COVID-19 and metabolic dysregulation: SARS-CoV-2 injures human exocrine and endocrine pancreas

Thu, 06/03/2021 - 17:55

Immunohistochemical staining of paraffin-embedded rat pancreas tissue with insulin antibody (red) and NKX6.1 antibody (green).

Jamshed Arslan, Pharm D, PhD

Humans rely on the pancreas for digesting food and generating energy from it. SARS-CoV-2-mediated damage to the exocrine pancreas is evident from the pancreatitis, pancreatic enlargement, and abnormal levels of digestive enzymes in severe COVID-19. Likewise, increased propensity for hyperglycemia, ketoacidosis, and diabetes in COVID-19 indicates an injury to the endocrine pancreas. As expected, the pancreas does express factors including ACE2 and TMPRSS2 that facilitate viral entry. However, the empirical data on whether and how the virus triggers pancreatic injury, especially to the β-cell, have been missing, until now!

A multinational team of researchers from Germany, Austria and Canada has shown that SARS-CoV-2-infection of the pancreatic β-cells brings about transcriptional and phenotypic changes that reduce insulin-secretory granules and impair the glucose-stimulated insulin secretion. Furthermore, the presence of viral nucleocapsid (N) protein in the pancreatic exocrine cells in people who died of COVID-19 supports the notion of SARS-CoV-2-induced metabolic dysregulation.

Fluorescent immunocytochemical staining of beta TC-6 mouse beta cell insulinoma cell line probed with insulin antibody followed by NorthernLights 557-secondary antibody.

Fluorescent immunocytochemistry image showing insulin expression in beta TC‑6 mouse beta cell insulinoma cell line using Rat anti-Insulin Monoclonal Antibody (MAB1417) then stained with the NorthernLights™ 557-conjugated Secondary Antibody (NL013) (red) and counterstained with DAPI (blue).

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Viral replication in human pancreas

The researchers first validated the expression pattern of all the previously reported isoforms of viral entry proteins (ACE2 and TMPRSS2) in human pancreatic tissue containing exocrine and endocrine cells. To show the ex vivo infection, pancreatic islets from human donors were first exposed to SARS-CoV-2 with/out Remdesivir, a polymerase inhibitor that blocks the virus. On days 3 and 5 post-infection, the viral Spike (S) and N proteins were detectable through immunohistology only in samples without Remdesivir treatment. Quantification further revealed that every infected islet had 20% N-positive cells at day 3, which increased to 34% at day 5. Since only a few infected cells were found to be stained with cleaved caspase-3 and pancreatic hormones C-pep/chromogranin A, the researchers concluded that infection and replication halts hormone expression, initially without significant apoptosis. Likewise, transmission electron microscopy revealed vacuolization and dilatation of the endoplasmic reticulum (ER)-Golgi body complex, indicating ER stress and Golgi body swelling.

The next experimental step sought to detect if the transcriptional signature of viral replication was present.

Immunohistochemical staining of human lung tissue with SARS Nucleocapsid antibody followed by HRP-conjugated secondary antibody, DAB chromogen, and counterstained with hematoxylin.

IHC image of tissue sections of SARS-CoV-2 infected human lung tissue using Rabbit anti-SARS Nucleocapsid Polyclonal Antibody (NB100-56576), followed by HRP-conjugated Secondary Antibody (VC003) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue).

Transcriptional and phenotypic changes in pancreas after SARS-CoV-2 infection

The researchers performed bulk-RNA sequencing and generated a Smart-seq2 expression heatmap to show an innate immune reaction (upregulated interferon-stimulated genes like IFITMs, IFI27, and OAS2) and loss of β-cell functions (downregulation of genes like Synaptotagmin 4, PASK, PEX6, and PLCXD3) in SARS-CoV-2-infected islets. To determine how these ex vivo changes translate to the human pancreas, the team stained various organs from four individuals who died of COVID-19 for viral N-protein. They co-stained the pancreas with a marker of adult β-cells, NKX6.1. Histology revealed that all postmortem samples had clusters of N/NKX6.1 double-positive cells closer to islets of Langerhans, indicating a localized viral spread that leads to hormone loss. The patient who died of pancreatic exocrine insufficiency had the highest viral load according to his nasopharyngeal swab. Elevated lipase levels in another patient pointed towards exocrine damage.

In other words, viral entry receptors (ACE2 and TMPRSS2) of the human pancreas allow SARS-CoV-2 infection and replication, which results in transcriptional and morphological changes indicative of exocrine and endocrine damage.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue probed with anti-NKX6.1 antibody, followed by HRP-DAB staining kit, and counterstained with hematoxylin. Lane view from a simple western with lysates from beta TC-6 mouse beta cell insulinoma cell line probed with anti-NKX6.1 antibody and HRP-conjugated secondary depicting a band specific for NKX6.1 at approximately 54 kDa.

(Left) Paraffin-embedded sections of human pancreas showing NKX6.1 expression detected using Goat Anti-NKX6.1 Antigen Affinity-purified Polyclonal Antibody (AF5857), followed by tissue staining with Anti-HRP-DAB Cell & Tissue Staining Kit (CTS008) (brown), and counterstaining with hematoxylin (blue). (Right) Simple Western lane view depicting lysates from beta TC‑6 mouse beta cell insulinoma cell line highlighting a specific band for NKX6.1 at ~54 kDa (as indicated) using Goat Anti-NKX6.1 Polyclonal Antibody (AF5857) followed by HRP-conjugated Secondary Antibody (HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Concluding remarks

This study provides the ex vivo and in vivo evidence of the SARS-CoV-2 replication in pancreas and its consequences. The postmortem data indicate that our knowledge of the frequency and association of extra-pulmonary infection and pre-existing conditions is limited. Further clinical research is warranted to overcome the limitations of the study, such as low postmortem sample size and compromised tissue quality due to autolytic necrosis.

Muller et al. (2021) used SARS Nucleocapsid Protein Antibody (NB100-56576)

Jamshed ArslanJamshed Arslan, Pharm D, PhD   
Dr Arslan is an Assistant Professor at Salim Habib University (formerly, Barrett Hodgson University), Pakistan. His interest lies in neuropharmacology and preparing future pharmacists.


Research in focus

Müller, J. A., Groß, R., Conzelmann, C., Krüger, J., Merle, U., Steinhart, J., Weil, T., Koepke, L., Bozzo, C. P., Read, C., Fois, G., Eiseler, T., Gehrmann, J., van Vuuren, J., Wessbecher, I. M., Frick, M., Costa, I. G., Breunig, M., Grüner, B., Peters, L., … Kleger, A. (2021). SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreasNature metabolism.

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