Glucose transport by epithelia prepared from harvested enterocytes

Yasuhiro Kimura; Marie van der Merwe; Stine Brandt Bering; Himabindu Penmatsa; Veronica G Conoley; Per Torp Sangild; Anjaparavanda P Naren; Randal K Buddington

doi:10.1007/s10616-013-9656-1

Glucose transport by epithelia prepared from harvested enterocytes

Yasuhiro Kimura, Marie van der Merwe, Stine Brandt Bering, Himabindu Penmatsa, Veronica G Conoley, Per Torp Sangild, Anjaparavanda P Naren, Randal K Buddington

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Abstract

Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer (14)C D-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell-cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5'-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of (14)C D-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli.

Original language	English
Journal	Cytotechnology
Volume	67
Issue number	1
Pages (from-to)	39-49
Number of pages	11
ISSN	0920-9069
DOIs	https://doi.org/10.1007/s10616-013-9656-1
Publication status	Published - Jan 2013

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Kimura et al_Cytotechnology_2015_Vol 67(1)_39-49Final published version, 1.3 MBLicence: CC BY

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title = "Glucose transport by epithelia prepared from harvested enterocytes",

abstract = "Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer (14)C D-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell-cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5'-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of (14)C D-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli.",

author = "Yasuhiro Kimura and {van der Merwe}, Marie and Bering, {Stine Brandt} and Himabindu Penmatsa and Conoley, {Veronica G} and Sangild, {Per Torp} and Naren, {Anjaparavanda P} and Buddington, {Randal K}",

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T1 - Glucose transport by epithelia prepared from harvested enterocytes

AU - Kimura, Yasuhiro

AU - van der Merwe, Marie

AU - Bering, Stine Brandt

AU - Penmatsa, Himabindu

AU - Conoley, Veronica G

AU - Sangild, Per Torp

AU - Naren, Anjaparavanda P

AU - Buddington, Randal K

N1 - CURIS 2015 NEXS 050

PY - 2013/1

Y1 - 2013/1

N2 - Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer (14)C D-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell-cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5'-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of (14)C D-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli.

AB - Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer (14)C D-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell-cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5'-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of (14)C D-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli.

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DO - 10.1007/s10616-013-9656-1

M3 - Journal article

C2 - 24166597

SN - 0920-9069

VL - 67

SP - 39

EP - 49

JO - Cytotechnology

JF - Cytotechnology

IS - 1

ER -

Glucose transport by epithelia prepared from harvested enterocytes

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