III- Intrinsic hyperstimulation of β-cells

1. Persistent hyperinsulinemic hypoglycemia of infancy: a model for β-cell proliferation and survival

Hyperinsulinism of infancy (HI), also known as persistent hyperinsulinemic hypoglycemia (PHH) of infancy, is a rare genetic disorder that occurs in approximately 1 in 50 000 live births. Most cases are caused by mutations in the subunits of the β-cell ATP-sensitive potassium channel (KATP channel), a minority of patients have glucokinase (GCK) or glutamate dehydrogenase mutations, and in 40%-50% of the patients, the genetic cause of the disease is still not known. The histologic appearance of the pancreata from affected children can be subdivided into 2 major forms: diffuse HI and focal HI. The diffuse HI is more common and bears some characteristics of nesidioblastosis such as the persistence of the neonatal-type β-cell distribution in older patients. The focal HI is generally easily recognized as a discrete region of adenomatous hyperplasia, whereas the rest of the pancreas appears normal for its age. Patients with genetic evidence of diffuse or focal HI who do not undergo surgery appear to enter clinical remission over a period of months to years. Whereas patients with diffuse HI progress to diabetes, patients with suspected focal HI glucose and insulin dynamics normalize. Glaser’s group studied the age-specific changes in human β-cell proliferation and apoptosis leading to the pancreatic remodeling normally seen in the postnatal period and compared them with pancreata from children with HI. They showed persistent increased β-cell proliferation and apoptosis in the HI pancreata, probably explaining the fetal-type β-cell distribution found in older patients with diffuse HI. The slow progressive decrease in insulin secretion observed clinically in these patients also suggested that the net rate of apoptosis was greater than that of proliferation leading to loss of β-cell mass (Benjamin Glaser, Lecture).19

2. Role of KATP channels

In the β cell, glucose metabolism turns on two distinct complementary sequences of events known as the triggering pathway and the metabolic amplifying pathway. The triggering pathway begins with the raising of the ATP/ADP ratio, which closes KATP channels, depolarizes the cell membrane, activates voltage-gated calcium channels (Ca2+ channels), and results in calcium influx, which in turn triggers exocytosis of insulin-containing granules (Figure 4).20 The metabolic amplifying pathway does not directly implicate KATP channels or any further rise in cytosolic global or subplasmalemmal [Ca2+]c, but augments the secretory response to the triggering Ca2+ signal by as yet unresolved mechanisms. Pancreatic KATP channels are hetero-octameric complexes of two separate proteins: the sulfonylurea receptor 1 (SUR1, ABCC8) subunit and the potassium channel Kir6.2 (KCNJ11) subunit. Antidiabetic agents that target KATP channels are a part of the treatment recommended by the American Diabetes Association and the European Association for the Study of Diabetes for T2D. Sulfonylureas (SUs), such as tolbutamide, gliclazide, and glibenclamide, inhibit KATP channel activity, causing membrane depolarization and triggering insulin secretion. Thus, SUs can uncouple metabolism from electrical activity and are widely used to treat T2D. However, many data have reported beneficial (eg, stimulation of β-cell proliferation and protection against β-cell death) as well as deleterious (eg, reduction in insulin content, increase in β-cell apoptosis, and reduction in GIIS) effects of SUs. Conversely, KATP-channel openers (KCOs), such as diazoxide, activate KATP channels, thereby electrically silencing the cell at elevated glucose concentrations and inhibiting insulin secretion. According to the current concept, protection against β-cell death is attributed to KCOs instead of channel blockers as they improve insulin secretion when applied during continuous hyperglycemia and can help in the restoration of cellular insulin content.

