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Deletion of PKCepsilon selectively enhances the amplifying pathways of glucose-stimulated insulin secretion via increased lipolysis in mouse beta-cells.

Cantley J, Burchfield JG, Pearson GL, Schmitz-Peiffer C, Leitges M, Biden TJ - Diabetes (2009)

Bottom Line: Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCepsilonKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCepsilon deletion.Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine beta-cells, and they suggest an interaction between PKCepsilon and lipolysis.These results further highlight the therapeutic potential of PKCepsilon inhibition to enhance GSIS from the beta-cell under conditions of lipid excess.

View Article: PubMed Central - PubMed

Affiliation: Diabetes and Obesity Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

ABSTRACT

Objective: Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of beta-cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase C epsilon (PKCepsilon) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCepsilon.

Research design and methods: We cultured islets isolated from PKCepsilon knockout (PKCepsilonKO) mice in palmitate prior to measuring GSIS, Ca(2+) responses, palmitate esterification products, lipolysis, lipase activity, and gene expression.

Results: The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated Ca(2+) influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCepsilonKO beta-cells was revealed under conditions in which functional ATP-sensitive K(+) channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCepsilonKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCepsilonKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCepsilon deletion.

Conclusions: Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine beta-cells, and they suggest an interaction between PKCepsilon and lipolysis. These results further highlight the therapeutic potential of PKCepsilon inhibition to enhance GSIS from the beta-cell under conditions of lipid excess.

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Enhanced incorporation of fatty acids into triglyceride stores in PKCεKO β-cells. A: Islets were cultured for 48 h with 0.4 mmol/l palmitate coupled to 0.92% BSA and 20 μCi/ml of [U-14C]palmitate. Islet lipid extracts were resolved by silica thin-layer chromatography, and incorporation of 14C-palmitate into neutral lipid pools was quantified by liquid scintillation spectrometry (n = 10–12). B: Diacylglycerol acyltransferase 1 (Dgat1) mRNA expression in islets after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). Data were expressed relative to wild-type control islets (n = 5). C–E: Islets from 12-week-old male mice, after palmitate or control culture, were lysed and proteins resolved by SDS-PAGE before immunoblots were performed for phospho-ACC Ser79 (p-ACC), ACC, phospho-AMPKα Thr172 (p-AMPKα), AMPKα, and β-actin (loading control). D and E: Densitometry quantification of Western blots; n = 4 animals from two independent islet preparations were used. Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
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Figure 4: Enhanced incorporation of fatty acids into triglyceride stores in PKCεKO β-cells. A: Islets were cultured for 48 h with 0.4 mmol/l palmitate coupled to 0.92% BSA and 20 μCi/ml of [U-14C]palmitate. Islet lipid extracts were resolved by silica thin-layer chromatography, and incorporation of 14C-palmitate into neutral lipid pools was quantified by liquid scintillation spectrometry (n = 10–12). B: Diacylglycerol acyltransferase 1 (Dgat1) mRNA expression in islets after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). Data were expressed relative to wild-type control islets (n = 5). C–E: Islets from 12-week-old male mice, after palmitate or control culture, were lysed and proteins resolved by SDS-PAGE before immunoblots were performed for phospho-ACC Ser79 (p-ACC), ACC, phospho-AMPKα Thr172 (p-AMPKα), AMPKα, and β-actin (loading control). D and E: Densitometry quantification of Western blots; n = 4 animals from two independent islet preparations were used. Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.

Mentions: Acute incubation of lipid-pretreated PKCεKO islets with [U-14C]palmitate had previously revealed a switch in glucose-regulated lipid partitioning, favoring lipid esterification over oxidation (18). That approach, however, did not address whether endogenous lipid pools were chronically altered by PKCε deletion. We therefore measured incorporation of [U-14C]palmitate into neutral lipid pools over 48 h of palmitate culture, which revealed equivalent 14C labeling of diacylglycerols and cholesteryl-esters in PKCεKO and wild-type islets (Fig. 4A). However, we did observe a significant increase in triglycerides derived from exogenous [U-14C]palmitate in PKCεKO islets (Fig. 4A). Despite this, oil red O staining of lipid droplets in chronic lipid-cultured PKCεKO β-cells was indistinguishable from that of wild-type β-cells (data not shown). Expression of the enzyme diacylglycerol acyltransferase (Dgat1), responsible for the formation of triglyceride from diacylglycerol, was unaltered in islets by palmitate culture and/or PKCε deletion (Fig. 4B), excluding this as a mechanism for the enhanced triglyceride esterification in PKCεKO islets.


