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Cited by 107Neuroinflammation and Neurodegeneration MechanismsDiabetes Treatment and ManagementAdipokines, Inflammation, and Metabolic Diseases

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GLP-1 secretion by microglial cells and decreased CNS expression in obesity

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Kappe, Camilla, Tracy, Linda M, Patrone, Cesare, Iverfeldt, Kerstin, Sjöholm, Åke·2012·Journal of Neuroinflammation
DOI 10.1186/1742-2094-9-276PMC PMC3546916
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Abstract

BACKGROUND: Type 2 diabetes (T2D) is a strong risk factor for developing neurodegenerative pathologies. T2D patients have a deficiency in the intestinal incretin hormone GLP-1, which has been shown to exert neuroprotective and anti-inflammatory properties in the brain. METHODS: Here we investigate potential sources of GLP-1 in the CNS and the effect of diabetic conditions on the proglucagon mRNA expression in the CNS. The obese mouse model ob/ob, characterized by its high levels of free fatty acids, and the microglia cell line BV-2 were used as models. mRNA expression and protein secretion were analyzed by qPCR, immunofluorescence and ELISA. RESULTS: We show evidence for microglia as a central source of GLP-1 secretion. Furthermore, we observed that expression and secretion are stimulated by cAMP and dependent on microglial activation state. We also show that insulin-resistant conditions reduce the central mRNA expression of proglucagon. CONCLUSION: The findings that microglial mRNA expression of proglucagon and GLP-1 protein expression are affected by high levels of free fatty acids and that both mRNA expression levels of proglucagon and secretion levels of GLP-1 are affected by inflammatory stimuli could be of pathogenic importance for the premature neurodegeneration and cognitive decline commonly seen in T2D patients, and they may also be harnessed to advantage in therapeutic efforts to prevent or treat such disorders.

