Abstract
Objectives
We constructed a recombinant oral GLP-1 analogue in Lactococcus lactis (L. lactis) and evaluated its physiological functions.
Results
In silico docking suggested the alanine at position 8 substituted with serine (A8SGLP-1) reduced binding of DPP4, which translated to reduced cleavage by DPP4 with minimal changes in stability. This was further confirmed by an in vitro enzymatic assay which showed that A8SGLP-1 significantly increased half-life upon DPP4 treatment. In addition, recombinant L. lactis (LL-A8SGLP-1) demonstrated reduced fat mass with no changes in body weight, significant improvement of random glycemic control and reduced systemic inflammation compared with WT GLP-1 in db/db mice.
Conclusion
LL-A8SGLP-1 adopted in live biotherapeutic products reduce blood glucose in db/db mice without affecting its function.
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Data availability
Data used to support the findings of this study are available from the corresponding author upon request.
Abbreviations
- CFU:
-
Colony forming units
- db/db:
-
C57BLKS/J-Lepr-/Lepr- mouse
- DPP4:
-
Dipeptidylpeptidase 4
- EK:
-
Enterokinase
- GLP-1:
-
Glucagon-like peptide 1
- LBP:
-
Live biotherapeutic products
- LEISS:
-
LEISSTCDA synthetic propeptide sequence
- LC–MS:
-
Liquid chromatography-mass spectrometry
- LL-empty:
-
Lactococcus lactis Transformed with empty plasmid
- LL- WT GLP-1:
-
Lactococcus lactis Transformed with wild type GLP-1 plasmid
- LL-A8SGLP-1:
-
Lactococcus lactis Transformed with A8SGLP-1 plasmid
- Usp45:
-
Unknown secreted protein of 45 kDa
- GSIS:
-
Glucose-stimulated insulin secretion
- RG:
-
Random glucose
- oGTT:
-
Oral glucose tolerance test
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Acknowledgements
We thank Dr. Jinkui Yang for providing the INS-1 cell line.
Funding
This study was supported by the General Program (Major Research Plan) of the National Natural Science Foundation of China [92057208], the China Postdoctoral Science Foundation [2021T140665], the Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine [FIRMC180304] and the National Natural Science Foundation of China [81770834].
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Material preparation and GLP-1 and A8SGLP-1 expression system construction was performed by MD. SY analysed the stability of amino acid mutations. HZ and MD finished the in vitro cell experiment. HZ, MD and SY carried out the animal experiments. MD and WJ conceived the project and analysed the results. The first draft of the manuscript was written by HZ and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Zhang, H., Dong, M., Yuan, S. et al. Oral glucagon-like peptide 1 analogue ameliorates glucose intolerance in db/db mice. Biotechnol Lett 44, 1149–1162 (2022). https://doi.org/10.1007/s10529-022-03288-1
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DOI: https://doi.org/10.1007/s10529-022-03288-1