Targeting the Pancreatic α-Cell to Prevent Hypoglycemia in Type 1 Diabetes

Abstract

Life-threatening hypoglycemia is a limiting factor in the management of type 1 diabetes. People with diabetes are prone to develop hypoglycemia because they lose physiological mechanisms that prevent plasma glucose levels from falling. Among these so-called counterregulatory responses, secretion of glucagon from pancreatic α-cells is preeminent. Glucagon, a hormone secreted in response to a lowering in glucose concentration, counteracts a further drop in glycemia by promoting gluconeogenesis and glycogenolysis in target tissues. In diabetes, however, α-cells do not respond appropriately to changes in glycemia and, thus, cannot mount a counterregulatory response. If the α-cell could be targeted therapeutically to restore its ability to prevent hypoglycemia, type 1 diabetes could be managed more efficiently and safely. Unfortunately, the mechanisms that allow the α-cell to respond to hypoglycemia have not been fully elucidated. We know even less about the pathophysiological mechanisms that cause α-cell dysfunction in diabetes. Based on published findings and unpublished observations, and taking into account its electrophysiological properties, we propose here a model of α-cell function that could explain its impairment in diabetes. Within this frame, we emphasize those elements that could be targeted pharmacologically with repurposed U.S. Food and Drug Administration-approved drugs to rescue α-cell function and restore glucose counterregulation in people with diabetes.

Figure 1

The dynamics of glucagon secretion do not reflect absolute glucose levels. Glucagon and insulin secretion were measured in perifusion experiments of isolated human islets stimulated with changes in glucose concentration. A: Glucagon secretion, to a drop in glucose concentration from 11 to 1 mmol/L, is transient. B: Glucagon and insulin secretion in response to stepwise increases in glucose concentration, the conventional way to determine glucose dependency in the field of islet biology. C: Glucagon and insulin secretion in response to stepwise decreases in glucose concentration. Notice that glucagon secretion does not continue to increase as glucose levels decrease. D and E: Glucagon secretion (green symbols) tightly follows the inverted first derivative of insulin secretion (blue line, calculated from data shown in B and C and adjusted for a 3-min lag). At low levels of insulin secretion (last 20 min in E, also see C), glucagon secretion no longer follows the change in insulin secretion. F: The concentration–response relationship of insulin secretion was very similar when glucose levels were increased (glucose up) or decreased (glucose down). Data are those shown in panels B and C, with data in C reversed and aligned in time to that for the increase in glucose. G: The concentration–response relationship of glucagon secretion depended very much on whether glucose levels were increased or decreased stepwise. Data are from experiments performed by Cabrera et al. (31,80) (n = 4 technical replicates).

Figure 2

β-Cell–derived serotonin inhibits glucagon secretion. A: Maximal projection of confocal images showing serotonin labeling in an islet in a human pancreatic section. Serotonin colocalizes with insulin. B: Serotonin is secreted in pulses form human islets maintained at 11 mmol/L glucose. C: In situ hybridization for the serotonin receptor 5HT_1F shows colocalization with transcripts for glucagon. D: Glucagon secretion in response to a drop in glucose concentration from 11 to 1 mmol/L is inhibited by the 5HT_1F receptor antagonist LY344864 (100 nmol/L) in human islets. E: Glucagon secretion in response to a drop in glucose concentration from 11 to 1 mmol/L is inhibited by the SSRI fluvoxamine (Flu; 500 nmol/L). F: Insulin-induced hypoglycemia was exacerbated in the presence of LY344864 (LY) (n = 12 mice per group). Veh, vehicle. G: Increases in glucagon plasma levels stimulated by decreasing glycemia with insulin (1 unit/kg) were inhibited in the presence of the 5HT_1F receptor agonist LY344864 (1 mg/kg, i.v.; n = 5 mice per group). H: Model of how β-cells release serotonin to regulate glucagon secretion in human islets. AC, adenylate cyclase; G_i, Galpha_i; GluR_2/3, iGluRs of the AMPA type composed by GluR₂ and GluR₃ subunits; VGCC, voltage-gated Ca²⁺ channels. Data were adapted from Almaça et al. (32).

