Synaptotagmins Tweak Functional β Cell Maturation

Abstract

Immature β cells secrete insulin at a lower glucose threshold compared to mature β cells. In this issue of Developmental Cell, Huang et al. (2018) show that the increase in glucose threshold during β cell maturation is achieved through balance between the Ca²⁺-sensitive synaptotagmin 7 and the Ca²⁺-insensitive synaptotagmin 4.

Functionally mature β cells regulate energy homeostasis by secreting exactly the right amount of insulin in response to any given blood glucose concentration. In type 1 diabetes mellitus (T1D) or advanced type 2 diabetes mellitus (T2D), β cells are permanently destroyed due to autoimmunity or metabolic overload, respectively, causing acute insulin deficiency and inability to regulate blood glucose levels. Thus, many research groups have focused on generating insulin-producing β cells in vitro from stem cells. Although remarkable progress has been made, full functional maturity in response to glucose has not yet been attained (Pagliuca et al., 2014; Rezania et al., 2014; Russ et al., 2015).

In rodents, functional β cell maturation occurs gradually between birth and weaning (Blum et al., 2012; Jermendy et al., 2011; Stolovich-Rain et al., 2015). Immature β cells secrete insulin in response to low (‘‘basal’’) glucose concentrations and have a limited ability to respond to blood glucose spikes (Blum et al., 2012). Conversely, mature β cells have lowered basal insulin secretion and respond to increases in glucose with a corresponding increase in insulin secretion (Figure 1A). How β cells ‘‘learn’’ during functional maturation to secrete the right amount of insulin is unclear. Addressing this question may be critical to stem cell therapies for diabetes, because immature β-like cells transplanted into humans may secrete excessive insulin at low blood glucose levels, exposing patients to the risk of hypoglycemia. In this issue of Developmental Cell, Huang et al. (2018) show that increased expression of the vesicle protein synaptotagmin 4 (Syt4) promotes functional β cell maturation by reducing insulin vesicle sensitivity to Ca²⁺ influx following exposure to glucose in the blood (Figure 1B).

Figure 1. The Ca²⁺-Insensitive Synaptotagmin Syt4 Mediates the Increase in Glucose-Stimulated Insulin Secretion Threshold during β Cell Maturation.

(A) Immature β cells display elevated insulin secretion in basal conditions (2.8 mM, blue) and have a blunted ability to respond to increases in glucose levels (16.7 mM, red). Both mature and immature cells do not respond to very low levels of glucose (0.5 mM, gray). (B) Schematic summarizing the mechanism reported in Huang et al. (2018). Ca²⁺ enters the cell through voltage-gated calcium channels (VGCCs). This is detected by the Ca²⁺-sensitive Syt7, which stimulates insulin secretion. Myt1, a repressor of Syt4, is expressed at this time. After maturation, Myt1 expression is decreased, allowing expression of the Ca²⁺-insensitive Syt4. Syt4 localizes to the ER and Golgi, where it prevents or reduces Syt7 sorting to insulin vesicles. Additionally, Syt4 localizes directly to vesicles and Syt7, interfering with the Ca²⁺-sensing abilities of Syt7 and insulin secretion.

Glucose-stimulated insulin secretion (GSIS) in β cells relies on the fusion of insulin-containing vesicles with the cell membrane in response to elevated glucose metabolism. In β cells, glucose is converted to ATP via glycolysis. The increased cytosolic ATP closes potassium channels, resulting in depolarization of the cell membrane and opening of voltage-gated calcium channels (VGCCs). These channels allow an influx of Ca²⁺ into the cell, stimulating insulin secretion through Ca²⁺-sensitive vesicle fusion with the cell membrane. By assaying depolarization-stimulated insulin secretion (DSIS) in immature and mature β cells, Huang et al. (2018) found that immature β cells displayed increased DSIS compared to adult islets, even in the presence of basal glucose levels. These results suggested that insulin granule fusion machinery in immature β cells has an increased sensitivity to Ca²⁺.

