Dysregulation of Glucagon Secretion by Hyperglycemia-Induced Sodium-Dependent Reduction of ATP Production

Abstract

Diabetes is a bihormonal disorder resulting from combined insulin and glucagon secretion defects. Mice lacking fumarase (Fh1) in their β cells (Fh1βKO mice) develop progressive hyperglycemia and dysregulated glucagon secretion similar to that seen in diabetic patients (too much at high glucose and too little at low glucose). The glucagon secretion defects are corrected by low concentrations of tolbutamide and prevented by the sodium-glucose transport (SGLT) inhibitor phlorizin. These data link hyperglycemia, intracellular Na⁺ accumulation, and acidification to impaired mitochondrial metabolism, reduced ATP production, and dysregulated glucagon secretion. Protein succination, reflecting reduced activity of fumarase, is observed in α cells from hyperglycemic Fh1βKO and β-V59M gain-of-function K_ATP channel mice, diabetic Goto-Kakizaki rats, and patients with type 2 diabetes. Succination is also observed in renal tubular cells and cardiomyocytes from hyperglycemic Fh1βKO mice, suggesting that the model can be extended to other SGLT-expressing cells and may explain part of the spectrum of diabetic complications.

Keywords: Fh1; diabetes; glucagon; sodium-glucose co-transport; succination.

Graphical abstract

Figure 1

Dysregulation of Glucagon Secretion in Fh1βKO Mice (A) Glucagon secretion in isolated islets from control (CTL; black) and normoglycemic (plasma glucose: <12 mM; gray) and diabetic (plasma glucose: >20 mM; red) Fh1βKO mice at 1 and 6 mM glucose. ^∗p < 0.05 versus 1 mM glucose; ^#p < 0.05 versus 1 mM glucose in normoglycemic Fh1βKO islets (n = 8–9 experiments using islets from 12 mice). (B) Islet glucagon content in normoglycemic and hyperglycemic Fh1βKO mice. ^∗p < 0.05 (n = 12 mice of each group, each measurement based on 12 islets). (C) Immunohistochemistry (IHC) for succination (2SC) in CTL and Fh1βKO islets. Scale bar, 50 μm. (D) Plasma fumarate levels in CTL and severely hyperglycemic (>20 mM) Fh1βKO mice (n = 22 CTL and n = 13 Fh1βKO mice). (E and F) Glucagon secretion in isolated islets from wild-type (NMRI) islets at 1 and 20 mM glucose and supplementing the extracellular medium with 5 mM Na₂-fumarate (E; n = 4 experiments using islets from three mice), or 5 mM dimethyl (dm)-fumarate (F; n = 12 experiments using islets from four mice). ^∗p < 0.05 versus 1 mM glucose; ^#p < 0.05 versus 20 mM glucose. All data presented as mean values ± SEM of indicated number of experiments. See also Figure S1.

Figure 2

Ablation of Fh1 in α Cells Recapitulates the Effects of Diabetes on Glucagon Secretion (A) IHC for 2SC (green), glucagon (red) and overlay (yellow) islets from CTL (above) and Fh1αKO mice. Note strong 2SC labeling of most glucagon-positive cells. Scale bar, 50 μm. Data are representative of four mice of each genotype. (B) Glucagon secretion in CTL and Fh1αKO islets measured during 1 hr static incubations at 1 and 6 mM glucose. ^∗p < 0.05 versus 1 mM glucose; ^#p < 0.05 versus corresponding group in CTL mice (n = 7–8 experiments using islets from three CTL and three Fh1αKO mice). Inset: glucagon secretion at 1 and 6 mM glucose after compensating for α cells that retain Fh1. (C) Glucagon content in islets from CTL and Fh1αKO mice. ^∗p < 0.05 (n = 3 mice for each genotype). Inset: glucagon content after compensating for α cells that retain Fh1. (D) Electron micrographs of α cells in a CTL islet (left), a normal α cell in an Fh1αKO islet (center) and an abnormal α cell in an Fh1αKO islet (right; n = 5 cells). Scale bar, 2 μm. All data presented as mean values ± SEM of indicated number of experiments. See also Figure S2.

