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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

A: Immunohistochemistry analysis of Kif5b expression in wild-type mice pancreas sections (left) and antibody specificity test (right). B: Western blot analysis of Kif5a/Kif5b/Kif5c expression in hypothalamus and isolated islets from mutant and control mice. C: Immunohistochemistry analysis of Kif5b expression level in islets from control (upper) and mutant mouse (lower). (A high-quality color representation of this figure is available in the online issue.)
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Figure 3: A: Immunohistochemistry analysis of Kif5b expression in wild-type mice pancreas sections (left) and antibody specificity test (right). B: Western blot analysis of Kif5a/Kif5b/Kif5c expression in hypothalamus and isolated islets from mutant and control mice. C: Immunohistochemistry analysis of Kif5b expression level in islets from control (upper) and mutant mouse (lower). (A high-quality color representation of this figure is available in the online issue.)

Mentions: Kif5b was mainly expressed in pancreatic islets (endocrine portion) with little expression in exocrine acinar glands (Fig. 3A, left). The positive staining was absent after preincubating the anti-Kif5b antibody with the 18-aa synthesized peptides (Fig. 3A, right). To test the efficiency of RIP-2-Cre mediated pancreatic β-cell specific deletion of exon 2 of Kif5b allele, we examined the protein expression levels of Kif5a, Kif5b, and Kif5c in hypothalamus and isolated islets from both mutant and wild-type mice (Fig. 3B). Inactivation of Kif5b by Cre-induced ablation resulted in a reduction of Kif5b protein level in islets, whereas both Kif5a and Kif5c were not detectable in islets although they are highly expressed in hypothalamus, consistent with an earlier finding that their expression is neuronal specific (29). Although RIP2-Cre displayed a low level of expression in the hypothalamus (24), we could not detect significantly decreased Kif5b expression in the hypothalamus due to Cre expression. In contrast, Western blot results showed that Kif5b level in the isolated islets was reduced by at least 80% in mutant mice, compared with the wild-type. Considering the fact that RIP-2 Cre is only expressed in ∼80–90% of β-cells and that β-cells account for 75% of islet cells, the Western blot result indicated that Kif5b was absent in most β-cells in the mutant mice islets. The knockout efficiency was further demonstrated by immunohistochemistry analysis of Kif5b expression in pancreatic sections under identical conditions. A significant decrease of Kif5b expression was observed in the islets from mutant mice compared with those in wild-type mice (Fig. 3C). Taken together, these results suggested that RIP-2 Cre mediated ablation of exon 2 in Kif5b allele had efficiently occurred, leading to significantly reduced Kif5b expression in the islets of mutant mice.


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)

A: Immunohistochemistry analysis of Kif5b expression in wild-type mice pancreas sections (left) and antibody specificity test (right). B: Western blot analysis of Kif5a/Kif5b/Kif5c expression in hypothalamus and isolated islets from mutant and control mice. C: Immunohistochemistry analysis of Kif5b expression level in islets from control (upper) and mutant mouse (lower). (A high-quality color representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3012189&req=5

Figure 3: A: Immunohistochemistry analysis of Kif5b expression in wild-type mice pancreas sections (left) and antibody specificity test (right). B: Western blot analysis of Kif5a/Kif5b/Kif5c expression in hypothalamus and isolated islets from mutant and control mice. C: Immunohistochemistry analysis of Kif5b expression level in islets from control (upper) and mutant mouse (lower). (A high-quality color representation of this figure is available in the online issue.)
Mentions: Kif5b was mainly expressed in pancreatic islets (endocrine portion) with little expression in exocrine acinar glands (Fig. 3A, left). The positive staining was absent after preincubating the anti-Kif5b antibody with the 18-aa synthesized peptides (Fig. 3A, right). To test the efficiency of RIP-2-Cre mediated pancreatic β-cell specific deletion of exon 2 of Kif5b allele, we examined the protein expression levels of Kif5a, Kif5b, and Kif5c in hypothalamus and isolated islets from both mutant and wild-type mice (Fig. 3B). Inactivation of Kif5b by Cre-induced ablation resulted in a reduction of Kif5b protein level in islets, whereas both Kif5a and Kif5c were not detectable in islets although they are highly expressed in hypothalamus, consistent with an earlier finding that their expression is neuronal specific (29). Although RIP2-Cre displayed a low level of expression in the hypothalamus (24), we could not detect significantly decreased Kif5b expression in the hypothalamus due to Cre expression. In contrast, Western blot results showed that Kif5b level in the isolated islets was reduced by at least 80% in mutant mice, compared with the wild-type. Considering the fact that RIP-2 Cre is only expressed in ∼80–90% of β-cells and that β-cells account for 75% of islet cells, the Western blot result indicated that Kif5b was absent in most β-cells in the mutant mice islets. The knockout efficiency was further demonstrated by immunohistochemistry analysis of Kif5b expression in pancreatic sections under identical conditions. A significant decrease of Kif5b expression was observed in the islets from mutant mice compared with those in wild-type mice (Fig. 3C). Taken together, these results suggested that RIP-2 Cre mediated ablation of exon 2 in Kif5b allele had efficiently occurred, leading to significantly reduced Kif5b expression in the islets of mutant mice.

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