Age-Dependent Decline in the Coordinated [Ca2+] and Insulin Secretory Dynamics in Human Pancreatic Islets

Abstract

Aging is associated with increased risk for type 2 diabetes, resulting from reduced insulin sensitivity and secretion. Reduced insulin secretion can result from reduced proliferative capacity and reduced islet function. Mechanisms underlying altered β-cell function in aging are poorly understood in mouse and human islets, and the impact of aging on intraislet communication has not been characterized. Here, we examine how β-cell [Ca²⁺] and electrical communication are impacted during aging in mouse and human islets. Islets from human donors and from mice were studied using [Ca²⁺] imaging, static and perifusion insulin secretion assays, and gap junction permeability measurements. In human islets, [Ca²⁺] dynamics were coordinated within distinct subregions of the islet, invariant with islet size. There was a marked decline in the coordination of [Ca²⁺] dynamics, gap junction coupling, and insulin secretion dynamics with age. These age-dependent declines were reversed by pharmacological gap junction activation. These results show that human islet function declines with aging, which can reduce insulin action and may contribute to increased risk of type 2 diabetes.

Figure 1

Human islets show a wide range of [Ca²⁺] activity and coordination. A: Representative false color map of [Ca²⁺] activity and coordination in C57/B6 WT and Cx36^−/− islets (left) with time courses from four individual cells (i–iv) indicated in these islets (right). [Ca²⁺] activity is represented by presence of false color, with each color representing a separate region of [Ca²⁺] coordination, as indicated in legend above. B: Representative [Ca²⁺] activity and coordination maps in human islets from donors where high coordination similar to mouse islets is observed (left), as in A, with time courses from four individual cells indicated in these islets (right). C: Representative [Ca²⁺] activity and coordination maps in human islets from donors where low coordination is observed (left), as in A, with time courses from four individual cells indicated in these islets (right). D: Area of [Ca²⁺] activity normalized to islet size (active area) averaged over islets from WT mice, Cx36^−/− mice, and all human donors. E: Largest area of coordinated [Ca²⁺] activity normalized to islet size (max. coordinated area) averaged over islets from WT mice, Cx36^−/− mice, and all human donors. F: Absolute largest area of coordinated [Ca²⁺] activity plotted as a function of islet size (left) or binned by islet size (right). G: Largest area of coordinated [Ca²⁺] activity normalized to islet size plotted as a function of islet size (left) or binned by islet size (right). Data in D–G is displayed as mean ± SEM, averaged over n = 4 WT mice, n = 8 Cx36^−/− mice, and n = 40 human donors (four to six islets each). Vertical scale bars in A–C (right) indicate 20% fluorescence change.

Figure 2

Age-dependent decline in [Ca²⁺] coordination in human islets. A: Area of [Ca²⁺] activity (active area) normalized to islet size as a function of donor age (left) and averaged over donors less than (black) or greater than (red) the median age of 40 years (right). B: Area of coordinated [Ca²⁺] activity normalized to islet size as a function of donor age (left) and averaged over donors less than or greater than the median age, as in A (right). C: Absolute area of coordinated [Ca²⁺] activity as a function of donor age (left) and averaged over donors less than or greater than the median age, as in A (right). D: [Ca²⁺] oscillation duty cycle (plateau fraction) of largest coordinated area as a function of donor age (left) and averaged over donors less than or greater than the median age, as in A (right). In left panels, each data point represents a single donor, with outliers (ROUT test) indicated by empty circles. Solid line indicates linear regression, dashed lines indicate 95% CIs, and P values indicate the significance of a correlation. In right panels, data are displayed as mean ± SEM averaged over n = 40 donors (four to six islets per donor), with P values indicating the significance of differences between indicated groups (Student t test).

Figure 3

Age-dependent decline in Cx36 gap junction coupling in human islets. A: Cx36 gap junction function, as assessed through FRAP (rate of fluorescence recovery) as a function of donor age (left) and averaged over donors less than (black) or greater than (red) the median age of 40 years (right). B: Absolute area of coordinated [Ca²⁺] activity as a function of Cx36 gap junction function in islets from each donor (left) or in islets of each donors that shows lower than median or greater than median Cx36 gap junction function (right). In left panels, each data point represents a single donor. Solid line indicates linear regression, dashed lines indicate 95% CIs, and P values indicate the significance of a correlation. In right panels, data are displayed as mean ± SEM averaged over n = 32 donors (three to four islets per donor), with P values indicating the significance of differences between indicated groups (Student t test).