Figure 3

Hyperglycemia-Induced Changes in Glucagon Secretion Are Corrected by Modulation of K_ATP Channels (A) IHC for succination (2SC) in CTL and βV59M islets. Scale bar, 50 μm. (B and C) Glucagon secretion in hyperglycemic Fh1βKO (B) and βV59M (C) islets measured during 1 hr static incubations at 1 and 6 mM glucose in the absence or presence of a low concentration (10 μM) of the K_ATP channel blocker tolbutamide. ^∗p < 0.05 versus 1 mM glucose (n = 7–8 experiments using islets from at least three Fh1βKO and three βV59M mice). (D) As in (B) and (C), but experiments obtained from CTL littermates of (n = 6 experiments/6 mice) Fh1βKO and βV59M mice (n = 6–12 experiments/6 mice). ^∗p < 0.05 versus 1 mM glucose, ^#p < 0.05 versus 1 mM glucose without tolbutamide. (E) [Ca²⁺]_i measurements in α cells, using the calcium dye fluo4, within CTL (black) or hyperglycemic Fh1βKO (red) islets at 1 and 20 mM glucose as indicated. In both genotypes α cells were identified by the [Ca²⁺]_i response to adrenaline. (F) Histogram summarizing effects of increasing glucose on [Ca²⁺]_i, measured using fluo4, in α cells from normoglycemic CTL (black) and hyperglycemic Fh1βKO (red) islets as indicated. Data are presented as area under the curve. ^∗p < 0.05 versus 1 mM glucose in CTL mice. ^#p < 0.05 versus 1 mM glucose in Fh1βKO mice. Data are from indicated number of cells (n) in three islets from one CTL mouse and five islets from two Fh1βKO mice, respectively. All data presented as mean values ± SEM of indicated number of experiments. See also Figure S7.

Figure 4

Hyperglycemia Results in Intracellular Acidification of α Cells (A) Histogram summarizing basal α cell intracellular pH (pH_i) measured using the pH indicator seminaphthorhodafluor (SNARF) in α cells in islets from CTL (black) and hyperglycemic Fh1βKO (red) mice. Number of cells indicated below the respective bars in the histogram. ^∗p < 0.05 versus CTL. (B) pH_i measured in α cells in islets from CTL (black) and hyperglycemic Fh1βKO (red) mice at 3 mM and 20 mM glucose. The traces have been offset to reflect the true difference in fluorescence ratios (F_650/550) between CTL and Fh1βKO α cells. Measurements were performed in acutely isolated intact islets. (C) Net effect of 20 mM glucose on pH_i (ΔF_650/550) in α cells from CTL (black) and nearly normoglycemic (gray) or hyperglycemic (red) Fh1βκO mice. Number of cells (n) indicated above the respective bars in the histogram. ^∗p < 0.05 versus CTL; ^#p < 0.05 versus basal (at 3 mM glucose). (D) Dose-dependent acidification of wild-type α cells in response to ethyl-isopropyl amiloride (EIPA) measured using the pH indicator SNARF. (E) Histogram summarizing the effect of EIPA on α cell pH_i in wild-type islets. Number of cells indicated above the respective bars in the histogram. ^∗p < 0.05 versus basal (no EIPA). (F) Effect of the non-metabolizable glucose analogue α-methyl-D-glucopyranoside (αMDG) on cytoplasmic Na⁺ ([Na⁺]_i) in wild-type α cells in the absence (black) and the presence (red) of phlorizin. Glucose (1 mM) was present throughout. [Na⁺]_i was measured using Sodium Green. Fluorescence (F) values have been normalized to that at 1 mM glucose (F₀). (G) Effect of αMDG on [Na⁺]_i in the absence (black) and presence (red) of phlorizin in wild-type islets, as indicated. Data in (A) and (B) normalized to Sodium Green fluorescence under basal conditions (1 mM glucose; n = 59 cells) ^∗p < 0.05 versus 1 mM glucose; ^#p < 0.05 versus αMDG in the absence of phlorizin. Data are presented as mean values ±SEM of indicated number of experiments (n). In (B), (D), and (F), representative single-cell traces have been selected for display. See also Figures S2 and S3.

