Hyperglycemia-induced cathepsin L maturation: Implications for diabetic comorbidities and COVID-19 susceptibility

Reviewer #1 (Public Review):

Summary:
The study by He et al. investigates the relationship of an increased susceptibility of diabetes patients to COVID-19. The paper raises the possibility that hyperglycemia-induced cathepsin L maturation could be one of the driving forces in this pathology, suggesting that an increased activity of CTSL leads to accelerated virus infection rates due to an elevated processing of the SARS-CoV-2 spike protein.

In a clinical case-control study, the team found that the severity of corona infections was higher in diabetic patients, and their CTSL levels correlated well with the progression of the disease. They further showed an increase in CTSL activity in the long term as well as acute hyperglycemia. SARS-CoV-2 increasingly infected cells that were cultured in serum from diabetic patients, the same was observed using high glucose medium. No effect was observed in the medium with increased concentrations of insulin. CTSL knockout abolished the glucose-dependent increase in infection.

Increased glucose levels did not correlate with an increase in CTSL transcription. Rather He et al. could show that high glucose levels led to CTSL translocation from the ER into the lysosome. It was the glucose-dependent processing of the protease to its active form which promoted infection.

Strengths:
It is a complete study starting from a clinical observation and ending on the molecular mechanism. A strength is certainly the wide selection of experiments. The clinical study to investigate the effect of glucose on CTSL concentrations in healthy individuals sets the stage for experiments in cell culture, animal models, and human tissue. The effect of CTSL knockout cell lines on glucose-induced SARS-CoV2 infection rates is convincing. Finally, the team used a combination of Western blots and confocal microscopy to identify the underlying molecular mechanisms. The authors manage to keep the diabetic condition at the center of their study and therefore extend on previous knowledge of glucose-induced CTSL activation and their consequences for COVID-19 infections. By doing so, they create a novel connection between CTSL involvement in SARS-CoV2 infections and diabetes.

Weaknesses:
The authors suggest that hyperglycemia as a symptom of diabetes leads to an increased infection rate in those patients. Throughout their study, the team focuses on two select symptoms of a diabetic condition, hyperglycemia and hyperinsulinemia. The team acknowledges in the discussion that there could be various other reasons. Hyperglycemia can lead to metabolic acidosis and a shift in blood pH. As CTSL activity is highly dependent on pH, it would have been crucial to include this parameter in the study.

The study rarely differentiates between cellular and extracellular CTSL activity. A more detailed explanation for the connection between the intracellular CTSL and serum CTSL in diabetic individuals, presumably via lysosomal exocytosis, could be helpful with regard to the final model to give a more complete picture.

In the early result section, an effect of hyperglycemia on total CTSL concentrations is described, but the data is not very convincing. Over the course of the manuscript, the hypothesis shifts increasingly towards an increase in protease trans-localization and processing to the active form rather than a change in total protease amounts. The overall importance of CTSL concentrations remains questionable.

Reviewer #2 (Public Review):

Summary:
In this study, the authors hypothesized that individuals with diabetes have elevated blood CTSL levels, which facilitates SARS-CoV-2 infection. The authors conducted in vitro experiments, revealing that elevated glucose levels promote SARS-CoV-2 infection in wild-type cells. In contrast, CTSL knockout cells show reduced susceptibility to high glucose-promoted effects. Additionally, the authors utilized lung tissue samples obtained from both diabetic and non-diabetic patients, along with db/db diabetic and control mice. Their findings indicate that diabetic conditions lead to an elevation in CTSL activity in both humans and mice.

Strengths:
The authors have effectively met their research objectives, and their conclusions are supported by the data presented. Their findings suggest that high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum to the lysosome, potentially contributing to diabetic comorbidities and complications.

Weaknesses:
1. In Figure 1e, the authors measured plasma levels of COVID-19 related proteins, including ACE2, CTSL, and CTSB, in both diabetic and non-diabetic COVID-19 patients. Notably, only CTSL levels exhibited a significant increase in diabetic patients compared to non-diabetic patients, and these levels varied throughout the course of COVID-19. Given that the diabetes groups encompass both male and female patients, it is essential to ascertain whether the authors considered the potential impact of gender on CTSL levels. The diabetes groups comprised a higher percentage of male patients (61.3%) compared to the non-diabetes group, where males constituted only 38.7%.

2. Lines 145-149: "The results showed that WT Huh7 cell cultured in high glucose medium exhibited a much higher infective rate than those in low glucose medium. However, CTSL KO Huh7 cells maintained a low infective rate of SARS-CoV-2 regardless of glucose or insulin levels (Fig. 3f-h). Therefore, hyperglycemia enhanced SARS-CoV-2 infection dependent on CTSL." However, this evidence may be insufficient to support the claim that hyperglycemia enhances SARS-CoV-2 infection dependent on CTSL. The human hepatoma cell line Huh7 might not be an ideal model to validate the authors' hypothesis regarding high blood glucose promoting SARS-CoV-2 infection through CTSL.

3. The Abstract and Introduction sections lack effective organization.