Clinical Observations on the Link between Diabetes Mellitus and COVID-19
Since the beginning of the pandemic, doctors in different countries have noted that some patients with COVID-19 developed diabetes mellitus against the background of coronavirus infection. For example, in April 2020, doctors in Singapore first diagnosed diabetic ketoacidosis and diabetes mellitus in a 37-year-old man infected with the SARS-CoV-2 coronavirus. In October 2020, German doctors reported that a 19-year-old man had insulin-dependent diabetes 7 weeks after suffering from COVID-19. In November 2020, American doctors published the results of the treatment of an 8-year-old patient who developed complications from COVID-19: the child developed hyperglycemia, ketosis, metabolic acidosis, and diabetic ketoacidosis.
The American Diabetes Association explains, based on data from the Centers for Disease Control and Prevention, that people with diabetes are at risk for severe COVID-19 due to high blood glucose levels associated with coronavirus infection. Hyperglycemia develops in over 50% of people with type I diabetes diagnosed with COVID-19. Diabetic ketoacidosis occurs in almost a third of these patients.
An international team of scientists from the universities of Stanford (USA), Basel (Switzerland), Jikei (Japan) conducted a study to establish whether the severity of COVID-19 is a cause or a consequence of diabetes. Experts have studied the mechanism of infection with the SARS-CoV-2 coronavirus in pancreatic endocrine cells that produce insulin. Scientists investigated the production of insulin by infected β-cells and the factors behind their programmed death in COVID-19.
Study Design
Scientists have compared how pancreatic cells function in people who have died from severe COVID-19, people who have not been infected with coronavirus, and died from other causes: cancer, stroke, head injury, or cardiac arrest. Experts infected healthy cells with the SARS-CoV-2 coronavirus and observed which pancreatic cells are most susceptible to infection. Scientists have investigated the mechanics of the penetration of the coronavirus into cells and the factors contributing to illness. The clinical isolate SARS-CoV-2 of the pandemic variant D614G was used for the experiments.
Scientists focus on the expected limited associated with research. First, all the experiments were conducted on the tissues of a small number of healthy donors and donors who died from COVID-19. Secondly, histological analysis of sections of these tissues gives only an approximate assessment of infection factors and only a general characteristic of the susceptibility of cells to infection. Third, the production of insulin by infected cells was evaluated in the laboratory. They cannot be correlated with glucose and insulin levels in donors’ blood since they have not been tested accordingly. The scientists emphasize that additional studies of the correlation between laboratory and clinical indicators will help to understand better the impact of SARS-CoV-2 on people with severe coronavirus infection.
Research Results
Insulin-Producing Cells Have More Coronavirus Infection Factors Than Other Types of Pancreatic Cells
First, scientists found out which types of pancreatic cells are most predisposed to infection. The largest population of endocrine cells of the pancreas is β-cells that produce insulin. There are about half as many cells that produce glucagon (α-cells) and somatostatin (δ-cells).
In earlier studies, it was found that the SARS-CoV-2 coronavirus requires not only the cellular receptor ACE2 but also the transmembrane serine protease TMPRSS2, the transferrin receptor TFRC, and neuropilin NRP1 to enter the cell. Moreover, the coronavirus can enter the cell bypassing ACE2 and using only TFRC and NRP1. However, cell infection with coronavirus via ACE2 is possible only with the participation of TFRC and NRP1.
By the amount of ACE2, TMPRSS2, TFRC, and NRP1 in the cells, the scientists evaluated the susceptibility of pancreatic cells to infection. Experts have performed joint staining of these proteins with insulin (a marker of β-cells) or glucagon, a marker of α-cells. The scientists used cells from healthy donors not infected with the coronavirus.
As a result, there were no significant differences in the expression of the ACE2 and TMPRSS2 proteins between α-cells and β-cells. However, β-cells had significantly more NRP1 and TFRC proteins than α-cells. This dependence is visible on graph B:
To confirm the result for the NRP1 protein, scientists used a particular antibody against it to determine the presence of the protein in cells. The result was the same: there was more NRP1 in β-cells than in α-cells.
Based on the data obtained, the scientists concluded that the β-cells of the pancreas, which produce insulin, contain more factors contributing to infection with the coronavirus than α-cells that synthesize glucagon.
Coronavirus Infects Cells That Produce Insulin More Strongly Than Cells That Produce Glucagon or Somatostatin
In the next experiment, scientists tested their hypothesis about the increased susceptibility of β-cells of the pancreas, which produce insulin, to infection with the SARS-CoV-2 coronavirus.
For the experiment, experts took islet accumulations of pancreatic endocrine cells from healthy donors. These clusters contain:
- α-cells that produce glucagon;
- β-cells that produce insulin;
- δ-cells that produce somatostatin;
- CD31 endothelial cells, which line the inner surface of the pancreatic vessels.
Cluster cells were infected with SARS-CoV-2. After 2 days and 6 days after infection, scientists assessed the degree of disorder of each type of cell. The researchers specially dyed the infected tissue to determine which cell types have the highest concentration of coronavirus proteins. One staining revealed the SARS-CoV-2 (NP) nucleocapsid protein, which contains the viral genome and its protein coat. Another staining showed the presence of the SARS-CoV-2 (SP) spike protein.
