Regulation of Blood Glucose

Introduction

In healthy people, blood glucose is tightly regulated to ensure normal body function. Blood glucose is maintained at a narrow range within 3.5 to 8.00 mmol/L despite the varying demand of food intake, physical activities and fasting (1). When the blood glucose is too low, it can lead to altered states of consciousness (2). On the other side, when the blood glucose is above the normal level, it can lead to diabetes, a disease that is associated with many complications (2). The blood glucose homeostasis can be achieved by involving various hormones, such as insulin, glucagon, epinephrine, cortisol and growth hormone (GH)(3). Under most circumstances, the hormones that play a major role are Insulin and Glucagon (3). The two hormones are produced by α (alpha) and β (beta) cells respectively, that clustered together forming island-like structure, so-called islet of Langerhans in the pancreas. These islets comprise only 1-2% of the whole pancreatic mass (4).

Although the hormones come from the same islet, they work by opposing each other to achieve normal blood glucose. Insulin works as anabolic hormones, while glucagon works as a catabolic hormone (3). In between meals or at sleep, when blood glucose concentrations are low, glucagon is released from α -cell to stimulate hepatic glycogenolysis (4). In addition to that, glucagon also promotes hepatic and renal gluconeogenesis to prevent further decrease in blood glucose during prolonged fasting (4). In contrast, insulin works after food intake, when the blood glucose concentration rises. After binding to its receptor on muscle and adipose tissue, insulin allows uptake of excess glucose in the bloodstream for storage, thus, lowers blood glucose concentration (4). Insulin also promotes glycogenesis and lipogenesis (3, 4).

Diabetes Mellitus

When there is a failure in blood glucose homeostasis, diabetes occurred. Diabetes is a chronic disease characterised by high blood sugar (hyperglycaemia) (5). Diabetes is caused by defects in insulin production, insulin action or both (5). The chronic hyperglycaemia in Diabetes is associated with devastating health complications that can occur acutely or chronically (5). The complications of diabetes may range from acute life-threatening hyperglycaemia with ketoacidosis, to failure of different organ systems (i.e. kidney failure, peripheral neuropathy with risk of amputations, and nephropathy and sexual dysfunction) (5). It is estimated that 422 million adults have diabetes globally (6). The number is expected to rise to 552 million in 2030 (7). Data from the Quality and Outcomes Framework (QOF) showed that around 3.1 million people in England were diagnosed with diabetes mellitus in 2016-2017 (8).

Diabetes Mellitus is divided into two categories: type 1 and type 2 diabetes. Type 1 result from the complete absence of insulin secretion due to autoimmune destruction of insulin-producing beta–cells in pancreatic islets(5). The pathological process occurring in the organ can be identified by genetic and serological markers(5). In contrast, the more prevalent type, type 2 diabetes occurs in the combination of the declining insulin production, tissues resistance to insulin action, and eventually, pancreatic beta-cell failure (7). The classical symptoms of diabetes include polydipsia, polyphagia, polyuria, and weight loss (5).

Although Type 1 diabetes was historically encountered in children and adolescents, the prevalence has shifted in the past few decades, so that the age at symptomatic onset is not a restricting factor(9). The main characteristic of type 1 diabetes is the immediate need of exogenous insulin replacement, therefore, is needed as lifetime treatment (9). The discovery of insulin in 1921 by Banting et al. has become the most significant therapy for type 1 diabetes (9). Through regular blood glucose monitoring and administration of the exogenous insulin, the progression of acute and long-term diabetic complications such as ketoacidosis, diabetic retinopathy, nephropathy, and neuropathy can be delayed, but the medication does not eliminate the risk of complications.

Despite the successful management of diabetes with exogenous insulin, people with diabetes are at risk of developing a life-threatening condition of iatrogenic hypoglycaemia. The risk of hypoglycaemia is high in people who are taking exogenous insulin therapy (9). On average, people with type 1 diabetes may experience two episodes of symptomatic hypoglycaemia per week (10, 11).

