Objective
We will start by testing how PRL-2903 affects glucagon release in mice that lack the somatostatin receptor SSTR2. To do this, we’ll use a special strain of mice in which the SSTR2 gene has been deleted. These mice will be bred in our lab to produce offspring without SSTR2, along with normal (wild-type) littermates for comparison. We’ll isolate pancreatic islets from both groups and measure how much glucagon they release when exposed to somatostatin (SST), PRL-2903, or a control solution under both low- and high-glucose conditions. Comparing these results will tell us whether PRL-2903’s effects on glucagon depend on SSTR2. We’ll also confirm PRL-2903’s effects in human alpha-cells and explore other possible signaling pathways using proteomics.
We aim to also study how PRL-2903 works in human cells. We will treat purified human alpha-cells collected from donor pancreatic islets with PRL-2903. These alpha-cells will be separated from other islet cell types using cell sorting and then reassembled into small aggregates in the lab so we can study their function. We will expose these alpha-cell clusters to different doses of PRL-2903 under low-glucose conditions and measure how much glucagon they release, in the absence of delta-cells and somatostatin. This will help us determine whether PRL-2903 acts only by blocking the SST-SSTR2 pathway or if it also works through other signaling mechanisms. In addition, we will analyze both the proteins inside the cells and those they secrete using proteomics to identify molecules that might explain any SSTR2-independent effects of PRL-2903.
Background Rationale
One of the biggest challenges in managing type 1 diabetes (T1D) is that patients often lose the body’s natural ability to release glucagon during low blood sugar (hypoglycemia). Glucagon is a hormone produced in and secreted by pancreatic islet alpha-cells that raises blood sugar, working as a counterbalance to islet beta-cell secreted insulin. When alpha cells are malfunctioning and glucagon release abnormally, it can make insulin therapy risky because the body can’t easily recover from low blood sugar.
A hormone called somatostatin (SST), secreted by islet delta-cells, normally helps control glucagon production by signaling through a receptor on alpha-cells known as somatostatin receptor 2 (SSTR2). In T1D, this regulation seems to be defected. Instead of releasing less SST when blood sugar is low, the body may release too much, which further shuts down glucagon release when it’s needed most. Because of this, blocking SSTR2 has become an exciting potential therapy to help restore healthy glucagon responses and prevent dangerous drops in blood sugar. A drug called PRL-2903, which specifically blocks SSTR2, has been shown to boost glucagon release and speed up recovery from insulin-induced low blood sugar in animals. However, it is still unclear whether these effects depend entirely on SSTR2, since the drug has not yet been tested in animals missing that receptor.
We have created a “pseudo-islet” model in which we use purified human islet alpha- and beta-cells and reassemble them in controlled combinations. This allows us to see how the presence or absence of certain types of islet cells affects alpha-cell glucagon secretion. When the purified human alpha-cells were treated with the SSTR2 antagonist PRL-2903 in culture, their secretion of glucagon was boosted significantly. This result is surprising because, as an antagonist, PRL-2903 is expected to release the alpha-cells from the delta-cells inhibitory effect, but not to have its own influence on glucagon secretion. But our data suggested that it was capable of stimulating glucagon secretion in the absence delta cells or SST. We therefore question whether the glucagon-stimulatory effect is entirely dependent on blocking the interactions between SST and SSTR2.
Description of Project
People with type 1 diabetes (T1D) often struggle with low blood sugar because their bodies can’t release enough glucagon, the hormone that raises glucose levels when they drop too low. This loss of the body’s natural counter-response to hypoglycemia is one of the biggest challenges in insulin therapy.
Normally, a hormone named somatostatin (SST), secreted by islet delta cells, helps regulate glucagon release by signaling through a receptor on glucagon-producing alpha-cells called SSTR2. In T1D, this control system may become unbalanced — too much SST may be released when blood sugar is low, which can further suppress glucagon release and make hypoglycemia worse. Because of this, drugs that block SSTR2 (called SSTR2 antagonists) are being studied as possible add-on treatments to help restore normal alpha-cell activity and improve blood sugar recovery during hypoglycemic episodes.
In our earlier work, we found that a compound called PRL-2903, a known SSTR2 blocker, can trigger glucagon release from human alpha-cells even when delta-cells (which produce SST) are absent. This suggests PRL-2903 might act not only through the SST/SSTR2 pathway but possibly through other, unknown mechanisms.
