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Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects.

Swisa A, Granot Z, Tamarina N, Sayers S, Bardeesy N, Philipson L, Hodson DJ, Wikstrom JD, Rutter GA, Leibowitz G, Glaser B, Dor Y - J. Biol. Chem. (2015)

Bottom Line: However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood.Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway.This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

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Lkb1 deletion in β cells disrupts mitochondrial function.A, quantification of fluorescence intensity of TMRE (left) and MitoTracker Green (MTG, right) in islet cells from lox/lox and βLKB mice. Mice were 3 months of age, 2 months post-tamoxifen injection. The graph presents the mean of five mice in each genotype. 20–30 fields were imaged and averaged per mouse. The difference in fluorescence intensity of TMRE is translated to Δ 3 mV between lox/lox and βLKB cells. B, NAD(P)H production in response to 16.7 mm glucose. The plots represent the mean of NAD(P)H-derived fluorescence intensity in whole islets from lox/lox (n = 4) and βLKB (n = 4) mice measured by UV autofluorescence. Islets were perifused with 2.8 mm glucose for 12 min then with 16.7 mm for 15 min and back to 2.8 mm. Mice were 6 months old, 5 months post-tamoxifen injection. Right, mean of area under the curve (AUC) of the NAD(P)H plots. *, p < 0.05. C, cytosolic ATP/ADP ratio changes in islets in response to glucose. Glucose was changed during the experiment from 3 mm (G3) to 11 mm (G11). Note the significantly impaired response in βLKB1 islets. AU, arbitrary units. Data are from three wild type and three βLKB mice. D, amplitude of responsiveness presented in C. E, ATP/ADP ratio presented as % of islets responsive to glucose. *, p < 0.05. F, basal OCR measured by Seahorse XF24 analyzer in the presence of 2.8 mm glucose. ns, p > 0.05. G, OCR measured over time. Data are presented as -fold induction from basal OCR presented in F. Each plot represents the mean of 7 wells with 50 islets each from wild type (n = 3) or βLKB (n = 4) mice. Mice were 2.5 months old. Compounds injected at indicated times were glucose (20 mm), FCCP (1 μm), and rotenone plus antimycin A (R/A, 5 μm each). H, OCR in the presence of glutamine and leucine (Gln+Leu). Protocol is as described in G. Each plot represents the mean of 6 or 7 wells with 50 islets from wild type (n = 5) or βLKB (n = 4) mice. Mice were 6 month old. The assay were performed on Pdx1-CreERTM;LKB1 lox/lox mice, except for ATP measurements (C–E) that were performed on Ins1-Cre;LKB1lox/lox mice aged 10 weeks.
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Figure 4: Lkb1 deletion in β cells disrupts mitochondrial function.A, quantification of fluorescence intensity of TMRE (left) and MitoTracker Green (MTG, right) in islet cells from lox/lox and βLKB mice. Mice were 3 months of age, 2 months post-tamoxifen injection. The graph presents the mean of five mice in each genotype. 20–30 fields were imaged and averaged per mouse. The difference in fluorescence intensity of TMRE is translated to Δ 3 mV between lox/lox and βLKB cells. B, NAD(P)H production in response to 16.7 mm glucose. The plots represent the mean of NAD(P)H-derived fluorescence intensity in whole islets from lox/lox (n = 4) and βLKB (n = 4) mice measured by UV autofluorescence. Islets were perifused with 2.8 mm glucose for 12 min then with 16.7 mm for 15 min and back to 2.8 mm. Mice were 6 months old, 5 months post-tamoxifen injection. Right, mean of area under the curve (AUC) of the NAD(P)H plots. *, p < 0.05. C, cytosolic ATP/ADP ratio changes in islets in response to glucose. Glucose was changed during the experiment from 3 mm (G3) to 11 mm (G11). Note the significantly impaired response in βLKB1 islets. AU, arbitrary units. Data are from three wild type and three βLKB mice. D, amplitude of responsiveness presented in C. E, ATP/ADP ratio presented as % of islets responsive to glucose. *, p < 0.05. F, basal OCR measured by Seahorse XF24 analyzer in the presence of 2.8 mm glucose. ns, p > 0.05. G, OCR measured over time. Data are presented as -fold induction from basal OCR presented in F. Each plot represents the mean of 7 wells with 50 islets each from wild type (n = 3) or βLKB (n = 4) mice. Mice were 2.5 months old. Compounds injected at indicated times were glucose (20 mm), FCCP (1 μm), and rotenone plus antimycin A (R/A, 5 μm each). H, OCR in the presence of glutamine and leucine (Gln+Leu). Protocol is as described in G. Each plot represents the mean of 6 or 7 wells with 50 islets from wild type (n = 5) or βLKB (n = 4) mice. Mice were 6 month old. The assay were performed on Pdx1-CreERTM;LKB1 lox/lox mice, except for ATP measurements (C–E) that were performed on Ins1-Cre;LKB1lox/lox mice aged 10 weeks.