Figure 4

Age-dependent decline in insulin secretion dynamics. A: Representative perifusion time course of two human donors under indicated glucose stimulation protocol. The decline after 16.7 mmol/L glucose or 16.7 mmol/L glucose + IBMX is fitted to an exponential decay model (gray). B: Fold-change insulin secretion between 5.6 and 16.7 mmol/L as a function of donor age. C: Fold-change insulin secretion in the presence and absence of IBMX, averaged over donors greater than (red, columns 3 and 4) or the less than (black, columns 1 and 2) the median age of 40 years. D: Exponential decay rate of insulin secretion after transition from 16.7 to 5.6 mmol/L glucose as a function of donor age. E: Exponential decay rate of insulin secretion after transition from 16.7 to 5.6 mmol/L glucose in the presence and absence of IBMX, averaged over donors greater than or less than the median age, as in C. For data in B and D, each data point represents a single donor, solid line indicates linear regression, dashed lines indicate 95% CIs, and P values indicate the significance of a correlation. For data in C and E, data are displayed as mean ± SEM averaged over n = 76 donors, each with a single perifusion with 105 or 267 islet equivalents (IEQ) per donor, with P values indicating the significance of differences between indicated groups (Student t test).

Figure 5

Age-dependent decline in static insulin secretion. A: Insulin secretion at 2 mmol/L glucose, as measured by static assays, as a function of donor age (left) and averaged over donors less than (black) or greater than (red) the median age of 40 years (right). B: Insulin secretion at 20 mmol/L glucose as a function of donor age (left) and averaged over donors less than or greater than the median age (right), as in A. C: Stimulation index (fold change between 2 and 20 mmol/L glucose) as a function of donor age (left) and averaged over donors less than or greater than the median age (right), as in A. D: Insulin content as a function of donor age (left) and averaged over donors less than or greater than the median age (right), as in A. In A–D left panels, each data point represents a single donor, with outliers (ROUT test, based on B) indicated by empty circles. Solid line indicates linear regression, dashed lines indicate 95% CIs, and P values indicate the significance of a correlation. In A–D right panels, data are displayed as mean ± SEM averaged over n = 43 donors (each donor in duplicate), with P values indicating the significance of differences between indicated groups (Student t test).

Figure 6

Modafinil recovers the age-dependent decline in Cx36 function and [Ca²⁺] coordination. A: [Ca²⁺] activity (active area) in the presence and absence of modafinil (1 h incubation), averaged over donors greater than (red, columns 3 and 4) or the less than (black, columns 1 and 2) the median age of 40 years. B: Area of coordinated [Ca²⁺] activity normalized to islet size in the presence and absence of modafinil (1 h incubation), averaged over donors greater than or less than the median age, as in A. C: Absolute area of coordinated [Ca²⁺] activity in the presence and absence of modafinil (1 h incubation), averaged over donors greater than or less than the median age, as in A. D: Cx36 gap junction function, as assessed through FRAP (rate of fluorescence recovery) in the presence and absence of modafinil (24 h incubation), averaged over donors greater than or less than the median age, as in A. Data are presented as mean ± SEM, averaged over n = 8 (<40) and n = 9 (>40) donors (four to six islets per donor), with P values indicating the significance of differences between indicated groups (Student t test).

Figure 7

Model regarding age-dependent decline in human islet function. Aging disrupts Cx36 gap junction function, which disrupts the coordination of [Ca²⁺] oscillation coordination and insulin secretion dynamics, thereby contributing to glucose intolerance. In addition, aging can disrupt [Ca²⁺] activity and insulin secretion levels also contributing to glucose intolerance. The disruption to [Ca²⁺] activity may occur as a result of altered β-cell function in aging (dashed), and/or the disruption to Cx36 gap junction function and [Ca²⁺] coordination may impact [Ca²⁺] activity and thus insulin secretion (dashed).