-Contributed by Abi Kasberg, PhD

 

Attending my first Advanced Technologies & Treatments for Diabetes (ATTD) conference in Florence, Italy this March was a treat for the mind (and palate). Being new to the diabetes space, it was thrilling to learn about the latest insights and advancements that are being made in diabetes research to improve patient outcomes. The location didn’t hurt either as I reveled in the exquisite Italian cuisine, from pizza and pasta to wine; somehow everything tastes better in Italy.

The presentations at ATTD 2024 fell into three main areas of diabetes research:

1) Type 1 diabetes (T1D)

2) Type 2 diabetes (T2D)

3) Islet cell transplantation (ITx) & stem cell-derived islets

Within pancreatic islets, beta cells are responsible for producing and secreting insulin. In turn, insulin is a critical mediator of glucose uptake into the cells and blood glucose homeostasis. Coming from an interest in beta cell biomarkers, it became immediately clear that beta cell health and function are important in each of these fields of diabetes research. Because each of these fields are brimming with new developments, and for the sake of brevity, I selected one diabetes topic to discuss here: islet cell transplantation and stem cell-derived islets.

 

Islet Cell Transplantation


Pancreatic islets are attacked by the immune system in T1D, resulting in beta cell destruction, impaired insulin secretion, and dysregulated glucose uptake. One approach being utilized to replace the damaged islet cells in T1D is through islet cell transplantation (ITx). ITx is the engraftment of healthy donor pancreatic islet cells into the portal vein of the liver of a diabetic patient. The goal of ITx is to restore insulin production and maintain glucose homeostasis. An added bonus would be to achieve independence from exogenous insulin therapy.

ITx candidate eligibility can vary from transplant center to transplant center, yet typically includes individuals with severe and poorly controlled blood sugar levels despite efforts to maintain glucose control. ITx requires chronic immunosuppression and other accompanying therapies such as anticoagulation treatments. Despite these efforts, there is no guarantee that islet graft survival will be sustained, nor that insulin independence will be achieved. Important topics of ITx research presented at ATTD that caught my attention surrounded islet graft health, islet graft functionality and survival, and liver toxicity.

 

Islet Graft Health, Pre-transplantation

Healthy human pancreatic islets display rich vascularization and are composed of a heterogenous population of endocrine cell types (alpha cells, beta cells, delta cells, and epsilon cells). The delicate interplay between islet cells and surrounding vasculature is pivotal for their functionality as finely regulated, fully operational islets. Easy access to vasculature is essential for blood glucose sensing and insulin secretion into circulation. Due to the importance of islet cellular structure and composition on islet function, there must be a mix of healthy islet cell types in grafts to become functional islet transplants.

Given the importance of islet graft health for successful transplantation, it was surprising to hear from Dr. Peter Senior (University of Alberta) that donor islets are not typically vetted for islet cell functionality prior to transplantation. A main reason is that there is limited time to screen donor tissues prior to engraftment. Islet cell quality, purity, and morphology are analyzed, but otherwise the functionality of islet components such as beta cells is not typically measured prior to transplantation. This led me to wonder the following questions:

Is there a need for more thorough vetting of donor pancreatic islets prior to transplantation?

Given the mechanistic importance of beta cells on glucose homeostasis, could biomarkers of beta cell health such as C-peptide or intact proinsulin be used to assess donor beta cell functionality prior to transplantation? Could they accurately be measured in non-fasting donors? For example, blood samples obtained during routine infectious disease screenings could be further analyzed for beta cell biomarkers to gain a deeper read into donor beta cell health.

Is there a correlation between pre-transplant donor islet functionality with post-transplant longevity and successful engraftment of the islet graft?

 

Islet Graft Functionality & Survival, Post-transplantation  

Islet cell grafts are susceptible to death by ischemia, nutrient deprivation, or immune attack. Dr. Qizhi Tang (UCSF) described that islets are inherently highly immunogenic and express human leukocyte antigen (HLA) at high levels, attracting cytokines. This makes islets and islet grafts targets of the autoimmune system. Innate defects in immune tolerance, such as seen in T1D, can kill islet grafts. Additionally, islet grafts are vulnerable to immune activation due to the small volume of islet tissues that are infused.

