Human amylin proteotoxicity impairs protein biosynthesis, and alters major cellular signaling pathways in the heart, brain and liver of humanized diabetic rat model in vivo

Abstract

Introduction

Chronic hypersecretion of the 37 amino acid amylin is common in type 2 diabetics (T2D). Recent studies implicate human amylin aggregates cause proteotoxicity (cell death induced by misfolded proteins) in both the brain and the heart.

Objectives

Identify systemic mechanisms/markers by which human amylin associated with cardiac and brain defects might be identified.

Methods

We investigated the metabolic consequences of amyloidogenic and cytotoxic amylin oligomers in heart, brain, liver, and plasma using non-targeted metabolomics analysis in a rat model expressing pancreatic human amylin (HIP model).

Results

Four metabolites were significantly different in three or more of the four compartments (heart, brain, liver, and plasma) in HIP rats. When compared to a T2D rat model, HIP hearts uniquely had significant DECREASES in five amino acids (lysine, alanine, tyrosine, phenylalanine, serine), with phenylalanine decreased across all four tissues investigated, including plasma. In contrast, significantly INCREASED circulating phenylalanine is reported in diabetics in multiple recent studies.

Conclusion

DECREASED phenylalanine may serve as a unique marker of cardiac and brain dysfunction due to hyperamylinemia that can be differentiated from alterations in T2D in the plasma. While the deficiency in phenylalanine was seen across tissues including plasma and could be monitored, reduced tyrosine was seen only in the brain. The 50 % reduction in phenylalanine and tyrosine in HIP brains is significant given their role in supporting brain chemistry as a precursor for catecholamines (dopamine, norepinephrine, epinephrine), which may contribute to the increased morbidity and mortality in diabetics at a multi-system level beyond the effects on glucose metabolism.

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Abbreviations

AbetaPP:

Amyloid beta protein precursor

GABA:

Gamma-aminobutyric acid

HIP:

Rat model expressing pancreatic human amylin

hIAPP:

Human islet amyloid polypeptide

PCA:

Principal component analysis

PLS-DA:

Partial least squares discriminant analysis

VIP:

Variable importance in projection

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Acknowledgments

This work was supported by the National Institutes of Health (R01HL104129 to M.W.; R01HL118474 to F.D.), a Jefferson-Pilot Corporation Fellowship (to M.W.), National Science Foundation (CBET 1357600 to F.D.), Alzheimer’s Association (VMF-15-363458 to FD) and the Leducq Foundation (to M.W.).

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Correspondence to Florin Despa or Monte S. Willis.

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The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Additional information

Amro Ilaiwy and Miao Liu have contributed equally.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Fig. 1. Non-targeted cardiac metabolomics of HIP heart tissue at one year of age. A. Enrichment by pathway-associated metabolite sets. B. Location-Based Metabolite sets determined from t-test significant metabolites identified. Supplementary material 1 (PDF 3304 kb)

Supplemental Fig. 2. Non-targeted cardiac metabolomics of HIP brain tissue at one year of age Enrichment by A. Pathway-associated metabolite sets, B. Location-Based Metabolite sets, and C. Disease-associated metabolite sets in the brain determined from from t-test significant metabolites identified. Supplementary material 2 (PDF 4314 kb)

Supplemental Fig. 3. Non-targeted cardiac metabolomics of HIP liver tissue at one year of age. Enrichment by A. Pathway-associated metabolite sets and B. Location-Based Metabolite sets determined from from t-test significant metabolites identified. Supplementary material 3 (PDF 3213 kb)

Supplemental Fig. 4. Non-targeted cardiac metabolomics of HIP plasma at one year of age. Enrichment by A. Pathway-associated metabolite sets, B. Location-Based Metabolite sets, and C. Disease-associated metabolite sets in the blood determined from from t-test significant metabolites identified. Supplementary material 4 (PDF 4564 kb)

Supplemental Fig. 5. Pathway enrichment analysis of significant metabolite sets identified by non-targeted cardiac metabolomics. A. Heart, B. Brain, C. Liver, and D. Plasma. A-D indicates top pathways identified, along with the specific significant metabolites found that placed it in this category. Supplementary material 5 (PDF 3869 kb)

11306_2016_1022_MOESM6_ESM.pdf

Supplemental Fig. 6. Significantly altered HIP-unique metabolites found by non-targeted metabolomics analysis of the heart using a recently published type 2 diabetes model (Kouzu et al. 2015). Data was normalized to the strain-matched wildtype control, followed by an ANOVA analysis of all four groups and included significantly altered metabolites found in both studies (see supplemental materials and methods for details, Supplemental Table 2 for data used in analysis). A. HIP significant cardiac metabolites, not altered in T2D model (indicated by *). B. HIP significant cardiac amino acids, not altered in T2D model (indicated by *). C. Comparison of HIP transgenic model (current study) and T2D model used in this analysis recently published (Kouzu et al. 2015). Boxes indicate similarly altered metabolites in both HIP and T2D models. A One-Way ANOVA was performed, with Holm-Sidak post hoc comparisons made to the wildtypeHIPTg+. Significance was defined as p < 0.05. Data is mean ± SEM. Supplementary material 6 (PDF 723 kb)

Supplementary material 7 (XLSX 456 kb)

Supplementary material 8 (XLSX 33 kb)

Supplementary material 9 (PPTX 50 kb)

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Ilaiwy, A., Liu, M., Parry, T.L. et al. Human amylin proteotoxicity impairs protein biosynthesis, and alters major cellular signaling pathways in the heart, brain and liver of humanized diabetic rat model in vivo. Metabolomics 12, 95 (2016). https://doi.org/10.1007/s11306-016-1022-9

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Keywords

  • Amylin
  • Metabolomics
  • Neurodegenerative disease
  • Cardiomyopathy
  • Diabetes
  • Proteotoxicity