miRNAs are small noncoding RNAs that play important roles in the regulation of β cell function, including proliferation and survival (27
). To our knowledge, their potential involvement in compensatory β cell expansion had not previously been investigated. The present study revealed changes in the expression of a subset of islet miRNAs that correlate with the adaptation of β cell mass during different stages of rat pregnancy. In fact, the peak of β cell mass expansion at day 14 of gestation corresponded to maximal upregulation of miR-144 and miR-451 and maximal downregulation of miR-218 and miR-338-3p. These expression changes were reversed at the end of pregnancy, when β cell mass returned to pregestation levels. At least part of these modifications in miRNA expression caused adaptations in cellular functions, with a potential effect on islet β cell number. Indeed, miR-338-3p downregulation promoted proliferation and protected rat β cells against apoptosis, and a rise of miR-451 was associated with improved resistance of rat β cells to proapoptotic stimuli. Conversely, upregulation of miR-338-3p triggered apoptosis, mimicking the reduction of β cells observed at the end of pregnancy (13
). The effect on apoptosis was also confirmed in human β cells, but anti–miR-338-3p treatment was not sufficient to trigger replication of these cells. This result was somehow expected, because proliferation of human β cells is an extremely rare event (33
); even exendin, estradiol, and the GRP30 ligand G1, which are capable of decreasing miR-338-3p expression under the same experimental conditions, failed to elicit proliferation. Thus, the potential role of miR-338-3p on human β cell proliferation will need to be reevaluated once better approaches to assess replication of these cells are developed.
Interestingly, knockdown of miR-338-3p triggered proliferation of rat β cells, but not of α cells. The precise causes of this cell-selective effect will need to be addressed in future studies. The global miRNA expression profile of primary α and β cells is not yet available, and it is possible that miR-338-3p is expressed at different levels in glucagon-secreting cells than in insulin-secreting cells or that the function of this miRNA is counterbalanced by other miRNAs. Another attractive possibility is that the signaling pathways elicited upon miR-338-3p downregulation are not operating in α cells. Indeed, we showed that at least part of the effect of anti–miR-338-3p on β cells was mediated through induction of Igf1r, a receptor that seems to not be involved in α cell proliferation (53
Despite these proliferative and antiapoptotic effects, miR-338-3p downregulation and miR-451 overexpression did not significantly affect insulin content and glucose-induced insulin secretion, which indicates that upon changes in the level of these miRNAs, the β cells retained a fully differentiated phenotype. Taken together, these results are consistent with these miRNAs contributing to maternal adaptations in β cell mass that occur during gestation. Modulation of the level of miR-218 or miR-144 in vitro did not result in detectable modifications in the activities of rat islet cells or INS832/13 cells. However, a contribution of these miRNAs to β cell adaptation in vivo cannot be excluded at this stage.
In view of its multiple effects on β cell activities, we attempted to elucidate the mode of action of miR-338-3p. Blockade of this miRNA had a strong effect on a set of genes playing key roles in β cell proliferation and survival. Birc5
(also known as Survivin
) and Foxm1
were among the most interesting genes upregulated in β cells upon blockade of miR-338-3p: the former has antiapoptotic and proproliferative properties, and the latter promotes β cell proliferation (19
). Both have been shown to increase in islets from animal models characterized by β cell expansion and regeneration, such as pregnant mice at day 14 of gestation and ob/ob
). Other adaptations in gene expression consistent with the increased proliferative capacity and improved survival of β cells included elevated levels of Igf1r
and a rise in the antiapoptotic protein Bcl2.