The authors next looked at transcriptional changes during β cell maturation, focusing on vesicular genes that could mediate β cell sensitivity to Ca²⁺. They identified an increase in the expression of Syt4, a member of the Synaptotagmin family of genes. Synaptotagmins are part of the SNARE complex of proteins, which regulates Ca²⁺-activated vesicle exocytosis in secreting cells, such as neurons and endocrine cells (Pang and Südhof, 2010). Among the 16 mammalian synaptotagmins, eight (Syt1–3, Syt5–7, Syt9, and Syt10) can bind to and are activated by Ca²⁺, and the rest (Syt4, Syt8, and Syt11–16) are insensitive to Ca²⁺. Ca²⁺-sensitive Syts promote vesicle exocytosis in response to Ca²⁺, and Ca²⁺-insensitive Syts inhibit it (Pang and Südhof, 2010). In β cells, the common Ca²⁺-sensitive Syt responsible for insulin vesicle fusion is Syt7. Based on their findings, Huang and colleagues hypothesized that the upregulation of the Ca²⁺-insensitive Syt4 during maturation may curb the glucose threshold to GSIS by suppressing Syt7-mediated vesicle fusion in response to Ca²⁺. This was supported by super-resolution structured-illumination microscopy showing that Syt4 is co-localized with Syt7 in insulin vesicles, the Golgi, and ER. Elegant mouse genetics experiments confirmed the hypothesis: islets from adult Syt4 null mice exhibited immature-like high insulin secretion at low glucose levels, whereas islets from neonatal mice overexpressing Syt4 had low basal insulin secretion reminiscent of mature β cell GSIS. Importantly, in vitro experiments in a human β cell line, as well as in human pluripotent stem-cell-derived β cells, corroborated the results.

Intriguingly, precocious expression of Syt4, while reducing the GSIS threshold in immature β cells, also impacts their health later on: 10 days after birth, at the time when mouse β cells normally start to show mature GSIS (Blum et al., 2012), Syt4-overexpressing cells appear to undergo partial de-differentiation, evident by dampened GSIS, altered expression of several maturation genes, and distorted islet architecture. Why precocious GSIS maturation in neonatal β cells causes damage later in life remains to be seen; it will be interesting to follow up on the authors’ suggestion that factors secreted from immature β cells control normal islet morphogenesis, gene expression, and proliferation. It is also still unclear how Syt4 expression is temporally regulated. Huang et al. (2018) show that pancreas-specific inactivation of all three Myt transcription factors (Myt1, Myt1L, and St18) results in an upregulation of Syt4 in neonatal β cells and precocious functional GSIS, suggesting that Myt transcription factors suppress Syt4 in immature β cells. However, it remains to be determined whether this regulation is direct.

In conclusion, Huang et al. (2018) provide convincing evidence that Syt4 is a regulator of the mature β cell insulin secretory response; its increase during functional β cell maturation reduces basal insulin secretion and facilitates mature GSIS by competing with the Ca²⁺-sensitive Syt7 (Figure 1B). Interestingly, this role of Syt4 in β cells closely resembles its function in neurons, where it binds to Syt1 (a paralog of Syt7) to reduce Ca²⁺-induced vesicle secretion (Littleton et al., 1999). In neurons, Syt4 is involved in activity-dependent synapse maturation (Yoshihara et al., 2005). It would be interesting to explore whether the GSIS-maturing function of Syt4 also involves an activity-dependent ‘‘learning.’’ This may explain why functional GSIS maturation in human pluripotent-derived β cells is only achieved after transplantation in vivo, where secretion-coupled feedback may be present via either direct innervation or indirect fluctuations in the surrounding glucose levels, but not in vitro, where the islet-like clusters are isolated from their natural environment and glucose levels remain constant. Although it remains to be seen whether Syt4 truly drives maturation at the level of gene expression or is a marker of it, its involvement in β cell maturity has exciting implications for diabetes therapeutics. Directed differentiation protocols for in vitro generation of β cells from human stem cells have stalled at the maturation step, but Huang et al. (2018) provide a crucial piece of this puzzle.