Healthy beta cells synthesize the insulin precursor proinsulin. Under healthy conditions, intact proinsulin undergoes cleavage to produce two fragments that are secreted into circulation: C-peptide and insulin. C-peptide has a longer half-life than insulin and is commonly measured to indirectly quantify insulin secretion levels for the evaluation of beta cell health. Dr. Senior shared fasting plasma C-peptide data illustrating that approximately 70% of islet cell transplant recipients sustained graft survival for longer than 5 years. Surviving portal vein islet cell transplants can persist for up to 20 years. Still, many patients will receive multiple ITx over their lifetime.

An additional measurement that is used to estimate beta cell health following ITx is the BETA-2 composite score (Forbes et al. 2016). The BETA-2 algorithm incorporates fasting plasma glucose, fasting C-peptide, HbA1c, and insulin dose to approximate beta cell functionality (Forbes et al. 2016).

There are limitations with the current algorithms and scores commonly used to monitor beta cell health following ITx:

  1. Dependence on blood glucose levels

The BETA-2 score is limited by its dependence on blood glucose levels, which can fluctuate due to diet, exercise, medications, hormone fluctuations, or other factors (“Good to Know,” 2018). While it is important to consider C-peptide levels in the context of blood glucose concentrations, the BETA-2 score may not accurately reflect the health of the islet graft in individuals with hypoglycemia (Forbes et al. 2016).

  1. Exogenous insulin treatment can reduce endogenous insulin secretion

Treatment with exogenous insulin may induce beta cell rest, which lowers the demand for endogenous insulin secretion from the islet (Brown and Rother 2008). Low C-peptide levels or BETA-2 scores may provide inaccurate readouts on islet graft function when measured on individuals receiving exogenous insulin therapy. As such, C-peptide measurements may be more reflective of endogenous insulin secretion rather than beta cell health, particularly when exogenous insulin therapy is used.

  1. Indirect measurement of islet graft functionality

The BETA-2 score provides a readout on glucose control and does not provide a focused picture on the functional capacity of the islet graft itself. Additional facets of islet graft functionality, such as insulin processing, insulin secretion dynamics, beta cell mass, and insulin sensitivity may not be captured with the BETA-2 score or C-peptide levels alone.

  1. Additional factors that impact islet graft survival are not captured with these scores, such as immune response and graft viability.

 

These limitations have highlighted the need for more biomarkers and algorithms to better detect early islet graft failure. Prompt detection of graft failure would enable earlier and quicker interventions with additional islet infusions or exogenous insulin therapies to improve the management of glucose homeostasis and health outcomes of the patient.

Serum intact proinsulin is an additional biomarker of beta cell dysfunction (Pfützner and Forst 2011). Under healthy conditions, intact proinsulin is enzymatically cleaved into insulin and the C-peptide byproduct, which are secreted from the cell (Fig. 1A). However, when beta cells experience stress or dysfunction, intact proinsulin is secreted from the beta cell without undergoing processing, resulting in a measurable accumulation of intact proinsulin in the blood (Fig. 1B) (Pfützner et al. 2008). Studies in T1D indicate that circulatory intact proinsulin in relation to C-peptide levels (PI:C ratio) serves as an early biomarker of beta cell stress (Sims et al. 2023). Elevated PI:C levels predicts disease progression in T1D, and hence can be used to identify candidates who may display greater response to Teplizumab immunotherapy (Sims et al. 2023).

Figure 1: In beta cells, preproinsulin is transported from the endoplasmic reticulum to the golgi apparatus where disulfide bonds are added to form intact proinsulin. (A) In a healthy beta cell, intact proinsulin undergoes cleavage in secretory vesicles to produce insulin and C-peptide. Insulin and C-peptide are secreted from the cell to enter the circulatory system. (B) In a beta cell experiencing stress or dysfunction, intact proinsulin fails to undergo proper cleavage in the secretory vesicles and is released from the beta cell to enter circulation.  Figure created with BioRender.com

Given the ability of the PI:C ratio to reflect beta cell dysfunction, Dr. Senior stated that it would be interesting to get a feel for how PI:C levels change in response to ITx, both pre-transplantation and post-transplantation. The following questions come to mind when considering islet graft health and survival post-ITx:

Is there a need for better indicators of beta cell health status prior to graft deterioration? Would a biomarker of beta cell dysfunction, such as PI:C, be valuable in detecting islet graft failure, especially if it were able to do so earlier and with a greater sensitivity than C-peptide or the BETA-2 score?