Glucose is the main source of metabolic fuel for the brain. Because the brain cannot synthesise glucose nor store the glucose more than few minutes as glycogen, it critically relays on the glucose supply from the circulation. Hence, it is important to maintain enough glucose within the circulation (11). At normal arterial blood glucose concentration, the rate of blood glucose transport to the brain is higher than the rate of brain glucose metabolism (11). However, as the arterial blood glucose drop below the normal physiological level, the transport of glucose to the brain becomes restricted for the use of metabolism and survival (11). The effects of hypoglycaemia range from acute symptoms (i.e. anxiety, palpitations, tremor, sweating, and hunger) and neurological impairment (i.e. cognitive dysfunction, seizures and coma) (11). These symptoms serve as a protective mechanism, allowing diabetes patient to be aware of dropping in blood glucose level (11). However, as the duration of the disease increases, many patients become asymptomatic or may not recognise the hypoglycaemic symptoms, thus manifesting the syndrome hypoglycaemia unawareness (11).

In the UK currently, most type 1 diabetes patients are prescribed with multiple daily doses of insulin (MDI). However, when the therapy fails to achieve the target HbA1c in patients or when the attempt results in the patient experiencing disabling hypoglycaemia, alternative management can be recommended (12). Several advances have been developed to maximise exogenous insulin therapy, such as continuous subcutaneous insulin infusions (CSII/insulin pump). CSII is a device that provides a flexible and regular insulin injection throughout the day. For patients who fail on achieving HbA1c on MDI, CSII has shown to be effective in decreasing HbA1c(10). In contrast, the ability of CSII to prevent hypoglycaemic events remains debatable with some reports showed that the rate of hypoglycaemia is higher than in the patients on MDI (9, 10).

To promote a better quality of life and independence from exogenous insulin, vascularized pancreas transplant may be an option. This type of treatment is an invasive treatment which involves transplanting a healthy pancreas (from a deceased donor) to a diabetic patient (13). Although pancreas transplant provides an excellent improvement on glycaemic control, this does not guarantee long-term insulin-free life to the patient, with the rate of pancreatic grafts survival in the first and fifth years is respectively 84 and 60% (13). In addition to that, the procedure is related to high morbidity in which the patient may suffer from acute or chronic organ failure and multiple infections due to continuous administration of immunosuppressant medication (13).

Islet Cell Transplantation

The effort to prevent diabetes complications and improve the quality of life of the patients have spanned beyond the administration of exogenous insulin and invasive pancreas transplantation, to a more stable and subtle way of cell-based therapy, namely beta-cell replacement therapy/allogenic islet transplantation.

In 1967, Lacy and Kostianovsky pioneer the first experiment on isolating numbers of metabolically active islet of Langerhans from rat pancreas using the collagenase (14). The experiment provided evidence that it is possible to isolate intact islets and the function of islets in response to high glucose would still be maintained (14). Thus, the research became the fundamental basis for future research of islet transplantation as a therapeutic tool to correct hyperglycaemia in diabetic patients (14).  Subsequent studies by Kemp et al. showed that transplantation of pancreatic islets could ameliorate diabetes in rats (15). They replicate the method of pancreatic islet isolation using collagenase digestion from Lucy’s experiment and inject it into the portal vein of the diabetic rat (15). The result was supported by Lucy’s group experiment in non-human primates (16).

The first clinical trial on human islet transplantation was performed in the mid-1980 (17). The purpose of the trial was to prevent and reduce the severity of diabetes in patients with chronic pancreatitis who need pancreatectomy (17). Instead of using the conventional allogeneic transplantation, the trial was done through the auto-transplantation procedure on ten patients (17). Insulin independence was achieved in three patients for 1, 9 and 38 months (17). Trial on islet transplantation in human continued, with the maintenance of functional islet only for few years. In 1995, it was reported that the success rate for islet auto-transplantation two years after pancreatectomy was 70% in patients who received >300,000 islets (18). It was suggested that the insulin independence after islet transplantation is strongly correlated with the number of islets transplanted (18).

