Insulin

 

Description

Insulin is a peptide hormone produced by beta (β) cells of the pancreatic islets considered to be the main anabolic hormone of the body. Insulin is synthesized as a single-chain precursor, preproinsulin, that contains an N-terminal signal sequence and a connecting peptide (C-peptide) linking the A and B chains of the insulin molecule (proinsulin) (Steiner, Cunningham et al. 1967, Dodson and Steiner 1998). Within the endoplasmic reticulum of the β cell, proinsulin is exposed to several specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin; insulin and free C peptide are packaged in the Golgi into secretory granules which accumulate in the cytoplasm for release when systemic glucose level increase. Insulin is not only a biomarker used in the identification of pathologies associated with glucose metabolism such as Type 1 and Type 2 diabetes; the secretion of insulin rises with age, which is related to both increasing body fat mass and ageing itself (Thorens, Rodriguez et al. 2019). Similarly, levels of circulating C peptide are being considered as potential biomarkers for certain cancers and atherosclerotic disease (Murphy, Cross et al. 2015, Cardellini, Farcomeni et al. 2017, Khan, Sobki et al. 2018). Figure 1 demonstrates that while the A and B chains of HUM and RHE insulin have the same sequences, MAR insulin has significant aminoacid differences with HUM insulin. We found one Luminex kit (Biorad) that had reactivity with MAR samples, but no reagents able to recognize MAR C peptide.

 

Alignment

Protein alignment for human, rhesus macaque and marmoset insulin:

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Protein alignment for marmoset, owl monkey, and squirrel monkey insulin:

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References

  • Cardellini, M., A. Farcomeni, M. Ballanti, M. Morelli, F. Davato, I. Cardolini, G. Grappasonni, S. Rizza, V. Guglielmi, O. Porzio, C. Pecchioli, R. Menghini, A. Ippoliti and M. Federici (2017). "C-peptide: A predictor of cardiovascular mortality in subjects with established atherosclerotic disease." Diab Vasc Dis Res 14(5): 395-399.
  • Dodson, G. and D. Steiner (1998). "The role of assembly in insulin's biosynthesis." Curr Opin Struct Biol 8(2): 189-194.
  • Khan, H. A., S. H. Sobki, A. Ekhzaimy, I. Khan and M. A. Almusawi (2018). "Biomarker potential of C-peptide for screening of insulin resistance in diabetic and non-diabetic individuals." Saudi J Biol Sci 25(8): 1729-1732.
  • Murphy, N., A. J. Cross, W. Y. Huang, V. Rajabzadeh-Heshejin, F. Stanczyk, R. Hayes and M. J. Gunter (2015). "A prospective evaluation of C-peptide levels and colorectal adenoma incidence." Cancer Epidemiol 39(2): 160-165.
  • Steiner, D. F., D. Cunningham, L. Spigelman and B. Aten (1967). "Insulin biosynthesis: evidence for a precursor." Science 157(3789): 697-700.
  • Thorens, B., A. Rodriguez, C. Cruciani-Guglielmacci, L. Wigger, M. Ibberson and C. Magnan (2019). "Use of preclinical models to identify markers of type 2 diabetes susceptibility and novel regulators of insulin secretion - A step towards precision medicine." Mol Metab 27S: S147-S154.

 

Status

There were technical difficulties producing a recombinant MAR insulin. Thus, mice were immunized with yeast-produced human insulin. Serological assays with plates coated with the immunizing antigen were successful, but no clones were produced after fusion of mouse splenocytes with myeloma cells. Thus, the decision was to test available anti-human insulin antibodies for crossreactivity against native MAR insulin. The selected anti-insulin antibodies were commercially available mAbs with reported binding to pig, rat, and mouse insulin. The rationale was that if an mAb was able to bind rat or mouse Insulin, there should be also binding to marmoset Insulin. Figure 1 shows the results of a sandwich ELISA using different capture/detection mAb combinations.

MWMIR-Insulin-figure01-20250120
Figure 1. ELISA assay for NWM Insulin. Different capture/detection mAbs were used to detect Insulin in plasma samples from NWM. A recombinant human Insulin was used as positive control.

The outcome of the ELISA assay suggested that the E2E3/2D11-H5 was the only combination able to bind to human Insulin, but it failed to recognize Insulin from any of the three NWM species tested. 

Since the concentration of Insulin in the NWM plasma samples was also unknown, we could not rule out that these mAbs were able to bind to NWM Insulin, but the amount of Insulin present was below the limit of detection. Thus, we performed immunohistochemistry assays with two of these antibodies using marmoset tissues obtained at necropsy. Figure 2 shows that the mAb 2D11-H5 recognizes marmoset Insulin, clearly identifying the Insulin-producing beta islets of Langerhans in pancreas sections; no signal was observed for 2D11-H5 on intestine or lymph node sections. Unfortunately, no signal was observed for the E2-E3 mAb.  
 

MWMIR-Insulin-figure02-20250120
Figure 2. Immunohistochemistry assay with anti-Insulin antibodies on formalin-fixed, paraffin-embedded marmoset tissue sections.

In summary, we have identified a mAb (2D11-H5) that binds to marmoset Insulin. We will continue to test other mAb for reactivity with marmoset Insulin. We will also initiate a new round of immunization with human Insulin linked to a protein carrier.