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Targeted inactivation of kinesin-1 in pancreatic β-cells in vivo leads to insulin secretory deficiency.

Cui J, Wang Z, Cheng Q, Lin R, Zhang XM, Leung PS, Copeland NG, Jenkins NA, Yao KM, Huang JD - Diabetes (2010)

Bottom Line: In this study, we examined the in vivo physiological role of Kinesin-1 in β-cell development and function.However, compared with controls, pancreas of Kif5b(fl/)⁻:RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in β-cells.In addition to being essential for maintaining glucose homeostasis and regulating β-cell function, Kif5b may be involved in β-cell development by regulating β-cell proliferation and insulin vesicle synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Hong Kong, Hong Kong.

ABSTRACT

Objective: Suppression of Kinesin-1 by antisense oligonucleotides, or overexpression of dominant-negative acting kinesin heavy chain, has been reported to affect the sustained phase of glucose-stimulated insulin secretion in β-cells in vitro. In this study, we examined the in vivo physiological role of Kinesin-1 in β-cell development and function.

Research design and methods: A Cre-LoxP strategy was used to generate conditional knockout mice in which the Kif5b gene is specifically inactivated in pancreatic β-cells. Physiological and histological analyses were carried out in Kif5b knockout mice as well as littermate controls.

Results: Mice with β-cell specific deletion of Kif5b (Kif5b(fl/)⁻:RIP2-Cre) displayed significantly retarded growth as well as slight hyperglycemia in both nonfasting and 16-h fasting conditions compared with control littermates. In addition, Kif5b(fl/)⁻:RIP2-Cre mice displayed significant glucose intolerance, which was not due to insulin resistance but was related to an insulin secretory defect in response to glucose challenge. These defects of β-cell function in mutant mice were not coupled with observable changes in islet morphology, islet cell composition, or β-cell size. However, compared with controls, pancreas of Kif5b(fl/)⁻:RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in β-cells.

Conclusions: In addition to being essential for maintaining glucose homeostasis and regulating β-cell function, Kif5b may be involved in β-cell development by regulating β-cell proliferation and insulin vesicle synthesis.

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Related in: MedlinePlus

Kif5bfl/−:RIP2-Cre mice exhibited reduced islet size in pancreas. A: Representative microphotographs of pancreatic sections from a control (left) and a mutant mouse (right) stained by insulin antibody showed the presence of multiples of small islets. B: Statistical analysis of the average size of islets in pancreatic samples from control (black bar) and mutant mice (white bar) (all islets in ten whole sections [>300 μm apart] from three wild-type mice and mutant mice were photographed and analyzed). C: Total islet area expressed as the percentage of total pancreas area. D: Number of islets per 10 mm2 of total pancreatic area. E: Histomorphometric analysis of the size and number of islets in these pancreatic samples from control and mutant mice. F: Mean islet cell size in control and mutant mice. G: Frequency of BrdU+ staining nuclei in islets as the percentage of total islet nuclei (59 and 62 islets corresponding to three wild-type and three mutant mice, respectively, were imaged and counted). Scale bar = 200 μm. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)
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Figure 8: Kif5bfl/−:RIP2-Cre mice exhibited reduced islet size in pancreas. A: Representative microphotographs of pancreatic sections from a control (left) and a mutant mouse (right) stained by insulin antibody showed the presence of multiples of small islets. B: Statistical analysis of the average size of islets in pancreatic samples from control (black bar) and mutant mice (white bar) (all islets in ten whole sections [>300 μm apart] from three wild-type mice and mutant mice were photographed and analyzed). C: Total islet area expressed as the percentage of total pancreas area. D: Number of islets per 10 mm2 of total pancreatic area. E: Histomorphometric analysis of the size and number of islets in these pancreatic samples from control and mutant mice. F: Mean islet cell size in control and mutant mice. G: Frequency of BrdU+ staining nuclei in islets as the percentage of total islet nuclei (59 and 62 islets corresponding to three wild-type and three mutant mice, respectively, were imaged and counted). Scale bar = 200 μm. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)

Mentions: The subcellular localization of insulin vesicles was then analyzed and found to not be affected significantly by the decreased Kif5b level. The cytoplasm of both wild-type and mutant β-cells were filled with insulin vesicles. Insulin vesicle numbers per square μm were determined by counting all insulin vesicles in randomly photographed β-cells (Fig. 7C). More insulin granules were found in Kif5b knockout β-cells compared with control cells. This phenomenon is consistent with the observation that insulin secretion by β-cells is affected. Interestingly, a number of small islets were observed in mutant mice as shown in the representative microphotographs in Figure 8A. Quantitation of the islet number and size confirmed that the average size of the islet in mutants was smaller than that of the wild-type mice (Fig. 8B). However, the total islet mass was not decreased in Kif5b deficient mice (Fig. 8C), concomitant with an increase of islet number (Fig. 8D). Histomorphometric analysis of the size and number of islets in these pancreatic specimens showed that the number of islets <4,000 μm2 was significantly increased in Kif5b conditional knockout mice. However, the number of islets >4,000 μm2 in mutant mice was comparable to that of wild-type control (Fig. 8E). No reduction of islet cell size was observed in Kif5b deficient mice (Fig. 8F); therefore, the major factor accounting for reduced islet size was reduced cell number in an islet. We analyzed islet cell proliferation rates in pancreatic sections obtained from 1-month-old mice by BrdU+ labeling. As shown in Figure 8G, islet cell proliferation was reduced by ∼50% relative to the wild-type control. There was no detectable increase in islet β-cell apoptotic rate based on TUNEL+ analysis (supplementary Fig. 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1078/DC1).