Refinement in the methods of islets isolation and purification continue for many years. In 2000, University of Alberta published a breakthrough procedure called Edmonton Protocol to treat type 1 diabetes patients with the used of specialised enzymes to remove islets from the pancreas of brain-dead donors (19). This therapy was initially exciting because the procedure is minimally invasive and does not require glucocorticoid-immunosuppressive medication (19). In this trial, 7 participants with type 1 diabetes received islets transplantation (19). Each recipient required more than one donor pancreas to be insulin independence with a median follow-up of 11.9 months with sustained C-peptide (19). The result from Edmonton studies had led to optimism for replacing whole organ pancreas transplantation as a treatment for diabetes. However, the initial promising shrinks as only less than 10% of patients remain exogenous insulin independent after five years in long-term clinical studies (20).

It should be noted that despite some drawback in islet transplantation such as the scarcity of donor islets, the treatment still offers major benefits for diabetic patients. Thus, islet transplantation becoming one of the recognised standard clinical therapies for type 1 diabetes in the UK. A follow-up study post islet-transplant showed that patients who still retained C-peptide positivity had decreased risk of disabling hypoglycaemia and long-term diabetes complications (21).

Causes of Graft Dysfunction

Studies in allograft islet transplantation recipients have shown loss of insulin independence after only a few years post-transplant, which becomes the main issue for clinical islet transplantation (22). Loss of insulin independence suggests greatly reduced and/or dysfunctional islet mass (22). Several factors may contribute to this problem including ischemic, hypoxic and inflammatory damage (22, 23).

Damage to the islet starts from the donor. Islet cells are mostly obtained from heart-beating, brain-dead (BD) cadaveric donors. Organs from heart-beating donors have better outcomes than organs from non-heart beating donors (23). However, due to physiological changes after brain death in the donors, significant destruction in islet cells from inflammatory events occurs (23). Report showed that the level of inflammatory cytokines is very high within the brain-dead donors’ systemic circulation, making it difficult to transplant their organs (23). This systemic elevation of inflammatory cytokines so-called ‘cytokine storm’, are an effective exposure for inducing beta-cell dysfunction and death (23).

Islets are susceptible to hypoxia starting from the onset of ischemia during graft isolation until graft revascularisation/transplantation (23, 24). Thus, to improve islet quality for transplantation, the problem regarding ischemic time needs to be addressed (23). Pancreatic beta-cells consume large amounts of oxygen to produce insulin due to the high demand of mitochondrial respiration (24). This high oxygen demand is supported with profuse oxygenated-blood supply (pO₂ of 40 mmHg) to pancreatic islets in healthy physiological condition (23, 25). Hence, when ischemic time increases, islets are prone to damage (23). Studies shown that prolonged cold ischemia time (interval between pancreas harvesting and islet isolation) of more than 8 hours result in reduced number and quality of functional islet (26, 27).

The vulnerability of islets to hypoxia does not end after the islet are transplanted. One day after transplantation, 70% of islets are still low oxygenated, contributing to beta-cells dysfunction (28). The ischemic period immediately after islet transplantation causes hypoxia with an average pO2 of 5-10 mmHg (<1% O₂) (25). This oxygen concentration is inadequate to meet the increased need for oxygen consumption rate of islets in the hyperglycaemic environment (transplant recipient) (25). In addition to the needs of oxygen by the mitochondria, endoplasmic reticulum (ER) also requires the presence of an oxygen molecule (24). ER requires the formation of three disulphide bonds to create correct folding of proinsulin, and this process needs molecular oxygen (24). Hence, lack of oxygen will lead to accumulation of unfolded proinsulin and other proteins that disrupts beta-cells function (24).

Adaptive response to hypoxia is regulated by hypoxia-inducible factor-1 (HIF-1), a transcription consisting of two subunits of HIF-1α and HIF-1β. HIF-1α induces the activation of other genes such as angiogenesis, oxygen transport, and growth factor (GF) signalling, which is important for beta-cell survival (24). HIF is activated when the oxygen level is low. Since islets are sensitive to hypoxia, a drop in oxygen tension after transplantation causing decreased in insulin production(25). A study showed that the lowest insulin production 3 days after transplant was observed during extreme hypoxia, which is demonstrated by the highest HIF-1α expression (25). Reduction in HIF-1α expression restores insulin production in the islet grafts (25). HIF-1α has also been reported to involve in hypoxia-induced apoptosis in isolated human pancreatic islets (29).

