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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 lipolysis is required for enhanced secretion in PKCεKO islets. Islets isolated from 12-week-old male mice were cultured for 48–72 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: Lipolysis was measured in islets, after palmitate culture, by detection of glycerol release in response to 20 mmol/l d-glucose (n = 7). B: GSIS in palmitate-cultured PKCεKO islets in the presence of the lipase inhibitor orlistat (0.2 mmol/l) or DMSO vehicle control (n = 9). C: Glucose-amplified insulin secretion measured under KATP channel–independent depolarizing conditions (25 mmol/l KCl and 100 μmol/l diazoxide) using palmitate-cultured PKCεKO islets in the presence of orlistat or DMSO control (n = 9). Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
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Figure 5: Enhanced lipolysis is required for enhanced secretion in PKCεKO islets. Islets isolated from 12-week-old male mice were cultured for 48–72 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: Lipolysis was measured in islets, after palmitate culture, by detection of glycerol release in response to 20 mmol/l d-glucose (n = 7). B: GSIS in palmitate-cultured PKCεKO islets in the presence of the lipase inhibitor orlistat (0.2 mmol/l) or DMSO vehicle control (n = 9). C: Glucose-amplified insulin secretion measured under KATP channel–independent depolarizing conditions (25 mmol/l KCl and 100 μmol/l diazoxide) using palmitate-cultured PKCεKO islets in the presence of orlistat or DMSO control (n = 9). Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.

Mentions: Mobilization of endogenous neutral lipid stores by lipolysis has been implicated in GSIS in rat β-cells (31,32), and these processes are glucose regulated in rat and mouse β-cells (33,34). Thus, lipolytic pathways may link the increased triglyceride formation with the enhanced lipid-dependent GSIS in PKCεKO islets. Therefore, we assayed lipolysis as glycerol generation because this metabolite is released from β-cells after lipolysis, rather than being recycled (35). Glucose-stimulated glycerol production from PKCεKO islets was significantly increased compared with wild-type islets after chronic lipid culture (Fig. 5A), suggesting that enhanced lipolysis might underlie the augmentation of GSIS observed under these conditions. To test this more directly, we assessed GSIS in the presence of the lipase inhibitor orlistat (0.2 mmol/l). Although without effect on wild-type islets, orlistat completely blocked the enhancement of GSIS and glucose-amplified insulin secretion otherwise observed using PKCεKO islets (Fig. 5B and C). This inhibition was dose dependent (supplementary Fig. A2).


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 lipolysis is required for enhanced secretion in PKCεKO islets. Islets isolated from 12-week-old male mice were cultured for 48–72 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: Lipolysis was measured in islets, after palmitate culture, by detection of glycerol release in response to 20 mmol/l d-glucose (n = 7). B: GSIS in palmitate-cultured PKCεKO islets in the presence of the lipase inhibitor orlistat (0.2 mmol/l) or DMSO vehicle control (n = 9). C: Glucose-amplified insulin secretion measured under KATP channel–independent depolarizing conditions (25 mmol/l KCl and 100 μmol/l diazoxide) using palmitate-cultured PKCεKO islets in the presence of orlistat or DMSO control (n = 9). Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Enhanced lipolysis is required for enhanced secretion in PKCεKO islets. Islets isolated from 12-week-old male mice were cultured for 48–72 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: Lipolysis was measured in islets, after palmitate culture, by detection of glycerol release in response to 20 mmol/l d-glucose (n = 7). B: GSIS in palmitate-cultured PKCεKO islets in the presence of the lipase inhibitor orlistat (0.2 mmol/l) or DMSO vehicle control (n = 9). C: Glucose-amplified insulin secretion measured under KATP channel–independent depolarizing conditions (25 mmol/l KCl and 100 μmol/l diazoxide) using palmitate-cultured PKCεKO islets in the presence of orlistat or DMSO control (n = 9). Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
Mentions: Mobilization of endogenous neutral lipid stores by lipolysis has been implicated in GSIS in rat β-cells (31,32), and these processes are glucose regulated in rat and mouse β-cells (33,34). Thus, lipolytic pathways may link the increased triglyceride formation with the enhanced lipid-dependent GSIS in PKCεKO islets. Therefore, we assayed lipolysis as glycerol generation because this metabolite is released from β-cells after lipolysis, rather than being recycled (35). Glucose-stimulated glycerol production from PKCεKO islets was significantly increased compared with wild-type islets after chronic lipid culture (Fig. 5A), suggesting that enhanced lipolysis might underlie the augmentation of GSIS observed under these conditions. To test this more directly, we assessed GSIS in the presence of the lipase inhibitor orlistat (0.2 mmol/l). Although without effect on wild-type islets, orlistat completely blocked the enhancement of GSIS and glucose-amplified insulin secretion otherwise observed using PKCεKO islets (Fig. 5B and C). This inhibition was dose dependent (supplementary Fig. A2).

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