We plan to test this idea in two ways. First, we’ll study how PRL-2903 affects glucagon release in pancreatic islets taken from mice that completely lack SSTR2. Second, we’ll test the drug in purified human alpha-cells and use proteomic analysis to look for other pathways that might explain its effects. By comparing how PRL-2903 works in these systems, we can determine whether it truly depends on SSTR2 or acts through additional signaling routes.
Understanding this distinction is important for drug development. If PRL-2903 works mainly through SSTR2, that would confirm the drug’s target and make it easier to predict its safety and effectiveness. If it also acts through other pathways, the off-target effects can lead to variability in how well the drug works or in potential side effects. Understanding these mechanisms will be crucial for moving forward with safe and effective therapies that can restore glucagon responses and prevent hypoglycemia in people with T1D. It will also reveal new aspects of alpha-cell biology and point to broader strategies for restoring alpha-cell function in T1D.
We’ll use two novel experimental models for the study: (a) mouse islets from SSTR2 knockout mice, and (b) human “pseudo-islets” made from purified alpha-cells that lack delta-cells. We will also apply advanced proteomic profiling to identify other proteins secreted by human alpha cells and the key proteins and pathways influenced by PRL-2903. This project is innovative because: It will directly test whether blocking SSTR2 increases glucagon by lifting SST’s inhibition on alpha-cells in both mice and humans; it addresses a key gap in understanding how SSTR2 antagonists might help treat hypoglycemia in insulin-treated diabetes, and it combines drug testing, physiology, and proteomic tools to uncover how these drugs regulate glucagon release.
Together, these studies will reveal how SSTR2 blockers work at the molecular level and could pave the way for safer, more effective treatments to prevent hypoglycemia in people with T1D.
Anticipated Outcome
We expect to demonstrate in Aim 1 that somatostatin, when added in the presence of low glucose during the glucose challenge assays, inhibits glucagon secretion from islets of wildtype mice but does not have any significant influence on glucagon secretion from mouse islets lacking its receptor SSTR2. We expect that PRL-2903 will show similar stimulatory effects on islets from both strains of mice, since we hypothesize that it does not act exclusively via SSTR2. If PRL-2903 acts exclusively via SSTR2, islets lacking SSTR2 will not show enhanced glucagon secretion in response to PRL-2903.
Under Aim 2, we’ll confirm our earlier findings that PRL-2903 stimulates glucagon secretion from purified human alpha-cells even when delta-cells and somatostatin are absent. We’ll test different doses of PRL-2903 to see how alpha-cells respond and expect its stimulatory effect to remain consistent within the dose range reported by others. We’ll also do proteomic analyses on both intracellular and secreted proteins to map out which signaling pathways are affected by PRL-2903 and identify other proteins secreted by alpha-cells in response to it.
If these studies work as expected, they will show that PRL-2903 stimulates glucagon release through mechanisms that do not rely on somatostatin or SSTR2. If our hypothesis does not hold up, the work will still provide valuable insights into how alpha-cells function. For example, if PRL-2903 does not increase glucagon secretion in SSTR2-lacking or delta-cell–depleted systems, that would confirm its effects depend on SSTR2. In that case, we will use our proteomic data to identify other PRL-2903–induced secreted proteins. Either way, these studies will deepen our understanding of alpha-cell regulation and glucose counter-regulation, offering useful clues for designing new glucagon-targeted therapies for type 1 diabetes.
Relevance to T1D
Figuring out whether PRL-2903 (or any drug that blocks SSTR2 for the treatment of hypoglycemia in T1D) works only through SSTR2 or also through other pathways is key to understanding how it really works and how best to develop it as a therapy. If its effects are truly SSTR2-dependent, that would confirm the drug is acting on its intended target and likely to be safer and more predictable. But if it also affects other pathways, the off-target effects can lead to variability in how well the drug works or in possible side effects. Understanding these mechanisms will be crucial for moving forward with safe and effective therapies that can restore glucagon responses and prevent hypoglycemia in people with T1D. It will also reveal new aspects of alpha-cell biology and point to broader strategies for restoring alpha-cell function in T1D.