V- Therapeutic perspectives

Diabetes is a major and increasing health problem worldwide which presently affects over 150 million people. Today’s treatment includes lifestyle changes (dietary adjustments, increased physical activity) with addition of metformin as a first-line pharmacological therapy. When this is insufficient, the choice until recently has been addition of sulfonylureas, glinides, α-glucosidase inhibitors, GLP-analogues and dipeptidyl IV inhibitors, thiazolidinediones, or insulin, or a combination thereof. These treatments offer improved glycemic control, but are also associated with significant advers eevents such as hypoglycemia, weight gain, and gastrointestinal discomfort. Moreover, especially at later disease stages, it becomes increasingly difficult to maintain physiological blood glucose levels and to avoid secondary macro- and microvascularcomplications, such as cerebrovascular accidents, myocardial infarction, and neuropathy. Therefore, other treatment options have received much attention in recent years. Below is an overview of therapeutic targets.


1. Insulin replacement therapy

Insulin replacement therapy stabilizes some of the metabolic disturbances of diabetes, but is not a cure and typically cannot prevent future severe chronic complications that stem from the hyperglycemic episodes produced by inaccurate insulin doses.


2. Pancreatic islet transplantation

Islet transplantation is a promising approach in attempts to replace conventional exogenous insulin therapy. However, its substantial benefits conferring insulin independence and ameliorating hyperglycemia diminish over the first five years in most patients. Furthermore, there is a limited supply of high-quality donor pancreases and the use of islets from multiple donors is typically required to achieve insulin-independence. Additionally, pancreas transplantation involves major risks associated wit hboth the surgery and the long-term post-transplant immunosuppressive treatment, so this procedure is seldom used to treat diabetes. An attractive alternative that circumvents such invasive complicated surgery is the transplant of donor allogeneic islets. Islets may be infused into the recipient portal vein by a minimally invasive technique. Furthermore, pancreatic islets have been reported to maintain their functionality and viability in culture, thereby allowing for pre-transplant ex vivo manipulation procedures and possibly eliminating the need for chronic use of powerful immunosuppressive agents. Infusion of islet suspensions via the portal vein results in their implantation into the recipient liver, yet the rate of islet engraftment is unfortunately low as a result of several mechanisms. Numerous gene delivery studies have been performed with the purpose of protecting the allografts from immune rejection, apoptosis and inflammation. Recently, adeno-associated viruses have emerged as excellent vectors to target pancreatic β-cells and also confer long-term expression.52 For example, gene delivery of VEGF to pancreatic islets improved graft survival and promoted revascularization (Kieffer, Lecture; Tudurí and Kieffer, unpublished).


3. The generation of functional β-cells

Alternative sources of functionally competent, insulin-secreting β-cells as substitutes for donor islets to meet the clinical need for transplantation therapy are under investigation. Interestingly, recent evidence of an inherent plasticity of mature pancreatic cells has fuelled interest in in vivo (re-)generation of β-cells, through β-cell self-replication, differentiation from putative precursor cells, or by reprogramming from endogenous cell types. The use of human embryonic stem cells or of induced pluripotent stem cells may constitute an unlimited source of replacement β-cells. However, to date, despite a number of established protocols, both the efficiency of the in vitro differentiation/programming and the function of the derived β-cells remain limited.

a. The use of enteroendocrine cells

Given the similar secretion patterns of GLP-1, GIP, and insulin, targeting insulin expression to either K- or L-cells of the intestine may be able to recapitulate meal-regulated physiological release of insulin, thereby eliminating the requirement for exogenous insulin. Importantly, enteroendocrine cells contain prohormone convertases, which are required to process proinsulin into mature bioactive insulin, and also secretory vesicles, suggesting that they could potentially process, store, and secrete insulin in a similar fashion to pancreatic β-cells. From these observations, ectopic expression of insulin from K-cells was performed and the experiments showed that the insulin produced was sufficient to maintain glucose homeostasis in the transgenic animals and to protect them from developing diabetes following β-cell damage.53 However, more investigations and improvements are needed before application in human subjects. Several studies have attempted to generate β-cells in vivo by ectopic expression of defined transcription factors in cells from the gut, given that the pancreas naturally develops from the gut. For example, adenoviral-induced overexpression of MafA (Ad-MafA) inrat intestinal cells in vivo promoted their differentiation into insulin-producing cells, resulting in increased plasma insulin levels and amelioration of hyperglycemia after STZ treatment. However, the insulin-producing intestinal cells did not secrete more insulin in response to an oral glucose load. Further investigation is clearly required to develop safe and effective vectors and approaches to make glucose-responsive insulin-producing cells (Kieffer, Lecture; Tudurí and Kieffer, unpublished).

b. Conversion of α-cells into functional β-cells

As described in Chapter I2c, forced Pax4 overexpression in glucagon-producing cells resulted in an increase of β-like cells.9 This indicates that a switch from α- to β-cell fate is possible. This discovery has led to the exciting possibility that Pax4 could be used for “reprogramming” of α-cells to β-cells. Recently, it was shown that following diphtheriatoxin−mediated ablation of >99% of β-cells in transgenic mice,54 the islet remnant mainly composed of α-cells was reprogrammed to upregulate β-cell transcription markers (as Pdx1 and Nkx6.1) and cells co-expressed glucagon and insulin transiently, before turning to the mature and functional β-cell fate. Thus α-cells can be reprogrammed to become β-cells. However, it is unclear whether the newly-formed β-cells are capable of replacing original, fully functional β-cells, as many mice in both studies are diabetic and this raises the question of the source of α-cells to be reprogrammed for use inhuman transplantation (Kaestner, Lecture; Bramswig and Kaestner, unpublished).


