ß-cell Jagged1 is sufficient but not necessary for islet Notch activity and insulin secretory defects in obese mice.

OBJECTIVE: Notch signaling, re-activated in ß cells from obese mice and causal to ß cell dysfunction, is determined in part by transmembrane ligand availability in a neighboring cell. We hypothesized that ß cell expression of Jagged1 determines the maladaptive Notch response and resultant insulin secretory defects in obese mice. METHODS: We assessed expression of Notch pathway components in high-fat diet-fed (HFD) or leptin receptor-deficient (db/db) mice, and performed single-cell RNA sequencing (scRNA-Seq) in islets from patients with and without type 2 diabetes (T2D). We generated and performed glucose tolerance testing in inducible, ß cell-specific Jagged1 gain-of- and loss-of-function mice. We also tested effects of monoclonal neutralizing antibodies to Jagged1 in glucose-stimulated insulin secretion (GSIS) assays in isolated islets. RESULTS: Jag1 was the only Notch ligand that tracked with increased Notch activity in HFD-fed and db/db mice, as well as in metabolically-inflexible ß cells enriched in patients with T2D. Neutralizing antibodies to block Jagged1 in islets isolated from HFD-fed and db/db mice potentiated GSIS ex vivo. To demonstrate if ß cell Jagged1 is sufficient to cause glucose tolerance in vivo, we generated inducible ß cell-specific Jag1 transgenic (ß-Jag1TG) and loss-of-function (iß-Jag1KO) mice. While forced Jagged1 impaired glucose intolerance due to reduced GSIS, loss of ß cell Jagged1 did not protect against HFD-induced insulin secretory defects. CONCLUSIONS: Jagged1 is increased in islets from obese mice and in patients with T2D, and neutralizing Jagged1 antibodies lead to improved GSIS, suggesting that inhibition of Jagged1-Notch signaling may have therapeutic benefit. However, genetic loss-of-function experiments suggest that ß cells are not a likely source of the Jagged1 signal.

Notch-mediated Ephrin signaling disrupts islet architecture and ß cell function.

Altered islet architecture is associated with ß cell dysfunction and type 2 diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed "reactivated" ß cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, ß cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in postnatal ß cells impaired glucose-stimulated insulin secretion and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased ß cell repulsion and impaired murine and human pseudoislet formation. Consistent with our mouse data, Notch and Ephrin signaling were increased in metabolically inflexible ß cells in patients with T2D. These studies suggest that ß cell Notch/Ephrin signaling can permanently alter islet architecture during a morphogenetic window in early life.

Osteocalcin promotes ß-cell proliferation during development and adulthood through Gprc6a.

Expanding ß-cell mass through ß-cell proliferation is considered a potential therapeutic approach to treat ß-cell failure in diabetic patients. A necessary step toward achieving this goal is to identify signaling pathways that regulate ß-cell proliferation in vivo. Here we show that osteocalcin, a bone-derived hormone, regulates ß-cell replication in a cyclin D1-dependent manner by signaling through the Gprc6a receptor expressed in these cells. Accordingly, mice lacking Gprc6a in the ß-cell lineage only are glucose intolerant due to an impaired ability to produce insulin. Remarkably, this regulation occurs during both the perinatal peak of ß-cell proliferation and in adulthood. Hence, the loss of osteocalcin/Gprc6a signaling has a profound effect on ß-cell mass accrual during late pancreas morphogenesis. This study extends the endocrine role of osteocalcin to the developmental period and establishes osteocalcin/Gprc6a signaling as a major regulator of ß-cell endowment that can become a potential target for ß-cell proliferative therapies.

A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure.

Leptin inhibition of bone mass accrual requires the integrity of specific hypothalamic neurons but not expression of its receptor on these neurons. The same is true for its regulation of appetite and energy expenditure. This suggests that leptin acts elsewhere in the brain to achieve these three functions. We show here that brainstem-derived serotonin (BDS) favors bone mass accrual following its binding to Htr2c receptors on ventromedial hypothalamic neurons and appetite via Htr1a and 2b receptors on arcuate neurons. Leptin inhibits these functions and increases energy expenditure because it reduces serotonin synthesis and firing of serotonergic neurons. Accordingly, while abrogating BDS synthesis corrects the bone, appetite and energy expenditure phenotypes caused by leptin deficiency, inactivation of the leptin receptor in serotonergic neurons recapitulates them fully. This study modifies the map of leptin signaling in the brain and identifies a molecular basis for the common regulation of bone and energy metabolisms. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Dissociation of the neuronal regulation of bone mass and energy metabolism by leptin in vivo.

The leptin regulation of bone remodeling, which has been documented through studies of loss-of-function mutations of this hormone or of its receptor in mice and humans, still raised several unanswered questions. For instance, it has been assumed but not formally demonstrated that this regulation occurs through neuronal means. Likewise, it has not been possible until now to dissociate the influence leptin exerts on appetite and energy expenditure from this function. We show here through mouse genetic studies that a deletion of the leptin receptor in neurons results in an increase in bone formation and bone resorption, resulting in a high bone mass as seen in leptin-deficient mice. In contrast, the same deletion in osteoblasts only does not influence bone remodeling. Furthermore, through the use of l/l mice, a model of gain of function in leptin signaling harboring a Y985L substitution in the leptin receptor, we show that leptin signaling inhibits bone mass accrual by up-regulating sympathetic activity independently of any change in appetite or energy expenditure. This work establishes that in vivo leptin regulates bone mass accrual by acting through neuronal means and provides a direct demonstration that this function of leptin can occur independently of its regulation of energy metabolism.

Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum.

Loss- and gain-of-function mutations in the broadly expressed gene Lrp5 affect bone formation, causing osteoporosis and high bone mass, respectively. Although Lrp5 is viewed as a Wnt coreceptor, osteoblast-specific disruption of beta-Catenin does not affect bone formation. Instead, we show here that Lrp5 inhibits expression of Tph1, the rate-limiting biosynthetic enzyme for serotonin in enterochromaffin cells of the duodenum. Accordingly, decreasing serotonin blood levels normalizes bone formation and bone mass in Lrp5-deficient mice, and gut- but not osteoblast-specific Lrp5 inactivation decreases bone formation in a beta-Catenin-independent manner. Moreover, gut-specific activation of Lrp5, or inactivation of Tph1, increases bone mass and prevents ovariectomy-induced bone loss. Serotonin acts on osteoblasts through the Htr1b receptor and CREB to inhibit their proliferation. By identifying duodenum-derived serotonin as a hormone inhibiting bone formation in an Lrp5-dependent manner, this study broadens our understanding of bone remodeling and suggests potential therapies to increase bone mass.