Figure 5

SGLT-Mediated Na⁺ Uptake Leads to Intracellular Acidification and Reduced ATP Production in the Hyperglycemic α Cell (A) Schematic of ex vivo experiments. Islets are isolated from mouse pancreas and incubated in 11–12 or 20 mM glucose for 48 hr. These concentrations approximate to fed plasma glucose levels before and after hyperglycemia develops in Fh1βKO mice (see Figures S1A and S1B). Figure was made using Servier medical ART. (B) Histogram summarizing basal pH_i measured using the pH indicator SNARF in α cells from wild-type islets incubated for 48 hr at 11 mM or 20 mM glucose, or 20 mM glucose plus 50 μM phlorizin. Number of cells (n) indicated below the respective bars in the histogram; ^∗p < 0.05 versus 11 mM glucose. ^#p < 0.05 versus 20 mM glucose. (C) Cytoplasmic ATP/ADP ratio at 1 and 20 mM glucose (indicated above recording) in α cells from wild-type islets cultured for 48 hr at 11 mM or 20 mM glucose, or 20 mM glucose plus 50 μM phlorizin. (D) Histogram summarizing the net effect of glucose on the cytoplasmic ATP/ADP ratio (ΔATP/ADP_c) in α cells from wild-type islets cultured at 11 or 20 mM glucose in the absence or presence of 50 μM phlorizin as indicated. ^∗p < 0.05 versus 1 mM glucose; ^#p < 0.05 versus 20 mM glucose in islets cultured at 11 mM glucose. (E) Glucagon secretion measured at 1 or 20 mM glucose in wild-type islets cultured for 48 hr at 12 mM, 20 mM glucose, or 20 mM glucose plus 50 μM phlorizin as indicated. ^∗p < 0.05 versus 1 mM glucose in respective group; ^#p < 0.05 versus 20 mM glucose in islets cultured at 20 mM glucose in the absence of phlorizin (n = 8 experiments using islets from six mice). (F) As in (E) using wild-type islets that were cultured for 48 hr at 12 mM glucose alone (white bars) or in the presence of 10 μM NHE inhibitor EIPA (gray bars) in the absence or presence of 10 μM tolbutamide as indicated. ^∗p < 0.05 versus 1 mM glucose in respective group; ^#p < 0.05 versus 20 mM glucose in islets cultured in the absence of EIPA; ^#p < 0.05 versus 1 mM glucose EIPA-incubated islets in the absence of tolbutamide (n = 3–8 experiments using islets from five mice). Data are presented as mean values ±SEM of indicated number of experiments (n). Traces in (C) represent average response in all cells. See also Figure S4.

Figure 6

Protein Succination Persists after Restoration of Normoglycemia (A) Glucagon secretion at 1 and 20 mM glucose in acutely isolated islets from CTL and hyperglycemic Fh1βKO mice. ^∗p < 0.05 versus 1 mM glucose (n = 9 experiments using islets from four mice of each genotype). (B) As in (A) but after 72 hr of culture at 12 mM glucose. ^∗p < 0.05 versus 1 mM glucose (n = 9 experiments for each genotype using islets from four CTL and four Fh1βKO mice). (C) Glucagon content in CTL and Fh1βKO islets either acutely isolated or after 72 hr of culture. ^∗p < 0.05 versus CTL. (D) Immunofluorescence for 2SC (green), glucagon (red), insulin (blue), and overlay (yellow) islets from CTL, hyperglycemic βV59M (diabetic), and normoglycemic βV59M mice (treated with glibenclamide). Note strong 2SC labeling of most glucagon-positive cells. Scale bar, 50 μm. (E) IHC for succination (2SC) in islets from non-diabetic (CTL) individuals and patients with type-2 diabetes (T2D). Scale bar, 50 μm. (F) Immunofluorescence for 2SC (green), glucagon (red), DAPI (blue), and overlay (yellow) in islets from patients diagnosed with T2D. Note strong 2SC labeling of most glucagon-positive cells. Scale bar, 50 μm. Data in (E) and (F) are representative of six donors for both the non-diabetic (CTL) and T2D groups. Data are presented as mean values ± SEM of indicated number of experiments (n). See Table S1 for details on the donors.

Figure 7

Protein Succination in Renal Tubular Cells and Cardiomyocytes from Hyperglycemic Fh1βKO Mice (A) IHC for 2SC in CTL (left) and diabetic Fh1βKO (right) hearts showing staining in a subset of cells in diabetic animals but not in the non-diabetic CTLs. Scale bar, 50 μm. (B) As in (A) but showing sections of kidney in CTL (left) and normoglycemic (middle) or hyperglycemic (right) Fh1βKO mice as indicated. (C) Schematic of the proposed relationship between hyperglycemia, impaired ATP production, and protein succination. The increased intracellular fumarate resulting from reduced activity of fumarase leads to protein succination. The sites of action of SGLT inhibitors and tolbutamide are indicated. Sections in (A) and (B) are representative of observations in >10 animals of both genotypes.