Mentions: Tamoxifen (Sigma, 20 mg/ml in corn oil) was injected subcutaneously to adult mice (1–2 months old). Two daily doses of 8 mg were used to achieve near total deletion of LKB1 in β cells, and animals were studied 2–16 months later. Because recombination occurred in utero in Ins1-Cre;LKB1lox/lox mice (30), these animals were used at younger ages (8–12 weeks) as indicated in Fig. 4. Glyburide and Nifedipine were injected intraperitoneally at the indicated doses. Measurements of blood glucose and serum insulin were performed as described elsewhere (31). The joint ethics committee (Institutional Animal Care and Use Committees) of the Hebrew University and Hadassah Medical Center and the United Kingdom Home Office (PPL 70/06608) approved the study protocol for animal welfare. The Hebrew University is an AAALAC International-accredited institute.


Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects.

Swisa A, Granot Z, Tamarina N, Sayers S, Bardeesy N, Philipson L, Hodson DJ, Wikstrom JD, Rutter GA, Leibowitz G, Glaser B, Dor Y - J. Biol. Chem. (2015)

Lkb1 deletion in β cells disrupts mitochondrial function.A, quantification of fluorescence intensity of TMRE (left) and MitoTracker Green (MTG, right) in islet cells from lox/lox and βLKB mice. Mice were 3 months of age, 2 months post-tamoxifen injection. The graph presents the mean of five mice in each genotype. 20–30 fields were imaged and averaged per mouse. The difference in fluorescence intensity of TMRE is translated to Δ 3 mV between lox/lox and βLKB cells. B, NAD(P)H production in response to 16.7 mm glucose. The plots represent the mean of NAD(P)H-derived fluorescence intensity in whole islets from lox/lox (n = 4) and βLKB (n = 4) mice measured by UV autofluorescence. Islets were perifused with 2.8 mm glucose for 12 min then with 16.7 mm for 15 min and back to 2.8 mm. Mice were 6 months old, 5 months post-tamoxifen injection. Right, mean of area under the curve (AUC) of the NAD(P)H plots. *, p < 0.05. C, cytosolic ATP/ADP ratio changes in islets in response to glucose. Glucose was changed during the experiment from 3 mm (G3) to 11 mm (G11). Note the significantly impaired response in βLKB1 islets. AU, arbitrary units. Data are from three wild type and three βLKB mice. D, amplitude of responsiveness presented in C. E, ATP/ADP ratio presented as % of islets responsive to glucose. *, p < 0.05. F, basal OCR measured by Seahorse XF24 analyzer in the presence of 2.8 mm glucose. ns, p > 0.05. G, OCR measured over time. Data are presented as -fold induction from basal OCR presented in F. Each plot represents the mean of 7 wells with 50 islets each from wild type (n = 3) or βLKB (n = 4) mice. Mice were 2.5 months old. Compounds injected at indicated times were glucose (20 mm), FCCP (1 μm), and rotenone plus antimycin A (R/A, 5 μm each). H, OCR in the presence of glutamine and leucine (Gln+Leu). Protocol is as described in G. Each plot represents the mean of 6 or 7 wells with 50 islets from wild type (n = 5) or βLKB (n = 4) mice. Mice were 6 month old. The assay were performed on Pdx1-CreERTM;LKB1 lox/lox mice, except for ATP measurements (C–E) that were performed on Ins1-Cre;LKB1lox/lox mice aged 10 weeks.
