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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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The amplifying pathways of GSIS are upregulated in PKCĪµKO islets. Aā€“C: Islets isolated from 12-week-old male mice were cultured for 48 h (chronic) in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: GSIS in response to 20 mmol/l glucose and inhibition by diazoxide (Dzx; 100 Ī¼mol/l; n = 7). B: KATP channelā€“independent glucose-amplified insulin secretion in the presence of 25mmol/l KCl and diazoxide (n = 5). C: Insulin secretion in response to nonnutrient secretogogues KCl (25 mmol/l) and forskolin (1 Ī¼mol/l; n = 3). D: Acute palmitate-potentiated GSIS from islets without chronic palmitate culture (n = 4). Data are the means Ā± SE. *P < 0.05; **P < 0.01. WT, wild type.
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Figure 2: The amplifying pathways of GSIS are upregulated in PKCĪµKO islets. Aā€“C: Islets isolated from 12-week-old male mice were cultured for 48 h (chronic) in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: GSIS in response to 20 mmol/l glucose and inhibition by diazoxide (Dzx; 100 Ī¼mol/l; n = 7). B: KATP channelā€“independent glucose-amplified insulin secretion in the presence of 25mmol/l KCl and diazoxide (n = 5). C: Insulin secretion in response to nonnutrient secretogogues KCl (25 mmol/l) and forskolin (1 Ī¼mol/l; n = 3). D: Acute palmitate-potentiated GSIS from islets without chronic palmitate culture (n = 4). Data are the means Ā± SE. *P < 0.05; **P < 0.01. WT, wild type.

Mentions: As shown previously (18), deletion of PKCĪµ did not alter acute GSIS from islets cultured under control conditions, but it selectively enhanced this response after lipid culture (Fig. 2A). GSIS from islets under all treatment conditions was inhibited by diazoxide (Fig. 2A), which maintains KATP channels in an open conformation, preventing membrane depolarization. These results indicate that the enhanced GSIS in PKCĪµKO islets was dependent on normal coupling of glucose metabolism with depolarization-dependent Ca2+ influx. To measure the amplification pathways, or KATP channelā€“independent secretion, GSIS assays were performed in the combined presence of diazoxide and 25 mmol/l KCl (9). The latter serves as a nonmetabolic depolarizing stimulus to activate voltage-gated Ca2+ channels and trigger insulin secretion under conditions in which the glucose-dependent initiation signal is inhibited by diazoxide. The secretory response to glucose in this protocol thus represents the amplification pathway (9), which was operative in all treatment groups (Fig. 2B). Moreover, after lipid culture, glucose-amplified insulin secretion was nearly doubled from PKCĪµKO islets relative to wild-type islets (Fig. 2B), whereas no significant difference between the two genotypes was observed under control culture conditions. These results suggest that an augmentation of the amplifying pathways is a major contributor to the overall enhancement of GSIS observed in lipid-cultured PKCĪµKO 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)

The amplifying pathways of GSIS are upregulated in PKCĪµKO islets. Aā€“C: Islets isolated from 12-week-old male mice were cultured for 48 h (chronic) in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: GSIS in response to 20 mmol/l glucose and inhibition by diazoxide (Dzx; 100 Ī¼mol/l; n = 7). B: KATP channelā€“independent glucose-amplified insulin secretion in the presence of 25mmol/l KCl and diazoxide (n = 5). C: Insulin secretion in response to nonnutrient secretogogues KCl (25 mmol/l) and forskolin (1 Ī¼mol/l; n = 3). D: Acute palmitate-potentiated GSIS from islets without chronic palmitate culture (n = 4). Data are the means Ā± SE. *P < 0.05; **P < 0.01. WT, wild type.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2712791&req=5

Figure 2: The amplifying pathways of GSIS are upregulated in PKCĪµKO islets. Aā€“C: Islets isolated from 12-week-old male mice were cultured for 48 h (chronic) in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont). A: GSIS in response to 20 mmol/l glucose and inhibition by diazoxide (Dzx; 100 Ī¼mol/l; n = 7). B: KATP channelā€“independent glucose-amplified insulin secretion in the presence of 25mmol/l KCl and diazoxide (n = 5). C: Insulin secretion in response to nonnutrient secretogogues KCl (25 mmol/l) and forskolin (1 Ī¼mol/l; n = 3). D: Acute palmitate-potentiated GSIS from islets without chronic palmitate culture (n = 4). Data are the means Ā± SE. *P < 0.05; **P < 0.01. WT, wild type.
Mentions: As shown previously (18), deletion of PKCĪµ did not alter acute GSIS from islets cultured under control conditions, but it selectively enhanced this response after lipid culture (Fig. 2A). GSIS from islets under all treatment conditions was inhibited by diazoxide (Fig. 2A), which maintains KATP channels in an open conformation, preventing membrane depolarization. These results indicate that the enhanced GSIS in PKCĪµKO islets was dependent on normal coupling of glucose metabolism with depolarization-dependent Ca2+ influx. To measure the amplification pathways, or KATP channelā€“independent secretion, GSIS assays were performed in the combined presence of diazoxide and 25 mmol/l KCl (9). The latter serves as a nonmetabolic depolarizing stimulus to activate voltage-gated Ca2+ channels and trigger insulin secretion under conditions in which the glucose-dependent initiation signal is inhibited by diazoxide. The secretory response to glucose in this protocol thus represents the amplification pathway (9), which was operative in all treatment groups (Fig. 2B). Moreover, after lipid culture, glucose-amplified insulin secretion was nearly doubled from PKCĪµKO islets relative to wild-type islets (Fig. 2B), whereas no significant difference between the two genotypes was observed under control culture conditions. These results suggest that an augmentation of the amplifying pathways is a major contributor to the overall enhancement of GSIS observed in lipid-cultured PKCĪµKO 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