Is PI:C more responsive to changes in beta cell function compared to C-peptide or the BETA-2 score?  Time-wise comparisons of PI:C compared to C-peptide levels and BETA-2 scores over the life of an islet graft may probe any lags in the ability of current diagnostic capabilities to reflect the health status of islet grafts.

Given that blood glucose management is the goal of ITx, how does the beta cell dysfunction biomarker PI:C correlate with glucose control?

 

Liver Toxicity and Health

The richly vascularized microenvironment of the liver provides a supportive and nourishing location for donor islet cells to engraft. In ITx, islet grafts are infused via catheter into the portal vein of the liver. In the liver, islet cells engraft to become functional, insulin-secreting islets capable of managing blood glucose levels. Due to the manipulation of the liver environment during ITx, it is important to monitor for and assess sustaining or damaging effects to the liver. Portal vein pressure must be monitored during islet cell infusion to prevent portal vein thrombosis. Collagen must be released following the infusion to prevent liver bleeding. It was stated at ATTD that there is a need for a deeper analysis into damaging or toxic effects that islet cell engraftment may have on the liver.

Does the local engraftment site of the liver become inflamed or injured during islet cell engraftment?

Are there any signs of liver toxicity following ITx, such as hepatocyte apoptosis, inflammation, or fibrosis?

Do immune responses to the engraftment into the liver correlate with decreased graft function or survival?

Does hepatic manifestations of graft-versus-host disease (GVHD) occur following ITx?

In cases of graft rejection, does the host liver tissue in proximity to the engraftment site sustain collateral damage?

Could screening liver health status in transplant recipients guide recipient selection?

 

 

Stem Cell-Derived Islets


There are many limitations and risks of ITx, listed below, that must be carefully considered when evaluating the benefits-to-risks of ITx compared to the continuation of T1D treatment.

  • Cadaveric donor islets are a limited resource.
  • Chronic immunosuppression
  • Risk of immune rejection
  • Increased risks of infections, cancers, neoplasia, nephrotoxicity, and anemia

To circumvent these limitations, research has advanced into the innovative field of stem cell-derived islets to treat diabetes. Stem cell technologies are being leveraged to derive islet-like cells from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Dr. Cristina Nostro (McEwen Stem Cell Institute) gave a striking presentation detailing the work that is being done to differentiate stem cells into islet-like cells by replicating the natural developmental time course of early endoderm to pancreatic islets. Protocols are now established that guide human PSC differentiation into islet-like cells.

Despite these advancements, there are many challenges and objectives to meet when differentiating stem cells into islet-like cells. Islet functionality, protection from immune rejection, scale-up protocols, optimized engraftment procedures, and safe removal of grafts must all be considered while developing this technology. Dr. Qizhi Tang described engineering efforts being explored with stem cell-derived beta cells to express immune modulatory molecules in response to immune attack. In cross-over from T1D immunotherapy, Teplizumab is being considered as a drug to target pathogenic T cells in encapsulated stem cell strategies to prevent graft rejection.  Dr. Jay Skyler (University of Miami) provided an excellent overview of stem cell approaches to T1D, including genetically edited pig islets and encapsulation strategies to avoid immunosuppression. Dr. Skyler also illuminated the importance of highly functional stem cell-derived islets, minimal cellular stress, and beta cell health.

Echoing these important characteristics of donor ITx, full islet structure and adequate vascularization are required for the successful differentiation of insulin-producing islets from stem cells. A mix of cell types are required to compose the islets and the cell types must be operational. Not only must beta-like cells secrete insulin and alpha-like cells secrete glucagon, these islet-like cells must also be responsive to environmental cues. For this technology to become clinically effective, insulin secretion from beta-like cells must occur in response to increasing blood glucose levels. Dr. Jose Oberholzer (University of Illinois) indicated there is a need for more beta cell functional biomarkers in stem cell-derived islet research to determine if the mechanistic function of induced stem cells are intact.