a. KATP channels, hyperstimulation and hyperexcitation of β-cells

Under physiological conditions, glucose stimulation of β-cell electrical excitation leads to insulin secretion. Islets from mice with loss-of-function mutations in KATP channels, such as Kir6.2−/− and Sur1−/− mice, are continuously hyperexcited even at low glucose concentrations that normally do not lead to hyperstimulation of insulin secretion. Neonatal mutant mice exhibit transient hyperinsulinemia and hypoglycemia, but islets from adults show a dramatic loss of insulin secretion at all glucose concentrations, and the animals are relatively hypoinsulinemic. In Kir6.2 [Gly132Ser] mice, which specifically lack β-cell KATP channels, loss of β-cell mass was reported, although hyperglycemia was apparently spontaneously improved and insulin content even increased in older mice. Conversely, β-cell–specific Kir6.2 [AAA] dominant-negative mice, which lose KATP channel activity in only about 70% of β cells, exhibit elevated circulating insulin levels that persists through adulthood, with essentially normal insulin content and islet morphology. Heterozygous Kir6.2+/− and Sur1+/− mice, with about 60% reduction in KATP density in every cell, show a similar phenotype. Complete lack of KATP activity has been reported in β cells from some HI patients, but the phenotype of many HI mutations would suggest that KATP is not always completely absent. On the contrary, gain-of-function mutations in mice expressing mutant KATP channels with reduced ATP sensitivity—either because of reduced ATP affinity (mutations in the ATP binding site) or through allosteric enhancement of open probability—only in pancreatic β cells (Kir6.2[ΔN2-30]) developed severe neonatal diabetes mellitus (NDM) and died. Indeed, KATP mutations that cause loss of ATP sensitivity are expected to maintain the membrane in a hyperpolarized state with hyposecretion of insulin. To overcome the drastic effect of the mutation, Nichols’ group used the inducible model strategy for the Rosa26-Kir6.2 [K185Q, ΔN30] transgene and observed that the resulting significant loss of ATP sensitivity led to strong glucose intolerance that progressed to severe diabetes. Growth retardation and dramatic reduction in insulin content, accompanied by profound loss of β-cell mass over time were also noticed as secondary effects. Importantly, syngeneic islet transplantation under the kidney capsule or chronic treatment with glibenclamide two days prior to onset of transgene expression induction prevented the development of diabetes and also maintained both pancreatic islet architecture and β-cell mass. This result clearly showed that secondary loss of insulin content and secretion was a consequence of systemic diabetes. Interestingly, glibenclamide was ineffectual once the disease had developed and insulin content was lost.21 One can suppose that exposure to episodic hyperglycemia may lead to similar secondary progression in human NDM patients, explaining that SUs can effectively trigger insulin secretion, but requirements tend to increase with length of disease and in some cases, SU therapy becomes ineffective (Colin Nichols, Lecture).20

b. KATP channels and oxidative stress

Glucolipotoxicity caused by chronic hyperglycemia and an excess of lipids is a main factor involved in the development of T2D. In fact, β cells often survive for a long period of time with exposure to high glucose and free fatty acid (FFA) concentrations that contribute to the slowly progressing impairment of β-cell function, including reduced β-cell mass and loss of insulin content. Numerous studies have reported that glucolipotoxicity might be related to oxidative stress which refers to a persistent imbalance between excessive ROS production and limited antioxidant defense, a situation that occurs in β cells during pathogenesis of diabetes. Briefly, the mitochondrial respiratory chain is a major source of ROS in β cells. The superoxide anion (O2 −) is tightly coupled to mitochondrial metabolism and is generated by a single electron reduction of molecular oxygen at the inner mitochondrial membrane mainly by complexes I and III. O2 − is a reactive molecule that is converted to less active hydrogen peroxide (H2O2) by superoxide dismutase (SOD) isoenzymes. H2O2 is further detoxified by CAT and GPxs. A growing amount of evidence indicates that ROS are involved in the maintenance of normal β-cell glucose responsiveness, as well as in the impairment of secretory capacity and cell viability, both parameters contributing to β-cell failure.22 This suggests that ROS may have different actions depending on whether cellular concentrations are either below or above a specific threshold, ie, signaling versus toxic effects. Rodent β cells are highly sensitive to oxidative stress because their antioxidant defenses are very low. Overexpression of antioxidant enzymes or antioxidant treatment protects β cells against the effects of nitric oxide (NO) and oxygen radicals and has beneficial effects on β-cell mass and insulin content in diabetic mice. Human βcells seem to be less prone to oxidative stress, possibly because they have greater CAT and SOD activity. Prediabetic and newly diagnosed T2D patients have increased oxidative stress and decreased antioxidant defense systems. Drews’ group showed that H2O2 can decrease the ATP/ADP ratio leading to the opening of β-cell KATP channels, hyperpolarization of the plasma membrane potential, and impaired insulin release. Interestingly, direct inhibition of KATP channels with the SU tolbutamide restored GIIS, suggesting that H2O2 interferes with β-cell metabolism whereas the secretory machinery of the cell remains intact. Importantly, Sur1–/– mice turned out to be less sensitive to ROS-induced inhibition of insulin secretion in vitro and streptozotocin (STZ)-induced diabetes in vivo. These mice showed an approximately 2-fold upregulation in SOD, CAT, and GPx activity that drastically reduced H2O2 – and NO-induced apoptotic cell death compared with Sur1+/+ mice. Similarly, gliclazide and tolbutamide treatment led to an increase in antioxidant enzyme activity, probably through Ca2+-dependent upregulation and attenuated ROS-induced apoptosis.23 Nevertheless, it is noteworthy that a global increase in apoptosis has been described for tolbutamide whereas gliclazide has no proapoptotic potency. This difference can be explained by the free radical–scavenging property of gliclazide. Figure 5 sums up the possible approaches to combat oxidative stress: reduction in ROS formation, upregulation of antioxidative enzymes, and application of antioxidants (Gisela Drews, Lecture).22

