Cells were treated as above where indicated. GK was detected in unreacted lysates and precipitated fractions by Western blot using an antibody to GK (Jetton and Magnuson, 1992).

Mentions: To assess the role of NOS in the regulation of GK by insulin, we examined whether insulin could stimulate NO production in β cells. βTC3 cells were loaded with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) (Kojima et al., 1999), an indicator dye whose fluorescence increases upon reaction with NO. A rapid increase in the fluorescence of DAF-FM was observed within minutes in insulin-treated cells compared with untreated cells (Fig. 1 A). This indicates that insulin treatment results in NO production on a time scale consistent with regulation of GK (Rizzo et al., 2002). We also examined the role of NOS activation in the regulation of GK association with secretory granules. Association of GK with secretory granules was analyzed in cells expressing a YFP-labeled GK (GK-YFP) and a CFP targeted to insulin granules by insertion into a proinsulin cDNA (Rizzo et al., 2002; Watkins et al., 2002) (Fig. 1 B). Association of GK-YFP with CFP-labeled granules can be assayed by selectively photobleaching the GK-YFP in a small region of the cell (Fig. 1 B, white box) and monitoring the FRAP to the CFP-labeled granules. Recovery of GK-YFP to CFP-labeled granules occurs at a faster rate in insulin-treated cells (Fig. 1 C), which indicates a net translocation of GK to the cytoplasm (Rizzo et al., 2002). A significant difference in the degree of fluorescence recovery between insulin-treated (5 min, 100 nM) and untreated cells could be reliably measured 2 s into recovery (Fig. 1 D). Inhibition of NOS activity using NG-nitro-l-arginine-methyl ester (L-NAME) blocked the stimulatory effects of insulin on GK-YFP FRAP, indicating a requirement for NO production. Furthermore, the effects of NOS inhibition were reversed by treatment with SNAP, an NO releasing agent. These results are consistent with a requirement for NO production in modulating GK association with secretory granules.


Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase.

Rizzo MA, Piston DW - J. Cell Biol. (2003)

Regulation of GK by insulin requires NO. (A) βTC3 cells were starved for 4 h before loading with DAF-FM and analysis using confocal microscopy. The change in DAF intensity is represented as the change in fluorescence/initial fluorescence (dF/F0) from the average of at least 10 cells treated with 100 nM insulin (indicated by the arrow, ▪) or left untreated (○). (B) FRAP measurements were taken in cells expressing GK-YFP (yellow) and proinsulin-CFP (cyan) by selectively photobleaching YFP fluorescence in a small region (white box) of the cell containing several granules. (C) Fluorescence recovery of GK-YFP to CFP-labeled insulin granules was measured after photobleaching GK-YFP in starved cells (○) and after insulin treatment (100 nM, 5 min, ▪). The bleaching period is indicated by the arrow and broken lines. (D) FRAP measurements show fluorescence intensity of granule-associated GK-YFP immediately after photobleaching (white bars) and 2 s after photobleaching (black bars) and expressed as the percentage of prebleached fluorescence intensity. Cells were starved previously in BMHH for 3 h before insulin treatment (5 min), pretreatment with L-NAME (5 mM, 10 min) before insulin treatment (100 nM, 5 min), and after treatment with SNAP (100 μM, 1 min). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (E) Cells expressing CFP-GK-YFP were examined for FRET by fluorescence microscopy as indicated. Statistical significance (P < 0.05 by ANOVA or t test as appropriate) is denoted by an asterisk. Cells were treated under the same conditions as in D. (F) Nitrosylated proteins were precipitated with neutravidin-agarose after biotinylation of S-nitrosylated proteins from cell lysates. Cells were treated as above where indicated. GK was detected in unreacted lysates and precipitated fractions by Western blot using an antibody to GK (Jetton and Magnuson, 1992).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172922&req=5

fig1: Regulation of GK by insulin requires NO. (A) βTC3 cells were starved for 4 h before loading with DAF-FM and analysis using confocal microscopy. The change in DAF intensity is represented as the change in fluorescence/initial fluorescence (dF/F0) from the average of at least 10 cells treated with 100 nM insulin (indicated by the arrow, ▪) or left untreated (○). (B) FRAP measurements were taken in cells expressing GK-YFP (yellow) and proinsulin-CFP (cyan) by selectively photobleaching YFP fluorescence in a small region (white box) of the cell containing several granules. (C) Fluorescence recovery of GK-YFP to CFP-labeled insulin granules was measured after photobleaching GK-YFP in starved cells (○) and after insulin treatment (100 nM, 5 min, ▪). The bleaching period is indicated by the arrow and broken lines. (D) FRAP measurements show fluorescence intensity of granule-associated GK-YFP immediately after photobleaching (white bars) and 2 s after photobleaching (black bars) and expressed as the percentage of prebleached fluorescence intensity. Cells were starved previously in BMHH for 3 h before insulin treatment (5 min), pretreatment with L-NAME (5 mM, 10 min) before insulin treatment (100 nM, 5 min), and after treatment with SNAP (100 μM, 1 min). Statistical significance from initial postbleach intensity (P < 0.05, t test) is denoted by an asterisk. (E) Cells expressing CFP-GK-YFP were examined for FRET by fluorescence microscopy as indicated. Statistical significance (P < 0.05 by ANOVA or t test as appropriate) is denoted by an asterisk. Cells were treated under the same conditions as in D. (F) Nitrosylated proteins were precipitated with neutravidin-agarose after biotinylation of S-nitrosylated proteins from cell lysates. Cells were treated as above where indicated. GK was detected in unreacted lysates and precipitated fractions by Western blot using an antibody to GK (Jetton and Magnuson, 1992).
Mentions: To assess the role of NOS in the regulation of GK by insulin, we examined whether insulin could stimulate NO production in β cells. βTC3 cells were loaded with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) (Kojima et al., 1999), an indicator dye whose fluorescence increases upon reaction with NO. A rapid increase in the fluorescence of DAF-FM was observed within minutes in insulin-treated cells compared with untreated cells (Fig. 1 A). This indicates that insulin treatment results in NO production on a time scale consistent with regulation of GK (Rizzo et al., 2002). We also examined the role of NOS activation in the regulation of GK association with secretory granules. Association of GK with secretory granules was analyzed in cells expressing a YFP-labeled GK (GK-YFP) and a CFP targeted to insulin granules by insertion into a proinsulin cDNA (Rizzo et al., 2002; Watkins et al., 2002) (Fig. 1 B). Association of GK-YFP with CFP-labeled granules can be assayed by selectively photobleaching the GK-YFP in a small region of the cell (Fig. 1 B, white box) and monitoring the FRAP to the CFP-labeled granules. Recovery of GK-YFP to CFP-labeled granules occurs at a faster rate in insulin-treated cells (Fig. 1 C), which indicates a net translocation of GK to the cytoplasm (Rizzo et al., 2002). A significant difference in the degree of fluorescence recovery between insulin-treated (5 min, 100 nM) and untreated cells could be reliably measured 2 s into recovery (Fig. 1 D). Inhibition of NOS activity using NG-nitro-l-arginine-methyl ester (L-NAME) blocked the stimulatory effects of insulin on GK-YFP FRAP, indicating a requirement for NO production. Furthermore, the effects of NOS inhibition were reversed by treatment with SNAP, an NO releasing agent. These results are consistent with a requirement for NO production in modulating GK association with secretory granules.

Bottom Line: Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO).Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK.GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 735 Light Hall, Nashville, TN 37232, USA.

ABSTRACT
Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic beta cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic beta cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

Show MeSH