Loss of tetraspanin-7 expression reduces pancreatic β-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism

Abstract

Background: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate β-cell L-type Ca²⁺ channel activity. However, the role of Tspan7 in pancreatic β-cell function is not yet fully understood.

Methods: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. β-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR.

Results: Tspan7 is expressed in insulin-containing granules of pancreatic β-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7^y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7^y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca²⁺ current was enhanced in Tspan7^y/- β-cells, but β-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca²⁺ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K⁺ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent β-cells, exocytotic Ca²⁺ sensitivity was reduced in a human β-cell line (EndoC-βH1) following Tspan7 KD.

Conclusion: Tspan7 is involved in the regulation of Ca²⁺ -dependent exocytosis in β-cells. Its function is more significant in human β-cells than their rodent counterparts.

Keywords: exocytosis; insulin; synaptotagmin; tetraspanin-7; β-cell.

FIGURE 1

Tetraspanin‐7 is expressed in pancreatic islets of Langerhans. (a) Immunofluorescent staining of human (upper) and mouse (lower) pancreas sections. Tetraspanin‐7 (TSPAN7 or Tspan7, red) is detected in β (Insulin, green)‐ and α (Glucagon, grey)‐cells. Scale bar: 30 μm. (b, c) Immunogold labelling of TSPAN7 (15 nm gold particles, black arrows), in glucagon (b) and insulin (c) containing vesicles in human islets. Insulin was labelled with 10 nm gold particles (white arrows). Scale bar: 500 nm.

FIGURE 2

In vivo metabolic phenotyping of Tspan7^y/− mouse model. (a–e) Ad libitum bodyweight (a), and plasma concentrations of glucose (b), insulin (c), C‐peptide (d) and proinsulin measured in Tspan7^y/− mice (red) and age‐matched (11.3 ± 0.3 wks) litter mate control (black). *p < 0.05 versus control and **p < 0.01 versus control. (f) Plasma glucose concentrations of Tspan7^y/− mice (red) and age‐matched litter mate control (black) measured during intraperitoneal glucose tolerance tests. Glucose bolus was injected at 0 min. N = 5 for control and 6 for Tspan7^y/−. Data are presented as mean value ± SEM.

FIGURE 3

Electrical activity and K_ATP‐channel conductance measured in Tspan7^y/− β‐cells. (a) Membrane potential recordings of control (upper trace, black) and Tspan7^y/− (lower trace, red) β‐cells within intact islets in response to changes in extracellular glucose (1 mM, 10 mM and 20 mM) or application of 200 μM tolbutamide, as indicated). (b) Summary of control (black) and Tspan7^y/− (red) β‐cell membrane potential measured at conditions indicated. N = 7 for control and 14 for Tspan7^y/− β‐cells at 1 mM glucose; N = 7 for control and 11 for Tspan7^y/− β‐cells at 10 mM glucose; N = 4 for control and 6 for Tspan7^y/− β‐cells at 20 mM glucose and N = 3 for control and 6 for Tspan7^y/− β‐cells when 200 μM tolbutamide was applied. *p < 0.05 versus control. (c, d), as in (b) but summarise the peak voltage (c) and firing frequency (d) of action potentials measured in control (black) and Tspan7^y/− (red) β‐cells at conditions indicated. N = 4 for control and 7 for Tspan7^y/− β‐cells at 10 mM glucose; N = 4 for control and 6 for Tspan7^y/− β‐cells at 20 mM glucose and N = 3 for control and 3 for Tspan7^y/− β‐cells when tolbutamide was applied. (e) Summary of K_ATP‐channel conductance measured in control (black) and Tspan7^y/− (red) β‐cells under the conditions indicated. N = 4 for control and 7 for Tspan7^y/− β‐cells at 1 mM and 10 mM glucose; N = 4 for control and 6 for Tspan7^y/− β‐cells at 20 mM glucose and N = 3 for control and 5 for Tspan7^y/− β‐cells when tolbutamide was applied. (f) Examples of K_ATP currents measured in control (black, middle trace) and Tspan7^y/− (red, bottom trace) β‐cells. K_ATP currents were triggered by ±10 mV excursions from −70 mV (200 ms, top) in voltage‐clamped β‐cells under the conditions indicated. All data are presented as mean value ± SEM.