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Figure 4: Lkb1 deletion in β cells disrupts mitochondrial function.A, quantification of fluorescence intensity of TMRE (left) and MitoTracker Green (MTG, right) in islet cells from lox/lox and βLKB mice. Mice were 3 months of age, 2 months post-tamoxifen injection. The graph presents the mean of five mice in each genotype. 20–30 fields were imaged and averaged per mouse. The difference in fluorescence intensity of TMRE is translated to Δ 3 mV between lox/lox and βLKB cells. B, NAD(P)H production in response to 16.7 mm glucose. The plots represent the mean of NAD(P)H-derived fluorescence intensity in whole islets from lox/lox (n = 4) and βLKB (n = 4) mice measured by UV autofluorescence. Islets were perifused with 2.8 mm glucose for 12 min then with 16.7 mm for 15 min and back to 2.8 mm. Mice were 6 months old, 5 months post-tamoxifen injection. Right, mean of area under the curve (AUC) of the NAD(P)H plots. *, p < 0.05. C, cytosolic ATP/ADP ratio changes in islets in response to glucose. Glucose was changed during the experiment from 3 mm (G3) to 11 mm (G11). Note the significantly impaired response in βLKB1 islets. AU, arbitrary units. Data are from three wild type and three βLKB mice. D, amplitude of responsiveness presented in C. E, ATP/ADP ratio presented as % of islets responsive to glucose. *, p < 0.05. F, basal OCR measured by Seahorse XF24 analyzer in the presence of 2.8 mm glucose. ns, p > 0.05. G, OCR measured over time. Data are presented as -fold induction from basal OCR presented in F. Each plot represents the mean of 7 wells with 50 islets each from wild type (n = 3) or βLKB (n = 4) mice. Mice were 2.5 months old. Compounds injected at indicated times were glucose (20 mm), FCCP (1 μm), and rotenone plus antimycin A (R/A, 5 μm each). H, OCR in the presence of glutamine and leucine (Gln+Leu). Protocol is as described in G. Each plot represents the mean of 6 or 7 wells with 50 islets from wild type (n = 5) or βLKB (n = 4) mice. Mice were 6 month old. The assay were performed on Pdx1-CreERTM;LKB1 lox/lox mice, except for ATP measurements (C–E) that were performed on Ins1-Cre;LKB1lox/lox mice aged 10 weeks.
Mentions: Tamoxifen (Sigma, 20 mg/ml in corn oil) was injected subcutaneously to adult mice (1–2 months old). Two daily doses of 8 mg were used to achieve near total deletion of LKB1 in β cells, and animals were studied 2–16 months later. Because recombination occurred in utero in Ins1-Cre;LKB1lox/lox mice (30), these animals were used at younger ages (8–12 weeks) as indicated in Fig. 4. Glyburide and Nifedipine were injected intraperitoneally at the indicated doses. Measurements of blood glucose and serum insulin were performed as described elsewhere (31). The joint ethics committee (Institutional Animal Care and Use Committees) of the Hebrew University and Hadassah Medical Center and the United Kingdom Home Office (PPL 70/06608) approved the study protocol for animal welfare. The Hebrew University is an AAALAC International-accredited institute.

Bottom Line: However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood.Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway.This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

Show MeSH
Related in: MedlinePlus