Several pharmaceutical companies are investigating the clinical utility of stem cell-derived islet transplantation as a treatment for diabetes. There are several ongoing studies in the clinical pipeline that are utilizing stem-cell derived beta cells, including VX-880 (Vertex Pharmaceuticals), VX-264 (Vertex Pharmaceuticals), and CTX211 (CRISPR Therapeutics). Given the importance of beta cell functionality during both the development of stem cell-derived islets and as a measurement of efficacy during clinical trials, I began to wonder about the types of beta cell biomarkers being utilized in these studies.

Which beta cell biomarkers are being measured in stem cell-derived islet clinical trials? Are they sufficient in identifying beta cell dysfunction?

Is the beta cell dysfunction biomarker intact proinsulin or PI:C being measured during the research and development of stem cell-derived islet therapies?

 

Summary


In summary, the presentations delivered at ATTD 2024 shed light on the importance and necessity of beta cell functional biomarkers across various areas of diabetes research. Of particular interest to this article, the vulnerable and delicate nature of donor islets and stem cell-derived islet cells underscores the importance of careful and precise monitoring of islet health. Early detection of beta cell dysfunction during stem cell-derived islet development and following engraftment will enable earlier interventions and optimized patient outcomes.

While C-peptide is arguably the most widely used beta cell biomarker, its effectiveness at directly and promptly depicting beta cell functional status in these contexts merits scrutiny. Emerging data has suggested the PI:C ratio is an effective tool in T1D immunotherapies at identifying initial stages of beta cell stress prior to disease progression. Given these advancements, investigations into the potential utility of intact proinsulin and PI:C may enhance our understanding of beta cell dysfunction in ITx and stem cell-derived islet technologies.

I extend my gratitude to the clinicians and scientists whose insightful presentations beautifully illustrated the status and scope of current diabetes research. I am especially appreciative of those who generously spared time to connect with me, patiently answering my many questions regarding the current diabetes landscape and the potential for advancements of beta cell biomarkers in diabetes research. Their expertise and dedication have greatly enriched my understanding and sparked further interest in this significant field.

 

 

 

 

Further Reading


Brown, R. J., & Rother, K. I. (2008). Effects of beta-cell rest on beta-cell function: A review of clinical and preclinical data. Pediatric Diabetes, 9(3 Pt 2), 14–22. https://doi.org/10.1111/j.1399-5448.2007.00272.x

Forbes, S., Oram, R. A., Smith, A., Lam, A., Olateju, T., Imes, S., Malcolm, A. J., Shapiro, A. M. J., & Senior, P. A. (2016). Validation of the beta-2 score: An improved tool to estimate beta cell function after clinical islet transplantation using a single fasting blood sample. American Journal of Transplantation, 16(9), 2704–2713. https://doi.org/10.1111/ajt.13807

Good to know: Factors affecting blood glucose. (2018). Clinical Diabetes : A Publication of the American Diabetes Association, 36(2), 202. https://doi.org/10.2337/cd18-0012

Pfützner, A., & Forst, T. (2011). Elevated intact proinsulin levels are indicative of beta-cell dysfunction, insulin resistance, and cardiovascular risk: Impact of the antidiabetic agent pioglitazone. Journal of Diabetes Science and Technology, 5(3), 784–793. https://doi.org/10.1177/193229681100500333

Pfützner, A., Weber, M. M., & Forst, T. (2008). A biomarker concept for assessment of insulin resistance, beta-cell function and chronic systemic inflammation in type 2 diabetes mellitus. Clinical Laboratory, 54(11–12), 485–490.

Sims, E. K., Geyer, S. M., Long, S. A., & Herold, K. C. (2023). High proinsulin:C-peptide ratio identifies individuals with stage 2 type 1 diabetes at high risk for progression to clinical diagnosis and responses to teplizumab treatment. Diabetologia, 66(12), 2283–2291. https://doi.org/10.1007/s00125-023-06003-5