As a result, it was found that the coronavirus mainly infects the β-cells of the pancreas, which produce insulin. In other types of cells, there were significantly fewer coronavirus particles. This result was observed both 2 days and 6 days after infection. Scientists believe that these data convincingly confirm the increased sensitivity of β-cells of the human pancreas to the SARS-CoV-2 coronavirus.
The researchers used data from a previous experiment to understand the reason for the high infection rate of β-cells. Since β cells contain much more of the NRP1 protein, through which the coronavirus enters the cell, scientists hypothesized that suppressing the activity of this protein would help block the infection.
This experiment used the molecule EG00229, which is a selective NRP1 antagonist. An earlier study published in November 2020 by an international group of scientists showed that this molecule binds to NRP1 and thus prevents coronavirus penetration into the cell. In the current study, this result was confirmed: pretreating pancreatic cells with EG00229 before infection with the coronavirus significantly reduced the rate of spread of the disease.
Thus, scientists have confirmed the critical role of the NRP1 protein in the susceptibility of pancreatic β-cells to the SARS-CoV-2 coronavirus. The dependences revealed in the experiment are illustrated in graphs C, D, E:
Coronavirus Enters the Pancreas of COVID-19 Patients and Stimulates Its Destruction
Scientists examined pancreatic samples from patients with COVID-19 who died from severe complications of the infection. It is important to note that none of these people had chronic or acute pancreatitis. Only one person had diabetes.
Histological analysis revealed the processes of pancreatic degradation in all donors with COVID-19. Signs of the destruction of the donor’s pancreas ranged from mild to extensive organ lesions:
- fibrosis – a proliferation of connective tissue and scarring;
- lipomatosis – an overgrowth of adipose tissue;
- autolysis – the self-destruction of cells under the influence of their enzymes;
- atrophy – a decrease in the size of the pancreas.
PCR analysis showed that the SARS-CoV-2 coronavirus was detected in the pancreas in 7 out of 9 donors. Using special staining, scientists found that 4 out of 7 of these people contain the coronavirus nucleocapsid in their β-cells. In the remaining 3 donors, staining did not reveal a nucleocapsid. Scientists see the reason for the absence in the extensive autolysis and atrophy of the pancreas in these three people.
The findings confirm previous evidence that the coronavirus preferentially infects β-cells, causing pancreatic dysfunction, leading to hyperglycemia and diabetes.
Coronavirus Suppresses Insulin Production and Kills Pancreatic Β-Cells
Scientists have evaluated the quantitative effect of the SARS-CoV-2 coronavirus on glucose-stimulated insulin production. To do this, they conducted a functional analysis of insulin production by β-cells in infected tissues.
The obtained data were compared with the production of insulin in the pancreas of healthy donors. As a result, it was found that β-cells infected with coronavirus produce approximately 2 times less insulin than healthy ones. The effect of lowering insulin production was partially offset by the treatment of infected cells with the EG00229 molecule, which prevents the penetration of coronavirus into cells.
The scientists then investigated how the coronavirus triggers programmed cell death in the pancreas. The researchers found that the programmed death signal is much higher in infected cells than in healthy cells. Moreover, the signal was increased in all types of infected cells. The scientists concluded that the programmed death caused by viral infection was independent of the cell type, although the percentage of β-cells dying was the highest.
A detailed study found that to start the process of programmed death in healthy cells, it is enough to treat them with a spike protein of the coronavirus. Experts have observed the start of this process as early as 30 minutes after cells were infected. Scientists report that these results support the original hypothesis that the SARS-CoV-2 coronavirus is the cause of the death of pancreatic β-cells, which leads to dysregulation and insulin secretion.
To clarify the mechanism of triggering programmed cell death, scientists analyzed the activity of regulatory kinases – unique proteins that control this process. Experts were interested in whether the binding of the coronavirus to the cellular receptor is sufficient to activate the pathways of programmed death.
Scientists observed that in response to the penetration of the coronavirus into the cell, kinases JNK1 and PAK1, associated with programmed β-cell death, were activated. To clarify the data obtained, specialists stained activated kinases in combination with the coronavirus spike protein in infected cells. The result was repeated.
Thus, a mechanism has been found by which the SARS-CoV-2 coronavirus kills pancreatic cells. Scientists believe this data opens up a new avenue for treating complications from COVID-19.
Conclusions
COVID-19 can increase glucose levels in patients, leading to hyperglycemia, diabetic ketoacidosis, and diabetes.
The mechanism of this complication of coronavirus infection is that the coronavirus, entering the pancreas, infects, first of all, β-cells that produce insulin. Infected cells significantly reduce insulin production, and the coronavirus spike protein triggers the process of programmed death in them. With a severe course of the disease, large areas of dead endocrine cells and overgrown connective and adipose tissue are found in the pancreas of deceased patients.
Scientists have discovered special kinase proteins that trigger the programmed death of infected cells in the pancreas. The discovery will help develop new ways to treat the complications of COVID-19.
Source
SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment