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Figure 1 The number of GLP-1-positive cells in the frontal cortex is reduced in an obese insulin-resistant mouse model. A. Representative images from the frontal cortex adjacent to the hippocampus of 36-week-old mice showing relative abundance of GLP-1-positive cells (brown) in C57 black control animals (top left panel) —increased magnification (100 X) of GLP-1-positive cells (top right panel) —as compared to obese insulin-resistant ob/ob mice (bottom panel). Scale bar: 80 μm. B. Statistical analysis showing a significant decrease in the number of GLP-1-positive cells/area—in the frontal cortex adjacent to the hippocampus—in obese insulin-resistant ob/ob mice as compared to healthy controls. Data from 4 C57 black mice and 4 ob/ob mice. Bars represent mean ± SEM. * P < 0.05 for a chance difference vs. C57 black mice. Statistical analysis was performed using Mann-Whitney rank sum test.
Figure 1 The number of GLP-1-positive cells in the frontal cortex is reduced in an obese insulin-resistant mouse model. A. Representative images from the frontal cortex adjacent to the hippocampus of 36-week-old mice showing relative abundance of GLP-1-positive cells (brown) in C57 black control animals (top left panel) —increased magnification (100 X) of GLP-1-positive cells (top right panel) —as compared to obese insulin-resistant ob/ob mice (bottom panel). Scale bar: 80 μm. B. Statistical analysis showing a significant decrease in the number of GLP-1-positive cells/area—in the frontal cortex adjacent to the hippocampus—in obese insulin-resistant ob/ob mice as compared to healthy controls. Data from 4 C57 black mice and 4 ob/ob mice. Bars represent mean ± SEM. * P < 0.05 for a chance difference vs. C57 black mice. Statistical analysis was performed using Mann-Whitney rank sum test.CC BY · PubMed Central
Figure 2 CNS proglucagon mRNA expression in the frontal cortex is reduced in an obese insulin-resistant mouse model. A. Proglucagon mRNA expression in obese ob/ob mice as compared to C57 black littermates. Data are from two groups of adult mice, in total 6 C57 black mice and 9 ob/ob mice, 6-9 months of age. B. TNFα mRNA expression in obese ob/ob mice as compared to C57 black littermates. Data from the same animals as in A. Proglucagon and TNFα mRNA expression was normalized with mRNA expression of GAPDH, which was used as an internal control. Bars represent mean ± SEM. *P < 0.05 for a chance difference. Statistical analysis was performed using Mann-Whitney rank sum test. C. Correlation between proglucagon mRNA and TNFα mRNA. Data from three C57 black mice and five ob/ob mice, 6 months of age; qPCR run in duplicate at two independent occasions. A.u.; arbitrary units.
Figure 2 CNS proglucagon mRNA expression in the frontal cortex is reduced in an obese insulin-resistant mouse model. A. Proglucagon mRNA expression in obese ob/ob mice as compared to C57 black littermates. Data are from two groups of adult mice, in total 6 C57 black mice and 9 ob/ob mice, 6-9 months of age. B. TNFα mRNA expression in obese ob/ob mice as compared to C57 black littermates. Data from the same animals as in A. Proglucagon and TNFα mRNA expression was normalized with mRNA expression of GAPDH, which was used as an internal control. Bars represent mean ± SEM. *P < 0.05 for a chance difference. Statistical analysis was performed using Mann-Whitney rank sum test. C. Correlation between proglucagon mRNA and TNFα mRNA. Data from three C57 black mice and five ob/ob mice, 6 months of age; qPCR run in duplicate at two independent occasions. A.u.; arbitrary units.CC BY · PubMed Central
Figure 3 Microglia express proglucagon mRNA and express and secrete GLP-1. GLP-1-positive cells in A, tissue sections from the murine cortex, and B, primary glial cell cultures, GLP-1-positive (green, top right panel) microglia (red, top left panel). Cells positive for GLP-1 are also positive for the microglia marker CD11b (bottom left panel), whereas the remaining glial cells in the culture (indicated by blue DAPI staining in the bottom right panel) are positive for neither GLP-1 nor CD11b. White arrows indicate cells double-positive for CD11b and GLP-1. C. BV-2 cells secrete GLP-1, and this secretion is stimulated in response to a 2-h treatment with 10 μM of the adenylyl cyclase activator forskolin. GLP-1 secretion was measured as pM and presented as percent of control. D. Expression of proglucagon is increased in response to a 2-h treatment with 10 μM forskolin. Proglucagon mRNA expression was normalized to mRNA expression of GAPDH, which was used as an internal control. Bars represent mean ± SEM, n = 3-4, in triplicates. * P < 0.05 for a chance difference vs. untreated controls. Statistical analysis was performed using Student’s t-test.
Figure 3 Microglia express proglucagon mRNA and express and secrete GLP-1. GLP-1-positive cells in A, tissue sections from the murine cortex, and B, primary glial cell cultures, GLP-1-positive (green, top right panel) microglia (red, top left panel). Cells positive for GLP-1 are also positive for the microglia marker CD11b (bottom left panel), whereas the remaining glial cells in the culture (indicated by blue DAPI staining in the bottom right panel) are positive for neither GLP-1 nor CD11b. White arrows indicate cells double-positive for CD11b and GLP-1. C. BV-2 cells secrete GLP-1, and this secretion is stimulated in response to a 2-h treatment with 10 μM of the adenylyl cyclase activator forskolin. GLP-1 secretion was measured as pM and presented as percent of control. D. Expression of proglucagon is increased in response to a 2-h treatment with 10 μM forskolin. Proglucagon mRNA expression was normalized to mRNA expression of GAPDH, which was used as an internal control. Bars represent mean ± SEM, n = 3-4, in triplicates. * P < 0.05 for a chance difference vs. untreated controls. Statistical analysis was performed using Student’s t-test.CC BY · PubMed Central
Figure 4 Microglial GLP-1 secretion is decreased in response to inflammatory stimuli.A. TNFα secretion is increased in response to 2-h and 24-h LPS treatment. TNFα secretion was measured as pg/ml and presented as percent of control. B. Expression of proglucagon is transiently increased in response to a 2-h treatment with LPS, with a clear trend toward reduced levels after 24 h. Proglucagon mRNA expression was normalized to mRNA expression of GAPDH, which was used as an internal control. C. Secretion of GLP-1 from BV-2 cells is inhibited by a 2-h treatment with 1 μg/ml LPS. GLP-1 secretion was measured as pM and presented as percent of control. Bars represent mean ± SEM, n = 3-4, in triplicates. * P < 0.05 for a chance difference vs. untreated controls. Statistical analysis was performed using Mann-Whitney rank sum test (A,B) and Student’s t-test (C).
Figure 4 Microglial GLP-1 secretion is decreased in response to inflammatory stimuli.A. TNFα secretion is increased in response to 2-h and 24-h LPS treatment. TNFα secretion was measured as pg/ml and presented as percent of control. B. Expression of proglucagon is transiently increased in response to a 2-h treatment with LPS, with a clear trend toward reduced levels after 24 h. Proglucagon mRNA expression was normalized to mRNA expression of GAPDH, which was used as an internal control. C. Secretion of GLP-1 from BV-2 cells is inhibited by a 2-h treatment with 1 μg/ml LPS. GLP-1 secretion was measured as pM and presented as percent of control. Bars represent mean ± SEM, n = 3-4, in triplicates. * P < 0.05 for a chance difference vs. untreated controls. Statistical analysis was performed using Mann-Whitney rank sum test (A,B) and Student’s t-test (C).CC BY · PubMed Central
Figure 5 Proglucagon gene expression in microglia is decreased by the free fatty acid palmitate. Microglia were exposed to 0.125 mM palmitate for 24 h. A. Proglucagon gene expression is significantly reduced in microglia exposed to palmitate (mimicking the diabetic milieu in vitro). B. GLP-1 secretion is not significantly reduced after treatment of microglia with palmitate. GLP-1 secretion was measured as pM and presented as percent of control. C. No significant effect on TNFα secretion is detected after a 2-h exposure to palmitate; however, TNFα secretion is reduced from microglia treated with palmitate for 24 h. TNFα secretion was measured as pg/ml and presented as percent of control. D. TNFα expression is significantly reduced in microglia exposed to palmitate. E. Treatment with palmitate increases the expression of chitinase-3 and arginase 1. F. Treatment with LPS decreases the expression of arginase 1 after 24 h. mRNA expression was calculated after normalizing to mRNA expression of GAPDH, used as an internal control. Bars represent mean ± SEM, n = 3, in triplicates. * and **P < 0.05 and P < 0.001, respectively, for chance differences vs. untreated controls. Statistical analysis was performed using Mann-Whitney rank sum test and Student’s t-test (C,D).
Figure 5 Proglucagon gene expression in microglia is decreased by the free fatty acid palmitate. Microglia were exposed to 0.125 mM palmitate for 24 h. A. Proglucagon gene expression is significantly reduced in microglia exposed to palmitate (mimicking the diabetic milieu in vitro). B. GLP-1 secretion is not significantly reduced after treatment of microglia with palmitate. GLP-1 secretion was measured as pM and presented as percent of control. C. No significant effect on TNFα secretion is detected after a 2-h exposure to palmitate; however, TNFα secretion is reduced from microglia treated with palmitate for 24 h. TNFα secretion was measured as pg/ml and presented as percent of control. D. TNFα expression is significantly reduced in microglia exposed to palmitate. E. Treatment with palmitate increases the expression of chitinase-3 and arginase 1. F. Treatment with LPS decreases the expression of arginase 1 after 24 h. mRNA expression was calculated after normalizing to mRNA expression of GAPDH, used as an internal control. Bars represent mean ± SEM, n = 3, in triplicates. * and **P < 0.05 and P < 0.001, respectively, for chance differences vs. untreated controls. Statistical analysis was performed using Mann-Whitney rank sum test and Student’s t-test (C,D).CC BY · PubMed Central

