Important advancements for discovering disease’s etiology and finding new treatments for it
Summary. In recent years, several studies have shown that a large number of people with long term duration of T1D still harbor insulin-producing β-cells. Other studies have found that very young children develop a more aggressive form of Type 1 Diabetes (T1D) than teenagers and adults; moreover, many people, especially among those who developed T1D as teenagers or adults, have “dormant” β-cells that can be reactivated in the laboratory. Collectively, these findings may speed up discovery of disease’s etiology and suggest that expansion of the remaining functional β-cells and/or reactivation of “dormant” β-cells might be a meaningful strategy to treat T1D.
Reading the footnotes will help clarify the concepts in this paper.
Background. Type 1 Diabetes is an autoimmune disease, triggered by environmental factors in genetic predisposed people (Etiology: genetic predisposed people and environmental factors are needed for T1D to develop, part 2. In Type 1 Diabetes, the insulin-producing β-cells, housed within the islets of Langheran are mistakenly recognized as foreign entities and killed by misguided immune cells infiltrating the islets. The CD8+ T leukocytes are the islets’ major immune cells infiltrate, and are regarded as the most prominent killers of the insulin-producing β-cells; yet, CD4+ leukocytes, B leukocytes and macrophages are also present in the infiltrate and take part in β-cells elimination (see Footnote i). Collectively, the infiltration by these leukocytes is referred to as insulitis and it is limited to the islets of Langheran (Reference 1). Many scientists believe that T1D progresses asymptomatically for years until the number of functional β-cells has become so low that they are no longer able to supply sufficient insulin to maintain normal blood glucose levels; indeed, some scientists think that it may take a loss of around 80% of functional cells to have the disease’s symptoms. Until recently, it was believed that the β-cells would, eventually, be wiped out completely, but recent studies from various groups have concluded that a large number of people with long term duration of T1D still harbor some insulin-producing β-cells (References 2-6) and/or “dormant” β-cells (References 7-9). Learning how to expand the functional β-cells that are left or how to make functional the dormant β-cells may help in finding a cure for the disease.
Insulin-producing β-cells in T1D long term patients. Studies reported by Liu and coworkers found serum C-peptide (Footnote ii) in 54 out of 141 (38%) patients with long-standing T1 D duration (from 7.4 to 31 years) meaning that they maintained the capacity to secrete small amounts of insulin (Reference 2). Research conducted on the Joslin Medalist cohort (Footnote iii) by Keenan and coworkers evaluated 411 patients with T1D duration of 50 years or more and showed that 67.4% of these patients had serum C-peptide either in the minimal (0.03– 0.2 nmol/L) or sustained range (more than 0.2 nmol/L), which implies residual endogenous insulin production. Moreover, postmortem analysis of pancreatic tissues from nine patients of the Medalist cohort indicated the presence of insulin in the tissues (Reference 3). Examination of 924 patients from two U.K. centers for insulin production was carried out by Oram and coworkers (Reference 4); the patients’ age ranged from 6 to 17 years at clinical diagnosis and the duration of diabetes was 11 to 27 years. Eighty percent of patients (740 of 924) had detectable endogenous urine C-peptide levels implying insulin production. The authors underlined that while most of the patients secrete very small amounts of insulin, some secrete endogenous insulin in clinically relevant amounts. A study by Davis and coworkers recruited 919 patients whose T1D duration was from 3 to 81 years (Reference 5). They assessed the frequency of residual insulin secretion by measurement of non-fasting serum C-peptide concentration. They found that 29% of the patients had detectable non-fasting C-peptide and that the frequency of C-peptide positivity was higher if diagnosis was made after age 18 compared to before age 18. In addition, 19% of those with undetectable non-fasting C-peptide were C-peptide positive upon stimulation testing. Despite the small amounts of insulin produced by the patients in the above studies, it is insufficient for maintaining blood sugar control. However, there are significant beneficial effects. Kuhtreiber and coworkers examined 1272 patients and found that C-peptide levels over 10pmol/L (a minuscule amount) had protective effects from complications such as nephropathy, neuropathy, foot ulcers and retinopathy. In contrast, low C-peptide levels resulted in a poor metabolic control and very low C-peptide was associated with severe events of hypoglycemia (it is not known why these dysfunctions occur).
It is not clear whether the remaining functional β-cells are protected by some factors, or have escaped the attack of autoimmunity, or are made freshly as they are killed; knowing this information would help in learning how to prevent T1D and/or expand these functional β-cells to allow a treatment/cure for those already with the disease.