Markers for beta-cell dysfunction

In 2012, Talchai et al. introduced a concept of beta-cell dedifferentiation as a mechanism of beta cell loss and dysfunction in type 2 diabetes. They defined beta-cell dedifferentiation as to when the beta-cell revert to their progenitor-like cell or convert to other pancreatic endocrine hormone-secreting cells (30, 31). The group provided evidence that the FoXO1, a transcription factor required for beta-cell to maintain its’ identity, is loss in insulin-resistant diabetic mice as well as when the beta-cell is under pathophysiological stress (30). They also found that some former beta-cells convert into alpha, delta, and pancreatic polypeptide (pp) cells (30).

The exact molecular mechanism of islet graft dysfunction still needs to be addressed. A study by Anderson et al. proposed that the islet graft dysfunction result from the loss of end-differentiated beta cell-marker, similar to the loss of beta-cell function in T2 Diabetes (30, 32). To further investigate beta-cell dysfunction/dedifferentiation is isolated human islet, some beta-cell phenotypic markers such as Nkx 6.1 and Ucn-3 can be utilized.

Nkx 6.1 is a homeodomain protein and an essential transcription factor required for the development of beta-cells (33). The gene is located centrally in the nuclei of beta-cell (33). A study reported that Nkx 6.1 null mice have a profound deficiency of beta-cell population and insulin content of only 2% of the wild-type mice, but no differences were found in the expression of other hormones in the islet (glucagon, somatostatin, pp) (34).  Furthermore, in a study where Nkx 6.1 gene is inactivated in beta cells of adult mice, the beta-cells switch into another alternative endocrine cell, the somatostatin-producing delta cells (35). These evidence highlighting the importance of Nkx 6.1 to maintain normal beta-cell function.

Urocortin (Ucn) 3 is a ligand for corticotropin-releasing factor (CRF2) receptor (36). CRF has an important role in regulating hypothalamic-pituitary-adrenal axis to respond to stress and the stress-associated behaviour (36). In the pancreas, CRF has been shown to modulate pancreatic hormone secretions, specifically for the insulin-producing pancreatic beta-cells (36). In adult mice, Ucn-3 was secreted in concomitant with high glucose concentration. A study found that Ucn-3 expresses in mature beta-cells cell and undetectable in islets from young embryos, suggesting that Ucn-3 is a strong phenotypic marker for mature beta-cells (37). Ucn-3 is also required for normal physiological activity of beta-cells, with In Ucn-3 knockout mice loss its ability to secrete insulin in high glucose period (32).

Loss of Ucn-3 as an end-differentiated phenotypic marker was reported in a case study of 2 islet transplant recipients despite the excellent graft function (32). While the co-expression of Ucn-3 can be found in beta-cells and glucagon-producing cell α-cells in normal control pancreas, the marker is absent in engrafted islet in islet transplant recipients (32). Loss of beta-cells end-differentiated marker in engrafted islet may explain despite sufficient islet engrafted, insulin independence cannot be maintained for a long period (32). Some cells with Ucn-3 also were identified to co-express both the insulin and glucagon (32). Thus, indicating beta-cell plasticity may also contribute to the abnormality of islet transplant (32).

Aims

The specific aims of this study are to: 1) To determine whether rotation culture of MIN6 pseudo-islet can prevent loss (maintain) of beta-cell genes and function, caused by hypoxia in static culture. 2) characterise beta-cell phenotypic changes in the isolated human islet.

Specific Objectives:

1. Establish MIN6 pseudo-islet model in different culture conditions (static and rotation) and quantify changes in phenotype between the two conditions based on the expression of beta-cell markers for dysfunction and dedifferentiation.
2. Assess the validity of the NIS Elements Software manual cell counting protocol
3. Quantify changes in isolated human islet phenotype, assessing ucn-3 / Nkx6.1 protein expression changes, using validated counting method.

The project is split into two hypotheses:

1. Loss of beta-cell function and gene expression, due to hypoxia in static culture, can be reversed/improved by rotation culture
2. Human islet isolation causes changes in mature beta-cell phenotype

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