Targeted inactivation of kinesin-1 in pancreatic β-cells in vivo leads to insulin secretory deficiency.

Cui J, Wang Z, Cheng Q, Lin R, Zhang XM, Leung PS, Copeland NG, Jenkins NA, Yao KM, Huang JD - Diabetes (2010)

Kif5bfl/−:RIP2-Cre mice exhibited reduced islet size in pancreas. A: Representative microphotographs of pancreatic sections from a control (left) and a mutant mouse (right) stained by insulin antibody showed the presence of multiples of small islets. B: Statistical analysis of the average size of islets in pancreatic samples from control (black bar) and mutant mice (white bar) (all islets in ten whole sections [>300 μm apart] from three wild-type mice and mutant mice were photographed and analyzed). C: Total islet area expressed as the percentage of total pancreas area. D: Number of islets per 10 mm2 of total pancreatic area. E: Histomorphometric analysis of the size and number of islets in these pancreatic samples from control and mutant mice. F: Mean islet cell size in control and mutant mice. G: Frequency of BrdU+ staining nuclei in islets as the percentage of total islet nuclei (59 and 62 islets corresponding to three wild-type and three mutant mice, respectively, were imaged and counted). Scale bar = 200 μm. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)
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Figure 8: Kif5bfl/−:RIP2-Cre mice exhibited reduced islet size in pancreas. A: Representative microphotographs of pancreatic sections from a control (left) and a mutant mouse (right) stained by insulin antibody showed the presence of multiples of small islets. B: Statistical analysis of the average size of islets in pancreatic samples from control (black bar) and mutant mice (white bar) (all islets in ten whole sections [>300 μm apart] from three wild-type mice and mutant mice were photographed and analyzed). C: Total islet area expressed as the percentage of total pancreas area. D: Number of islets per 10 mm2 of total pancreatic area. E: Histomorphometric analysis of the size and number of islets in these pancreatic samples from control and mutant mice. F: Mean islet cell size in control and mutant mice. G: Frequency of BrdU+ staining nuclei in islets as the percentage of total islet nuclei (59 and 62 islets corresponding to three wild-type and three mutant mice, respectively, were imaged and counted). Scale bar = 200 μm. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)
Mentions: The subcellular localization of insulin vesicles was then analyzed and found to not be affected significantly by the decreased Kif5b level. The cytoplasm of both wild-type and mutant β-cells were filled with insulin vesicles. Insulin vesicle numbers per square μm were determined by counting all insulin vesicles in randomly photographed β-cells (Fig. 7C). More insulin granules were found in Kif5b knockout β-cells compared with control cells. This phenomenon is consistent with the observation that insulin secretion by β-cells is affected. Interestingly, a number of small islets were observed in mutant mice as shown in the representative microphotographs in Figure 8A. Quantitation of the islet number and size confirmed that the average size of the islet in mutants was smaller than that of the wild-type mice (Fig. 8B). However, the total islet mass was not decreased in Kif5b deficient mice (Fig. 8C), concomitant with an increase of islet number (Fig. 8D). Histomorphometric analysis of the size and number of islets in these pancreatic specimens showed that the number of islets <4,000 μm2 was significantly increased in Kif5b conditional knockout mice. However, the number of islets >4,000 μm2 in mutant mice was comparable to that of wild-type control (Fig. 8E). No reduction of islet cell size was observed in Kif5b deficient mice (Fig. 8F); therefore, the major factor accounting for reduced islet size was reduced cell number in an islet. We analyzed islet cell proliferation rates in pancreatic sections obtained from 1-month-old mice by BrdU+ labeling. As shown in Figure 8G, islet cell proliferation was reduced by ∼50% relative to the wild-type control. There was no detectable increase in islet β-cell apoptotic rate based on TUNEL+ analysis (supplementary Fig. 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1078/DC1).

Bottom Line: In this study, we examined the in vivo physiological role of Kinesin-1 in β-cell development and function.However, compared with controls, pancreas of Kif5b(fl/)⁻:RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in β-cells.In addition to being essential for maintaining glucose homeostasis and regulating β-cell function, Kif5b may be involved in β-cell development by regulating β-cell proliferation and insulin vesicle synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Hong Kong, Hong Kong.

ABSTRACT

Objective: Suppression of Kinesin-1 by antisense oligonucleotides, or overexpression of dominant-negative acting kinesin heavy chain, has been reported to affect the sustained phase of glucose-stimulated insulin secretion in β-cells in vitro. In this study, we examined the in vivo physiological role of Kinesin-1 in β-cell development and function.

Research design and methods: A Cre-LoxP strategy was used to generate conditional knockout mice in which the Kif5b gene is specifically inactivated in pancreatic β-cells. Physiological and histological analyses were carried out in Kif5b knockout mice as well as littermate controls.

Results: Mice with β-cell specific deletion of Kif5b (Kif5b(fl/)⁻:RIP2-Cre) displayed significantly retarded growth as well as slight hyperglycemia in both nonfasting and 16-h fasting conditions compared with control littermates. In addition, Kif5b(fl/)⁻:RIP2-Cre mice displayed significant glucose intolerance, which was not due to insulin resistance but was related to an insulin secretory defect in response to glucose challenge. These defects of β-cell function in mutant mice were not coupled with observable changes in islet morphology, islet cell composition, or β-cell size. However, compared with controls, pancreas of Kif5b(fl/)⁻:RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in β-cells.

Conclusions: In addition to being essential for maintaining glucose homeostasis and regulating β-cell function, Kif5b may be involved in β-cell development by regulating β-cell proliferation and insulin vesicle synthesis.

Show MeSH
Related in: MedlinePlus