Dormant β-cells in T1D long term patients. Scientists at the University of Exeter (UK), in collaboration with colleagues at the University of Oslo, have undertaken studies to gain more insights into insulitis. The studies were performed to better understand the nature of immune infiltrating cells in inflamed islets of T1D patients given the fact that only less than 200 cases of insulitis have been analyzed worldwide in over 100 years. They have unraveled some new interesting features of T1D onset/progression and β-cells survival (Leete et al, Reference 7; and Arif et al. Reference 8). These investigators had access to a large collection of samples from organ donors (nPOD;USA), biopsies from living donors (Diabetes Virus Detection-study, abbreviated DiViD; Norway), and samples taken from patients after death (UK). Their results confirmed previous studies showing that cells of the immune system such as CD8+ T leukocytes (the most prominent infiltrate), CD4+ leukocytes, and a variable numbers of CD20+ B leukocytes, were present around and inside the islets of Langerhans. These investigators were surprised to find two different profiles of CD20+ B leukocytes insulitis in patients’ inflamed islets. Specifically, the group of children diagnosed prior to, or at age seven, had a consistently higher number of CD20+ B leukocytes (referred to as CD20Hi) in their inflamed islets than the group of children that developed the disease in their teens or after their teenage years (referred to as CD20Lo). Additionally, the younger group of kids had lost more β-cells and at a faster pace than the older children and consequently, developed a much more aggressive form of diabetes. These investigators pointed to important accessory roles played by the CD20+ B leukocytes in the destructive process of β-cells.
Another important discovery made in this study relates the number of islets containing insulin (ICIs) to the patients’ age at onset. These investigators found that patients diagnosed as teenagers or adults retained about 40% of ICIs, while patients diagnosed at age seven or younger had much less ICIs. Because many people develop T1D when they still have a relatively high β-cell mass, the inability of the β-cells to control glucose is likely related to β-cell dysfunction rather than to β-cell death. Furthermore, it is possible that part of the dysfunction is that these β-cells cannot release insulin into the blood stream. In support of their findings, they cite a recent paper by Krogvold and coworkers (Reference 9) regarding patients of the DiViD cohort, one of the cohorts also used by Leete and coworkers (Reference 7). This cohort was formed by six living adult patients who donated samples of their pancreases within days or weeks of T1D onset; moreover, Krogsvold and coworkers (Reference 9) added two pancreases from donors that died at disease onset, and as a control they used three non-diabetic people. Being adult pancreases, the DiViD cohort had a CD20Lo insulitis profile and a high content of ICIs. Upon islets removal from the pancreas, the β-cells from some patients still produced some insulin when challenged by high glucose, but those from other patients did not. Since the production of insulin in response to increasing glucose concentrations is regulated by numerous genes (and the proteins they express), these researchers wanted to investigate these genes. They showed that all the genes in the insulin pathway were expressed; however, their expression was reduced in most of the T1D patients. Restoration of normal gene expression should, in principle, help to restore proper insulin expression. In order to attempt normalizing insulin expression in response to glucose, these investigators cultured the islets three to six days in the laboratory under conditions mimicking those of their native pancreatic environment. In four out of eight cases they succeeded in normalizing insulin response to glucose.
These studies may open interesting opportunities to understanding the mechanism of T1D onset and to finding a treatment for it. Specifically, which signals are responsible to make β-cells “dormant”, instead of killing them? Finding an answer would allow preventing such signals to operate, or if the disease has already occurred, especially in the teenage years or beyond when up to 40% dormant β-cells may be retained, to develop treatments to awaken them and produce insulin.
(i) Both CD8+ T and CD4+ T cells are a subtype of white blood cells (Leukocyte) in the vertebrate immune system. The actions of the T cells are kept in check by another subtype of T cells called regulatory T cells (or Treg). It has been shown that in autoimmune diseases, such as T1D, Treg are defective. CD20+ B-cells are a subtype of white blood cells that produce antibodies. Macrophages are a subtype of white blood cells that digest foreign substances.
(ii)The Joslin 50-Year Medalist Study involves a large cohort of patients with T1D who have been treated with insulin for 50 years or longer.
(iii) In the β cells of the pancreas, a polypeptide chain of 110 amino acids (referred to as pre-pro-insulin) is first synthesized (Figure 1); pre-pro-insulin is then cleaved in several steps by proteases of the β cells to finally yield two fragments [A- chain (in red) and B-chain (in blue), of 21 and 30 amino acids, respectively). The A- and B- chain generate mature insulin by assembling and staying together by virtue of so-called disulfide bonds (shown in Figure 1 by the lines drawn between C-Cs). An A- and B- chain Connecting Peptide (hence the name C-Peptide), containing 31 amino acids (in orange), is also formed by the cleavage process; specifically, one molecule of C-Peptide is generated per molecule of insulin produced. The synthesis of insulin by the β-cells is a multi-step highly regulated process and more information can be found here.
A test to evaluate how much insulin the pancreas of a person is still producing (at any stage of the disease) is not performed by measuring the blood concentration of insulin, but rather that of C-Peptide; the reasons for doing it are several: e.g., insulin is degraded rather rapidly; in fact, insulin half-life is ~ 4–6 minutes (insulin’s half-life is the time for insulin concentration to become half), while C-peptide has a half-life of ~35 minutes. People develop antibodies to insulin (but not to C-Peptide) and they may interfere with insulin concentration determinations. In patients with diabetes insulin is injected (but C-Peptide is not) and that makes it very hard to determine how much insulin is produced by the body.
Figure 1. Insulin and C-Peptide; amino acids are given in a single letter code
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