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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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Lipase activities and gene expression in PKCεKO islets. A: Lipase activities of islet homogenates after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont) and subsequent stimulation with 20 mmol/l glucose for 1 h. Triglyceride or cholesteryl-ester lipid pools were provided as substrate, spiked with [3H]triolein or [14C]cholesteryl-oleate, respectively. Islets from n = 5 animals were used per genotype. B and C: mRNA expression of Hsl (Hsl), adipose triglyceride lipase (Atgl), adiponutrin (Adpn), α/β-hydrolase domain–containing protein 5 (Abhd5), translocator protein (Tspo), and lipoprotein lipase (Lpl). Islets were cultured in palmitate or control media, and data were expressed relative to wild-type control islets (n = 5). D: Western blot and quantification of Hsl and Atgl with 14-3-3β loading control; n = 3. □ = wild type, ■ = PKCεKO. Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
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Figure 6: Lipase activities and gene expression in PKCεKO islets. A: Lipase activities of islet homogenates after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont) and subsequent stimulation with 20 mmol/l glucose for 1 h. Triglyceride or cholesteryl-ester lipid pools were provided as substrate, spiked with [3H]triolein or [14C]cholesteryl-oleate, respectively. Islets from n = 5 animals were used per genotype. B and C: mRNA expression of Hsl (Hsl), adipose triglyceride lipase (Atgl), adiponutrin (Adpn), α/β-hydrolase domain–containing protein 5 (Abhd5), translocator protein (Tspo), and lipoprotein lipase (Lpl). Islets were cultured in palmitate or control media, and data were expressed relative to wild-type control islets (n = 5). D: Western blot and quantification of Hsl and Atgl with 14-3-3β loading control; n = 3. □ = wild type, ■ = PKCεKO. Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.

Mentions: We next measured glucose-stimulated lipase activities directly, using homogenates of islets derived from our different treatment groups. Triglyceride lipolysis was significantly enhanced in PKCεKO islets after palmitate culture and showed a similar, albeit nonsignificant, increase after control culture relative to wild-type islets (Fig. 6A). Cholesteryl-ester hydrolysis was 200-fold lower than that of triglyceride and was downregulated in control-cultured PKCεKO islets, relative to wild-type islets, but unaltered by genotype after palmitate culture (Fig. 6A).


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)

Lipase activities and gene expression in PKCεKO islets. A: Lipase activities of islet homogenates after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont) and subsequent stimulation with 20 mmol/l glucose for 1 h. Triglyceride or cholesteryl-ester lipid pools were provided as substrate, spiked with [3H]triolein or [14C]cholesteryl-oleate, respectively. Islets from n = 5 animals were used per genotype. B and C: mRNA expression of Hsl (Hsl), adipose triglyceride lipase (Atgl), adiponutrin (Adpn), α/β-hydrolase domain–containing protein 5 (Abhd5), translocator protein (Tspo), and lipoprotein lipase (Lpl). Islets were cultured in palmitate or control media, and data were expressed relative to wild-type control islets (n = 5). D: Western blot and quantification of Hsl and Atgl with 14-3-3β loading control; n = 3. □ = wild type, ■ = PKCεKO. 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 6: Lipase activities and gene expression in PKCεKO islets. A: Lipase activities of islet homogenates after culture for 48 h in the presence of 0.4 mmol/l palmitate coupled to 0.92% BSA (Palm) or BSA alone (Cont) and subsequent stimulation with 20 mmol/l glucose for 1 h. Triglyceride or cholesteryl-ester lipid pools were provided as substrate, spiked with [3H]triolein or [14C]cholesteryl-oleate, respectively. Islets from n = 5 animals were used per genotype. B and C: mRNA expression of Hsl (Hsl), adipose triglyceride lipase (Atgl), adiponutrin (Adpn), α/β-hydrolase domain–containing protein 5 (Abhd5), translocator protein (Tspo), and lipoprotein lipase (Lpl). Islets were cultured in palmitate or control media, and data were expressed relative to wild-type control islets (n = 5). D: Western blot and quantification of Hsl and Atgl with 14-3-3β loading control; n = 3. □ = wild type, ■ = PKCεKO. Data are the means ± SE. *P < 0.05; **P < 0.01. WT, wild type.
Mentions: We next measured glucose-stimulated lipase activities directly, using homogenates of islets derived from our different treatment groups. Triglyceride lipolysis was significantly enhanced in PKCεKO islets after palmitate culture and showed a similar, albeit nonsignificant, increase after control culture relative to wild-type islets (Fig. 6A). Cholesteryl-ester hydrolysis was 200-fold lower than that of triglyceride and was downregulated in control-cultured PKCεKO islets, relative to wild-type islets, but unaltered by genotype after palmitate culture (Fig. 6A).

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