2. β-Cell fate in defective cellular calcium regulation

The fundamental second messenger for insulin release is Ca2+ (Figure 4).20 Indeed, a number of nutrients, hormones, and pharmacological substances that influence insulin secretion alter Ca2+ uptake and/or Ca2+ influx. Whereas millimolar concentrations of Ca2+ are present in the extracellular space and equally high amounts of bound Ca2+ may be found in intracellular stores, the intracellular free Ca2+ concentration in the β-cell cytoplasm is kept below 100 nM under unstimulated conditions. This is achieved by various types of Ca2+-ATPases that pump out cytoplasmic Ca2+ and by Ca2+-buffering actions of many cytoplasmic Ca2+-binding proteins. When the β cell is stimulated, cytosolic Ca2+ increases as a result of either Ca2+ influx from the extracellular space or Ca2+ release from intracellular stores. The Na+/Ca2+ exchanger (NCX) is an antiporter membrane protein that removes calcium from cells. This electrogenic transporter uses the energy that is stored in the electrochemical gradient of Na+ by allowing Na+ to flow down its gradient across the plasma membrane in exchange for the countertransport of Ca2+ ions, with a stoichiometry of 3 Na+ for 1 Ca2+. It extrudes Ca2+ in parallel with the plasma membrane’s ATP-driven Ca2+ pump and as a reversible transporter, it also mediates Ca2+ entry in parallel with various ion channels. Four isoforms of the NCX (NCX1 to NCX4) have been cloned and the β cell expresses various NCX1 splice variants in a species-specific pattern (NCX1.3 and 1.7 in the rat; NCX1.2, 1.3, and 1.7 in the mouse), in variable and different proportions. Interestingly, overexpression of the exchanger led to the depletion of endoplasmic reticulum (ER) Ca2+ stores causing ER stress, reduction of β-cell growth, and finally, activation of β-cell death by apoptosis.24 On the contrary, Ncx1 heterozygous deficient mice (Ncx1+/−), with NCX1 downregulation, were reported to have an increased GIIS and an enhancement of both phases of insulin release, but no increased mitochondrial glucose metabolism. Insulin content was doubled in Ncx1+/− mice compared with Ncx1+/+ mice, accompanied by an enhancement in proinsulin staining. In adult mice, β-cell mass had risen 100% in Ncx1+/− mice compared with Ncx1+/+ mice; this was mainly due to β-cell proliferation resulting from the activation of the calcineurin/nuclear factor of the activated T-cell signaling pathway by increased cellular Ca2+. Finally, Ncx1+/− islets were protected against hypoxia. Thus, the effectiveness of Ncx1+/− islet transplantation was tested, because in clinical islet transplantation, up to 70% of the transplanted β-cell mass is destroyed in the early post-transplant period mainly due to prolonged hypoxia during the revascularization process. Interestingly, transplantation with Ncx1+/− islets was at least twice as efficient as with Ncx1+/+ islets. Data both on improvement of insulin production and islet transplantation imply that Ncx1 is a potential new therapeutic target (André Herchuelz, Lecture).25

3. β-Cell fate in mice with activating GCK mutations

GCK is the high-Km enzyme that phosphorylates glucose on its entry into the β cell (Figure 4).20 Persistent hyperinsulinemic hypoglycemia (PHH) due to a novel GCK mutation was recently described. The mutation led to a markedly increased affinity for glucose and to the subsequent increase in intracellular glucose flux and lower threshold for GIIS. Moreover, abnormally large pancreatic islets were reported that showed both proliferating and apoptotic β cells, probably resulting from increased intracellular glucose flux.26 This data in human was consistent with previous findings in murine models. In constrast, adult mice with tamoxifen-inducible deletion of GCK specifically in β cells developed severe hyperglycemia and hypoinsulinemia, consistent with the inability of mutant β cells to sense glucose and secrete insulin. Mutant mice also showed a dramatic drop in β-cell proliferation and increased β-cell apoptosis resulting in a 2-fold reduction in total β-cell mass 2 months after GCK deletion. As expected, increasing GCK activity stimulated the β-cell proliferation rate in a KATP channel/membrane depolarization–dependent way. This work suggests a role for GCK and glucose metabolism in the regulation of β-cell secretion and replication, such as a short pulse of glucose metabolism, as after a meal, would trigger β-cell secretion, while more persistent activation of the pathway would trigger β-cell replication. This implies that GCK activators could have beneficial effects on β-cell mass and on the contrary, that normalization of blood glucose in diabetic patients could lead to the suppression of the glucose metabolism–induced mitogenic effects on β cells (Yuval Dor, Lecture).27

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The hyperstimulated β cell: prelude to diabetes?
I- What is the role of epigenetics in insulin gene expression and insulin secretion and action
II- Impact of insulin resistance on β-cell function
III- Intrinsic hyperstimulation of β-cells
IV- Modulation of β-cell function by secretory products
V- Conclusion
Lectures during IGIS meeting