Figure 3

Glutamate receptor signaling forms a positive autocrine loop that amplifies glucagon secretion. A: Glutamate induced glucagon responses in human islets that could be blocked by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 μmol/L). The iGluR agonists kainate and AMPA (both 100 μmol/L) also elicited strong glucagon secretion. B: Insulin release was induced by high glucose (11 mmol/L; 11G) but not by kainate (representative of six islet preparations). C: Glucagon secretion from human islets in response to kainate was blocked by Ca²⁺ channel blockers and was strongly reduced in the absence of nominal Ca²⁺ in the solution. D: Ca²⁺ imaging of dispersed human β-cells (black traces) and α-cells (red traces) showed that only α-cells responded to glutamate. E: Glucagon secretion in response to a drop from 11 to 1 mmol/L glucose concentration, as measured by biosensor cells, was inhibited in the presence of CNQX (10 μmol/L). F, left: Hyperinsulinemic-hypoglycemic clamp to provide a constant hypoglycemic stimulus at ∼3 mmol/L blood glucose concentration was induced with insulin infusion in mice. Glucagon secretion in response to hypoglycemia was significantly diminished in mice after infusion of the iGluR antagonist NBQX (10 mg/kg; red symbols; n = 7) compared with saline-infused mice (black symbols; n = 3; repeated-measures ANOVA, P < 0.05). Bar indicates drug infusion. Notice that the glucose infusion rate needed to maintain glycemia after drug infusion was significantly larger in NBQX-treated mice than in saline-treated mice (not shown; n = 3; Student’s t test, P < 0.05). G: Model of how α-cells release glutamate to amplify glucagon secretion in human islets. GluR_2/3, iGluRs of the AMPA type composed by GluR₂ and GluR₃ subunits; VGCC, voltage-gated Ca²⁺ channels. Data werae adapted from Cabrera et al. (31).

Figure 4

Using living pancreas slices to study α-cell function. A: A human pancreas slice immunostained for endocrine markers (green) after a physiologic experiment shows widespread distribution of islets embedded in the exocrine pancreas. B and C: Confocal images showing islets immunostained for insulin and glucagon in slices from a donor without diabetes (B) and a donor with type 1 diabetes (C). D: Ca²⁺ imaging performed in a living pancreas slice from a mouse expressing the genetically encoded Ca²⁺ indicator GCamP6 in α-cells. The islet stands out by its backscatter (top left). The fluorescence intensity of GCamP6 (in pseudocolor scale) increases in response to a drop in glucose from 7 to 1 mmol/L (1G) or to KCl depolarization. E and F: The loss of β-cells seen in type 1 diabetes can be mimicked in the mouse by using streptozotocin. Notice the similarity of the islet in a slice from a streptozotocin-treated mouse to the islet from the type 1 diabetes donor in C (same settings as those in B and C). G and H: Using Ca²⁺ imaging to interrogate large numbers of α-cells reveals differential responses to lowering the glucose concentration and to serotonin (10 μmol/L). Panel G shows a heatmap of GCamP6 fluorescence intensity over time, with each row representing a single α-cell (total of 555 α-cells, pooled from 8 islets, 8 slices, and 4 mice). Scale shows dF/F of GCamP6 fluorescence intensity. F: Representative traces of data shown in G illustrating the heterogeneity of responses to serotonin in the α-cell population. Scale bars: 50 μmol/L.

Figure 5

Proposed model of regulation of glucagon secretion. α-Cells are activated by a lowering of glucose concentration. Under constant conditions, glucagon secretion wanes because voltage-gated channels inactivate and ionotropic glutamate receptors desensitize (red cloud). When glucose levels increase, β-cells secrete insulin and other paracrine factors that inhibit α-cells, among others, by hyperpolarizing the cell via GABA_A receptors. This allows voltage-gated ion channel and ionotropic glutamate receptors to recover from inactivation and desensitization (green star). In type 1 diabetes, α-cells are locked in the refractory state shown on the right because they cannot be reset by β-cell input. GluR_2/3, iGluRs of the AMPA type composed by GluR₂ and GluR₃ subunits; VGCC, voltage-gated Ca²⁺ channels.