FIGURE 4

Ca²⁺ current and exocytosis in Tspan7^y/− β‐cells. Tspan7 loss of function significantly increases Ca²⁺ current density in β cells (a, b). (a) Representative traces of control (black) and Tspan7^y/− (red) β‐cell Ca²⁺ current elicited by a 100‐ms depolarisation from −70 mV to 0 mV. (b) Summary of control (black) and Tspan7^y/− (red) β‐cell Ca²⁺ current density in relationship to membrane voltages. N = 7 for control and 5 for Tspan7^y/− β‐cells, **p < 0.01 versus control, paired comparison and Tukey. (c) Examples of exocytosis induced by membrane depolarisations from −70 mV to 0 mV (at the duration of 100 ms, 200 ms, 300 ms, 500 ms and 800 ms as indicated above the traces) of control (black) and Tspan7^y/− (red) β‐cells. (d) Summary of control (black) and Tspan7^y/− (red) β‐cell exocytosis (normalised to cell size, fF/pF) in relationship to pulse durations. N = 11 cells for control and 14 cells for Tspan7^y/− β‐cell. (e) Control (black) and Tspan7^y/− (red) β‐cell charges‐exocytosis relationship for pulses up to 100 ms. Corresponding durations of the pulses are as labelled. N = 7 cells for control and 5 cells for Tspan7^y/− β‐cells. (f) Insulin secretion measured from batch‐incubated control (black) and Tspan7^y/− (red) islets in response to glucose or tolbutamide (concentrations as indicated). Islets were isolated from mice aged 20 weeks and N ≥ 3 for control and Tspan7^y/− islets. **p < 0.01 and ***p < 0.001 versus 1 mM glucose alone of the same genotype. All data are presented as mean value ± SEM.

FIGURE 5

TSPAN7 KD affects exocytosis from human pancreatic β‐cells. (a) Gene expressions that changed significantly in human TSPAN7 KD pseudo‐islet. Volcano plot shows that genes involved in the pathways of secretion (yellow area) and regulation of transport (green area) were down‐regulated; markers of the apoptotic pathway (blue) were up‐regulated. TSPAN7 KD was demonstrated by the marked reduction in TSPAN7 expression (red). (b) Dynamic insulin secretion measured from perifused human pseudo‐islets of TSPAN7 KD (red) or control (Scramble, black) in response to different glucose concentrations and 20 mM K⁺ as indicated. Inset: area of the curve (AUC) of insulin secretion stimulated by 20 mM K⁺. N = 3 for control and TSPAN7 KD, *p < 0.05 versus insulin secretion at 1 mM glucose in the same group of pseudo‐islets; ^† p < 0.05 versus control (Scramble), Student's t‐test. (c) Examples of exocytosis of TSPAN7 KD EndoC‐βH1 (red) or control EndoC‐βH1 (Scramble, black) triggered by depolarisation from −70 to 0 mV with progressively increasing duration (as indicated). (d) Summary of control (black) and exocytosis (normalised to cell size, fF/pF) in relationship to pulse durations. N = 10 cells for control and 11 cells for TSPAN7 KD EndoC‐βH1 cells. (e) The relationship between depolarisation‐triggered charge influx and pulse durations of control (black) and TSPAN7 KD (red) EndoC‐βH1 cells. N = 10 cells for control and 11 cells for TSPAN7 KD EndoC‐βH1 cells. *p < 0.05 versus control, student's t‐test. (f) The relationship between charges and exocytosis of control (black) and TSPAN7 KD (red) EndoC‐βH1 cells. Corresponding durations of the pulses are as labelled. N = 10 cells for control and 11 cells for TSPAN7 KD EndoC‐βH1 cells. (g) Insulin secretion measured from batch‐incubated control (black) and TSPAN7 KD (red) EndoC‐βH1 cells in response to 1 and 20 mM glucose. Data are the summary of three independent biological repeats in technical triplicate. *p < 0.05 and ***p < 0.001 versus control insulin secretion at 1 mM glucose and ^†† p < 0.01 versus control insulin secretion at 20 mM glucose; two‐way ANOVA and Tuckey post hoc test. (h) Insulin content measured from control (black) and TSPAN7 KD (red) EndoC‐βH1 cells. Data are the summary of three independent biological repeats in technical triplicate. All data are presented as mean value ± SEM.

FIGURE 6

Changes in VGCC‐ and synaptotagmin‐encoding genes in EndoC‐βH1 following TSPAN7 KD. The expression of genes encoding TSPAN7 (TSPAN7), VGCCs (CACNA1A, CACNA1C and CACNA1D) and synaptotagmins (SYT4, SYT5 and SYT7) measured from control (transduced with Ad‐Scramble, Scramble, black) and TSPAN7 KD (red) EndoC‐βH1 cells. The levels of expression are normalised to that in control cells (as 100%). Error bars of the control cells condition reflect the variation of the control across the biological repeats. Data are summary of measurements made in two to four independent biological replicates. ***p < 0.0001 versus control, Student's t‐test.