Deletion of PKCepsilon selectively enhances the amplifying pathways of glucose-stimulated insulin secretion via increased lipolysis in mouse beta-cells.

Cantley J, Burchfield JG, Pearson GL, Schmitz-Peiffer C, Leitges M, Biden TJ - Diabetes (2009)

Enhanced incorporation of fatty acids into triglyceride stores in PKCεKO β-cells. A: Islets were cultured for 48 h with 0.4 mmol/l palmitate coupled to 0.92% BSA and 20 μCi/ml of [U-14C]palmitate. Islet lipid extracts were resolved by silica thin-layer chromatography, and incorporation of 14C-palmitate into neutral lipid pools was quantified by liquid scintillation spectrometry (n = 10–12). B: Diacylglycerol acyltransferase 1 (Dgat1) mRNA expression in islets after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). Data were expressed relative to wild-type control islets (n = 5). C–E: Islets from 12-week-old male mice, after palmitate or control culture, were lysed and proteins resolved by SDS-PAGE before immunoblots were performed for phospho-ACC Ser79 (p-ACC), ACC, phospho-AMPKα Thr172 (p-AMPKα), AMPKα, and β-actin (loading control). D and E: Densitometry quantification of Western blots; n = 4 animals from two independent islet preparations were used. Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
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Figure 4: Enhanced incorporation of fatty acids into triglyceride stores in PKCεKO β-cells. A: Islets were cultured for 48 h with 0.4 mmol/l palmitate coupled to 0.92% BSA and 20 μCi/ml of [U-14C]palmitate. Islet lipid extracts were resolved by silica thin-layer chromatography, and incorporation of 14C-palmitate into neutral lipid pools was quantified by liquid scintillation spectrometry (n = 10–12). B: Diacylglycerol acyltransferase 1 (Dgat1) mRNA expression in islets after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). Data were expressed relative to wild-type control islets (n = 5). C–E: Islets from 12-week-old male mice, after palmitate or control culture, were lysed and proteins resolved by SDS-PAGE before immunoblots were performed for phospho-ACC Ser79 (p-ACC), ACC, phospho-AMPKα Thr172 (p-AMPKα), AMPKα, and β-actin (loading control). D and E: Densitometry quantification of Western blots; n = 4 animals from two independent islet preparations were used. Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
Mentions: Acute incubation of lipid-pretreated PKCεKO islets with [U-14C]palmitate had previously revealed a switch in glucose-regulated lipid partitioning, favoring lipid esterification over oxidation (18). That approach, however, did not address whether endogenous lipid pools were chronically altered by PKCε deletion. We therefore measured incorporation of [U-14C]palmitate into neutral lipid pools over 48 h of palmitate culture, which revealed equivalent 14C labeling of diacylglycerols and cholesteryl-esters in PKCεKO and wild-type islets (Fig. 4A). However, we did observe a significant increase in triglycerides derived from exogenous [U-14C]palmitate in PKCεKO islets (Fig. 4A). Despite this, oil red O staining of lipid droplets in chronic lipid-cultured PKCεKO β-cells was indistinguishable from that of wild-type β-cells (data not shown). Expression of the enzyme diacylglycerol acyltransferase (Dgat1), responsible for the formation of triglyceride from diacylglycerol, was unaltered in islets by palmitate culture and/or PKCε deletion (Fig. 4B), excluding this as a mechanism for the enhanced triglyceride esterification in PKCεKO islets.

Bottom Line: Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCepsilonKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCepsilon deletion.Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine beta-cells, and they suggest an interaction between PKCepsilon and lipolysis.These results further highlight the therapeutic potential of PKCepsilon inhibition to enhance GSIS from the beta-cell under conditions of lipid excess.

View Article: PubMed Central - PubMed

Affiliation: Diabetes and Obesity Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

ABSTRACT

Objective: Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of beta-cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase C epsilon (PKCepsilon) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCepsilon.

Research design and methods: We cultured islets isolated from PKCepsilon knockout (PKCepsilonKO) mice in palmitate prior to measuring GSIS, Ca(2+) responses, palmitate esterification products, lipolysis, lipase activity, and gene expression.

Results: The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated Ca(2+) influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCepsilonKO beta-cells was revealed under conditions in which functional ATP-sensitive K(+) channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCepsilonKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCepsilonKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCepsilon deletion.

Conclusions: Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine beta-cells, and they suggest an interaction between PKCepsilon and lipolysis. These results further highlight the therapeutic potential of PKCepsilon inhibition to enhance GSIS from the beta-cell under conditions of lipid excess.

Show MeSH
Related in: MedlinePlus