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Metadata

Authors
Kappe, C., Tracy, L. M., Patrone, C., Iverfeldt, K., & Sjöholm, Å.
Affiliations
  • Stockholm University (SE)
  • Karolinska Institutet (SE)
Corresponding author
  • Tracy, Linda M
  • Iverfeldt, Kerstin
  • Sjöholm, Åke
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2012-12
Volume
9
Issue
1
Pages
276-276
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-9-276
PMC
PMC3546916
OpenAlex
W2112292567
Cited by
107
References
32
Citations by year
2026: 6 · 2025: 10 · 2024: 5 · 2023: 9 · 2022: 9 · 2021: 7 · 2020: 11 · 2019: 9 · 2018: 9 · 2017: 10 · 2016: 9 · 2015: 7 · 2014: 3 · 2013: 3
FWCI
2.09
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Gun och Bertil Stohnes Stiftelse
  • AFA Försäkring
  • Karolinska Institutet
  • Stockholms Läns Landsting
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Diabetes Treatment and Management
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adipokines, Inflammation, and Metabolic Diseases
Keywords
glucagon-like peptide-1, proglucagon, microglia, obesity, type 2 diabetes, neurodegeneration, insulin resistance, free fatty acids, cAMP signaling, microglial activation, neuroinflammation, cognitive decline
MeSH terms
Cyclic AMP, Animals, Arginase, Cell Line, Transformed, Central Nervous System, Chitinases, Disease Models, Animal, Gene Expression Regulation, Insulin Resistance, Male, Mice, Inbred C57BL, Mice, Obese, Obesity, Palmitates, Plant Proteins, Polysaccharides, RNA, Messenger, Transfection, Tumor Necrosis Factor-alpha, Microglia, Statistics, Nonparametric, CD11b Antigen, Mice, Glucagon-Like Peptide 1, Proglucagon

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