Carole Sourbier

Insulin has played an important role in the treatment of diabetes since its discovery in the early 1920s. Initially derived from animal sources, insulin production underwent significant changes with the advent of recombinant DNA technology, allowing for the development of insulin analogs and biosimilar products. Through scientific and technological advances, various types of insulin have been engineered to cater to diverse patient populations, improving the quality, safety, efficacy, and accessibility of these products. There are currently over 50 insulin products approved by the U.S. Food and Drug Administration (FDA), each tailored to meet specific therapeutic needs. This review traces the journey of insulin, from its discovery and purification to recombinant DNA technology, biosimilar developments, and recent advancements in formulation including ultra-rapid formulations and combination therapies. The review also examines the impact of structural and formulation modifications on the pharmacokinetics and pharmacodynamics of insulin, resulting in a range of insulin products with different time-action profiles (rapid-, short-, intermediate-, and long-acting insulins). These technological and manufacturing developments have improved the quality of insulin products available to the public and have made insulin therapy safer, more effective, and more adaptable to individual patient needs, greatly enhancing the management of diabetes over time and patient quality of life.

Insulin therapy is essential for helping diabetic patients manage their blood glucose levels. Over the years, various insulin analogs with distinct pharmacokinetic and pharmacodynamic profiles have been developed, offering a range of therapeutic options. Rapid-acting insulins, such as insulin lispro, insulin aspart and insulin glulisine, and long-acting or basal insulins such as insulin detemir and insulin degludec, are commonly used in clinical practice. Each analog features unique formulations and structural modifications compared to endogenous human insulin. As a result, methods used to assess their biological activity in vitro must be specifically optimized for each analog. Previously, we published protocols for evaluating the biological activity of insulin glargine, insulin aspart and insulin lispro using an in vitro cell-based assay. In this report, we present step-by-step protocols for assessing the biological activity of insulin glulisine, insulin detemir and insulin degludec using adaptations to the same in-cell western assay.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor and is characterized in 75% of the cases by an inactivation or mutation of the tumor suppressor gene von Hippel Linda...

Opioids are commonly prescribed for extended periods of time to patients with advanced clear cell renal cell carcinoma to assist with pain management. Because extended opioid exposure has been shown to affect the vasculature and to be immunosuppressive, we investigated how it may affect the metabolism and physiology of clear cell renal cell carcinoma. RNA sequencing of a limited number of archived patients’ specimens with extended opioid exposure or non-opioid exposure was performed. Immune infiltration and changes in the microenvironment were evaluated using CIBERSORT. A significant decrease in M1 macrophages and T cells CD4 memory resting immune subsets was observed in opioid-exposed tumors, whereas the changes observed in other immune cells were not statistically significant. Further RNA sequencing data analysis showed that differential expression of KEGG signaling pathways was significant between non-opioid-exposed specimens and opioid-exposed specimens, with a shift from a gene signature consistent with aerobic glycolysis to a gene signature consistent with the TCA cycle, nicotinate metabolism, and the cAMP signaling pathway. Together, these data suggest that extended opioid exposure changes the cellular metabolism and immune homeostasis of ccRCC, which might impact the response to therapy of these patients, especially if the therapy is targeting the microenvironment or metabolism of ccRCC tumors.

Insulin is a hormone produced by β-cells of the pancreas and controls the amount of sugar in the blood. Since its discovery over 100 years ago, insulin has been used as a life-saving treatment for people with diabetes. Historically, the biological activity or bioidentity of insulin products has been assessed using an in vivo model. However, reduction in animal experiments is a goal for many worldwide, and there is a need to develop in vitro bioassays to reliably test the biological activity of insulin products. This article describes an in vitro cell-based method to assess the biological activity of insulin glargine, insulin aspart, and insulin lispro in a step-by-step manner.

Immune checkpoint inhibitors (ICIs) have changed the therapeutic landscape for cancer patients, but diabetes, a rare, severe immune-related endocrinopathy, is linked to ICI therapy. It is unclear whether glycosylation of ICIs may play a role in the development of this adverse event and how the physiological effects of different ICIs on pancreatic cells should be evaluated. We used a mouse pancreatic organoid model to compare three PD-L1 blocking antibodies in the presence or absence of IFNγ using a metabolic bioanalyzer. Modulation of ICI glycosylation altered its metabolic effects on mouse pancreatic organoids, suggesting that this model could be used to monitor and compare ICIs and to study the mechanisms underlying the development of IC-mediated diabetes.

