Leaders in Pharmaceutical Business Intelligence (LPBI) Group

While the etiopathogenesis of type 1 and type 2 diabetes is different (Boitard, 2012, Muoio and Newgard, 2008), a paucity of functional β cell mass is a central feature in both diseases (Butler et al., 2003, Henquin and Rahier, 2011, Lysy et al., 2013). Currently there is considerable interest in developing safe approaches to replenish bioactive insulin in patients with diabetes by deriving insulin-producing cells from pluripotent cells (D’Amour et al., 2006, Kroon et al., 2008, Pagliuca et al., 2014, Rezania et al., 2014) or promoting proliferation of pre-existing β cells (Dor et al., 2004, El Ouaamari et al., 2013, Yi et al., 2013). While the former approach continues to evolve, several groups have focused on identifying growth factors, hormones, and/or signaling proteins to promote β cell proliferation (cited in El Ouaamari et al., 2013 and Dirice et al., 2014). Compared to rodents, adult human β cells are contumacious to proliferation and have been suggested to turnover very slowly, with the β cell mass reaching a peak by early adulthood (Butler et al., 2003, Gregg et al., 2012, Kassem et al., 2000). Attempts to enhance human β cell proliferation have also been hampered by poor knowledge of the signaling pathways that promote cell-cycle progression (Bernal-Mizrachi et al., 2014, Kulkarni et al., 2012, Stewart et al., 2015). While two recent studies have reported the identification of a small molecule, harmine (Wang et al., 2015), and denosumab, a drug approved for the treatment of osteoporosis (Kondegowda et al., 2015) to increase human β cell proliferation, the identification of endogenous circulating factors that have the ability to replenish insulin-secreting cells is attractive for therapeutic purposes. We previously reported (Flier et al., 2001) that compensatory β cell growth in response to insulin resistance is mediated, in part, by liver-derived circulating factors in the liver-specific insulin receptor knockout (LIRKO) mouse, a model that exhibits significant hyperplasia of islets without compromising β cell secretory responses to metabolic or hormonal stimuli (El Ouaamari et al., 2013). Here we report the identification of serpinB1 as a liver-derived secretory protein that promotes proliferation of human, mouse, and zebrafish β cells.

Identification of molecules that have the ability to enhance proliferation of terminally differentiated cells is a desirable goal in regenerative medicine, particularly in diabetes where β cell numbers are reduced. Here, we identified serpinB1 as an endogenous liver-derived secretory protein that stimulates human, mouse, and zebrafish β cell proliferation.

One interesting aspect of serpinB1 viewed as a secretory molecule is its lack of the classical hydrophobic signal peptide. Our data indicate that inflammation stimulates unconventional secretion of serpinB1 in a caspase-1-dependent manner. It is important to note, however, that the levels of several circulating cytokines in the LIRKO model are comparable to those observed in age-matched controls (El Ouaamari et al., 2013) and hence excludes systemic inflammation as a physiological factor triggering serpinB1 release in vivo. It is possible that the absence of insulin signaling in the liver interferes with caspase-1 activation and thus serpinB1 release. This notion is compatible with a previous report suggesting the suppressive role of insulin/IGF-1 in caspase-1 processing (Jung et al., 1996) and is consistent with increased levels of active caspase-1 in LIRKO-derived hepatocytes that are blind to insulin.

Since inhibition of proteases is SerpinB1’s reported biochemical function to date (Cooley et al., 2001), we postulated that the enhancing effect of SerpinB1 on β cell proliferation involves the intermediacy of a protease. Indeed, recombinant SerpinB1 proteins lacking the ability to inhibit protease activity were unable to enhance β cell proliferation in vitro. This observation suggests that SerpinB1 neutralizes a protease that would otherwise interfere with proliferation. In fact, the small-molecule inhibitors of elastases, GW311616A and sivelestat, directly enhanced proliferation of mouse and human insulin-producing cells. The parallel findings for GW311616A, sivelestat, and SerpinB1 make elastases strong candidates. While SerpinB1 action could be explained by its ability to modulate phosphorylation of key molecules (e.g., MAPK3, GSK3β/α, and PKA) of the insulin/IGF-1 growth/survival pathways, it is unclear how SerpinB1 precisely regulates these pathways. One possibility is that these pathways are activated through SerpinB1-mediated protease inhibition, particularly inhibition of elastase molecules known to be expressed in pancreatic β cells (Kutlu et al., 2009). This idea is consistent with previous reports suggesting the role for neutrophil elastase in modulating proteins in the insulin/IGF-1 signaling pathway (Bristow et al., 2008, Houghton et al., 2010, Talukdar et al., 2012). Elucidation of interactions with other proteases such as proteinase-3 and cathepsin G in the β cell and its potential role in regulating insulin sensitivity will further assist in deciphering the signaling pathways activated by SerpinB1. Alternative possibilities that require further investigation include interactions with protease-activated receptors (PARs), which are expressed in islets (J.S., A.E.O., and R.N.K., unpublished data).

Using zebrafish, we determined that serpinB1’s ability to potentiate β cell proliferation is conserved from fish to mammals. Moreover, in zebrafish we showed that serpinB1 can potentiate β cell proliferation in vivo analogous to the in vivo effects we observed in mouse and human islets. By ablating the β cells in zebrafish, we also observed that serpinB1 can stimulate β cell regeneration and warrants studies to examine its role during β cell development.

In sum, the identification of SerpinB1 as a conserved endogenous secretory protein that promotes proliferation of β cells across species constitutes an important step to achieve regeneration of functional β cells. While it is likely that additional factors will be identified, the next challenge will be to explore whether one or a combination of these factors can safely, specifically, and reversibly enhance human β cell mass with the long-term goal of restoring normoglycemia in patients with diabetes.