Compensatory β cell mass expansion during pregnancy is thought to be triggered by an increase in maternal hormones. Lactogens are generally recognized as key inducers of this process (15
). Indeed, pregnant mice lacking the prolactin receptor, which mediates the action of lactogenic hormones, develop gestational diabetes (14
). Despite increasing plasmatic levels of estradiol during pregnancy and the capacity of this hormone to increase replication and improve survival and function of insulin-secreting cells in vitro and in vivo (20
), the contribution of estrogens to adaptive β cell mass expansion had thus far remained elusive. Our study unveiled a possible role for estradiol in pregnancy-associated β cell mass expansion through repression of miR-338-3p. Reduction of miR-338-3p level in rat islet cells mimicked the phenotypic changes observed upon estradiol treatment and, conversely, overexpression of miR-338-3p prevented estradiol-induced proliferation. Our data indicate that the effect of estradiol is not mediated through conventional ERα/β receptors, but rather occurs through activation of GPR30 (46
). In fact, the effect of estradiol was cAMP-dependent and was blocked by PKA inhibitors. Moreover, the reduction of miR-338-3p expression observed upon estradiol treatment was mimicked by a GPR30 agonist, and silencing of the receptor by RNA interference eliminated the effect of the hormone. Moreover, GPR30 expression was strongly upregulated during pregnancy and correlated with β cell mass expansion. Thus, the effect of estradiol is likely to be restricted to pregnancy, and miR-338-3p levels are probably not influenced by the fluctuations of this hormone during oestrus. GPR30 knockout mice display alterations in blood glucose homeostasis (42
), but the potential role of this receptor in the control of β cell mass in vivo has not been investigated. Because of the broad tissue expression of GPR30, a definitive assessment of the contribution of this receptor in compensatory β cell mass expansion during pregnancy will necessitate the generation of β cell–specific knockouts.
A mechanism analogous to the one we uncovered for compensatory β cell mass expansion during pregnancy is likely to operate in other conditions characterized by insulin resistance, such as obesity. Indeed, miR-338-3p was downregulated in young prediabetic db/db
mice and in HFD-fed mice. Injection of estradiol in Zucker diabetic fatty rats has previously been reported to prevent β cell death by suppressing lipid synthesis in insulin-secreting cells (61
). However, it is unlikely that the reduction in miR-338-3p expression in obese animals is mediated by GPR30, because GPR30
mRNA levels were unchanged in the islets of HFD-fed mice. Additional experiments will be required to completely rule out a contribution of GPR30 to β cell mass compensation in obesity models. Because GPR30 is expressed in several tissues, and general knockout mice display metabolic perturbations (42
), the generation of β cell–specific GPR30 knockout mice will be required to assess the role of GPR30 in compensatory β cell mass expansion in obesity models. Our data suggest that in nonpregnant animals, the level of miR-338-3p may be controlled by incretins. GLP1 was able to downregulate miR-338-3p expression in isolated rat islet cells. Moreover, the level of miR-338-3p was significantly increased in the islets of Glp1r–/–Gip1r–/–
mice. A recent study demonstrated IL-6–induced production of GLP1 by α cells in pancreatic islets of HFD-fed mice (63
). This obesity-induced adaptation process triggered by local production of GLP1 was fully compatible with the miRNA-dependent mechanism of β cell mass expansion described herein. The beneficial effects elicited by GLP1 on β cells rely on the activation of an autocrine loop that results in a rise in Igf1r
). These key events in the signaling cascade triggered by GLP1 could be reproduced by miR-338-3p downregulation. Moreover, overexpression of this miRNA prevented the increase in Igf1r
and the enhanced proliferation rate observed in the presence of GLP1, which suggests that a reduction in miR-338-3p level is an important requirement for incretins to achieve full beneficial effects on β cells. Our observations indicate that the adaptive changes in β cell mass in response to insulin resistance associated with pregnancy and obesity are activated through distinct receptors, but that the downstream signaling pathways they elicit converge and culminate in overlapping modifications in gene expression. Other factors known to induce β cell proliferation, such as pituitary adenylate cyclase–activating polypeptide (PACAP; ref. 64
) and glucose (65
), trigger a rise in cAMP levels. Thus, it is tempting to speculate that at least part of the proliferative effects of these molecules may be achieved by reduction of miR-338-3p expression.
In conclusion, we here unveiled an analogy between the effects of estrogens and incretins on a subset of miRNAs involved in compensatory β cell mass expansion occurring during pregnancy and obesity. Our findings have relevant implications for understanding the physiological adaptation of β cells to increased insulin demand. Detailed knowledge of the mechanisms controlling the level and activity of the miRNAs differentially expressed during pregnancy may open the way to new therapeutic strategies, with the ultimate goal being to prevent and treat diabetes by promoting β cell mass regeneration.