Background: The mechanism of action of romidepsin and other histone deacetylase inhibitors is still not fully explained. Our goal was to gain a mechanistic understanding of the RAS-linked phenotype associated with romidepsin sensitivity. Methods: The NCI60 dataset was screened for molecular clues to romidepsin sensitivity. Histone acetylation, DNA damage, ROS production, metabolic state (real-time measurement and metabolomics), and gene expression alterations (transcriptomics) were determined in KRAS-WT versus KRAS-mutant cell groups. The search for biomarkers in response to HDACi was implemented by supervised machine learning analysis on a 608-cell transcriptomic dataset and validated in a clinical dataset. Results: Romidepsin treatment induced depletion in acetyl-CoA in all tested cell lines, which led to oxidative stress, metabolic stress, and increased death—particularly in KRAS-mutant cell lines. Romidepsin-induced stresses and death were rescued by acetyl-CoA replenishment. Two acetyl-CoA gene expression signatures associated with HDACi sensitivity were derived from machine learning analysis in the CCLE (Cancer Cell Line Encyclopedia) cell panel. Signatures were then validated in the training cohort for seven HDACi, and in an independent 13-patient cohort treated with belinostat. Conclusions: Our study reveals the importance of acetyl-CoA metabolism in HDAC sensitivity, and it highlights acetyl-CoA generation pathways as potential targets to combine with HDACi.

The immune checkpoint programmed death-ligand 1 (PD-L1) is expressed on the cell surface of tumor cells and is key for maintaining an immunosuppressive microenvironment through its interaction with the programmed death 1 (PD-1). Clear cell renal cell carcinoma (ccRCC) is a highly immunogenic cancer characterized by an aberrant aerobic glycolytic metabolism and is known to overexpress PD-L1. Multiple immunotherapies have been approved for the treatment of ccRCC, including cytokines and immune checkpoint inhibitors. Recently the intrinsic role of PD-L1 and interferon gamma (IFNγ) signaling have been studied in several types of tumor cells, yet it remains unclear how they affect the metabolism and signaling pathways of ccRCC. Using metabolomics, metabolic assays and RNAseq, we showed that IFNγ enhanced aerobic glycolysis and tryptophan metabolism in ccRCC cells in vitro and induced the transcriptional expression of signaling pathways related to inflammation, cell proliferation and cellular energetics. These metabolic and transcriptional effects were partially reversed following transient PD-L1 silencing. Aerobic glycolysis, as well as signaling pathways related to inflammation, were not induced by IFNγ when PD-L1 was silenced, however, tryptophan metabolism and activation of Jak2 and STAT1 were maintained. Our data demonstrate that PD-L1 expression is required to mediate some of IFNγ’s effect in ccRCC cells and highlight the importance of PD-L1 signaling in regulating the metabolism of ccRCC cells in response to inflammatory signals.

Immune cells express an array of inhibitory checkpoint receptors that are upregulated upon activation and limit tissue damage associated with excessive response to pathogens or allergens. Mouse leukocyte immunoglobulin like receptor B4 (LILRB4), also known as glycoprotein 49B (gp49B), is an inhibitory checkpoint receptor constitutively expressed in myeloid cells and upregulated in B cells, T cells, and NK cells upon activation. Here, we report that expression of LILRB4, which binds Zika virus (ZIKV), was increased in microglia and myeloid cells infiltrating the brains of neonatal mice with ZIKV-associated meningoencephalitis. Importantly, while C57BL/6 mice developed transient neurological symptoms but survived infection, mice lacking LILRB4/gp49B (LILRB4 KO) exhibited more severe signs of neurological disease and succumbed to disease. Their brains showed increased cellular infiltration but reduced control of viral burden. The reduced viral clearance was associated with altered NK cell function in the absence of LILRB4/gp49B. In naive animals, this manifested as reduced granzyme B responses to stimulation, but in ZIKV-infected animals, NK cells showed phenotypic changes that suggested altered maturation, diminished glucose consumption, reduced IFN-γ and granzyme B production, and impaired cytotoxicity. Together, our data reveal LILRB4/gp49B as an important regulator of NK cell function during viral infections.

Graft-versus-host disease (GvHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (HSCT). Definitive diagnosis of GvHD is invasive and biopsies of involved tissues pose a high risk of bleeding and infection. Our previous studies in a chronic GvHD mouse model demonstrated that alloreactive CD4 ⁺ T cells are distributed to target organs ahead of overt symptoms, meanwhile CD4 ⁺ T cell activation is tied to increased glycolysis. Thus, we hypothesized that metabolic imaging of glycolysis would allow non-invasive detection of insipient GvHD in target organs infiltrated by glycolytic effector memory CD4 ⁺ T cells. We metabolically characterized CD4 ⁺ T cell subsets on day 14 post-transplant before the onset of chronic GvHD in a pre-clinical mouse model and performed ¹³ C hyperpolarized magnetic resonance imaging (MRI) to quantify glycolytic activity in the liver of mice over the course of the disease. Intracellular metabolic screening and ex vivo metabolic profiling of CD4 ⁺ T cell subsets at day 14 confirmed that activated CD4 ⁺ T cells were highly glycolytic. Concurrently, hyperpolarized ¹³ C-pyruvate MRI of the liver showed high conversion of pyruvate to lactate, indicative of increased glycolytic activity, that distinguished allogeneic from syngeneic HSCT recipients prior to the development of overt chronic GvHD. Furthermore, single cell sequencing of T cells in patients undergoing allogeneic HSCT indicated that similar metabolic changes may play a role in acute GvHD, providing a rationale for testing this imaging approach in the clinical post-HSCT setting. Our imaging approach is amenable to clinical translation and may allow early, non-invasive diagnosis of GvHD.