4. Incretin-based therapy

In 2005, the treatment choices for T2D were expanded by the introduction of incretin-based therapy, considering the pleiotropic antidiabetic effects of GLP-1. A challenge in the development has been the short half-life of GLP-1 (1-2 minutes), due to the rapid inactivation through truncation by removal of the N-terminal dipeptide end by DPP-4. To overcome this shortcoming of native GLP-1 as a therapeutic agent, two strategies have been explored and developed in clinical practice: GLP-1 receptor agonists and DPP-4 inhibitors.
■ The GLP-1 receptor agonists have high affinity for the receptors and are largely resistant to inactivation by DPP-4. They have therefore the ability to achieve longstanding GLP-1 receptor activation. There are two kinds of GLP-1 receptor agonists: exendin-4−based compounds (Exenatide (ByettaR, Amylin/Lilly) and lixisenatide (Sanofi Aventis)) and true GLP-1 analogues (liraglutide (VictozaR, Novo Nordisk)). Both drugs reduce blood glucose and lower hemoglobin A1c in diabetic subjects, with concurrent improvements in insulin secretion and reduction in plasma glucagon. Ways to prolong the half-lives of GLP-1 receptor agonists are under investigation, so as to avoid repeated administration.
■ DPP-4 inhibitors inhibit the catalytic site of DPP-4 and prevent the inactivation of endogenous GLP-1. They also increase GIP levels, along with insulin secretion, reduce glucagon secretion and improve blood glucose. DPP-4 inhibitors are all orally active small molecules: sitagliptin (JanuviaR, Merck) was introduced clinically in 2006, followed by vildagliptin (GalvusR, Novartis, 2008), saxagliptin (OnglyzaR, BMS/AstraZeneca, 2009) and (in Japan only so far) alogliptin (Takeda) (D’Alessio,Lecture; D’Alessio, unpublished).
Incretin-based therapy can be used in several indications in patients with T2D with insufficient glycemic control, as monotherapy or in combination with metformin,sulfonylurea, thiazolidinediones, and insulin. Several recent studies have shown that patients treated with metformin and incretin-based therapy have considerably less hypoglycemia and no weight gain (DPP-4 inhibitors) or weight loss (GLP-1 receptor agonists), compared with treatment with metformin and sulfonylurea which is associated with high incidences of hypoglycemia and weight gain. However, there are still no long-term studies with incretin-based therapies that include cardiovascular hard end-point data, although data from clinical studies have shown high safety with very low risk of adverse events, and the cost of the treatment is considerably higher than that of sulfonylurea.
One potential new indication for incretin-based therapy is T1D, since GLP-1 may preserve and even possibly restore β-cell function, as well as potentially expanding the β-cell mass and also inhibiting glucagon secretion. Administration of pharmacologic amounts of GLP-1 to hyperglycemic T1D subjects with minimal β-cell function caused a 3-4 mM drop in blood glucose coincident with a 40%-50% decrease in plasma glucagon.
An important potential future development of incretin-based therapy is also the development of compounds that stimulate the release of endogenous GLP-1. The cellular mechanisms responsible for nutrient-regulated GLP-1 secretion are all potential targets, such as sugar, amino acids, fat, main macronutrients (the “preload concept,” ie, ingestion of a small amount of macronutrients prior to ingestion of the main meal may augment incretin hormone secretion during the main meal). An alternative approach is gene delivery systems to express GLP-1 in β-cells, and this has resulted in increased β-cell proliferation and protection against STZ-induced diabetes in injected mice. Finally, manipulation of proglucagon processing, like adenovirus-mediated prohormone convertase overexpression in islet cells,55 can lead to the conversion of hyperglycemic glucagon-secreting α-cells into hypoglycemic GLP-1−secreting cells, a tactic that might be useful in the context of diabetes (Kieffer, Lecture; Tudurí and Kieffer, unpublished). Such alteration of proglucagon processing was observed invivo after islet injury and could explain, at least in part, the high circulating concentrations of GLP1 and glucagon following bariatric surgery (D’Alessio, personal communication; Madsen, Lecture; Jensen et al, unpublished).
■ Based on the knowledge that high glucagon contributes to hyperglycemia in T2D, attempts have been undertaken to develop glucagon receptor antagonists for the treatment. However, this approach to treat diabetes has not been successfully developed yet. Moreover, acute administration of glucagon stimulates energy expenditure and inhibits food intake. Thus an approach to treat T2D would be to stimulate the glucagon receptors provided that the hyperglycemic effect of glucagon is prevented. This may be achieved by simultaneous stimulation of GLP-1 receptors(Ahren, Lecture; Ahren, unpublished).

5. Other incretins and bioactive gastro-entero-pancreatic hormones

Other incretins and bioactive gastro-entero-pancreatic hormones can improve insulin secretion as GIP receptor agonists. However, GIP stimulates glucagon secretion, so inhibition of GIP receptors, rather than stimulation, would be a therapeutic target. Oxyntomodulin analogues are also potential therapeutic agents since oxyntomodulin is released together with GLP-1 and is an agonist for both GLP-1 and glucagon receptors. PYY3-36 alone or in combination with oxyntomodulin, cholecystokinin, and ghrelin receptor antagonism could also be used (Ahren, Lecture).

“Pancreatic α-cells and glucagon—neglected metabolic actors”
I- Birth and death of the α-cell
II- Regulation of glucagon expression
III- Regulation of glucagon secretion
IV- Role of glucagon in metabolism
V- Therapeutic perspectives
VI- Conclusion
Lectures during IGIS meeting and unpublished reviews