Harpreet K. Brar

Prostaglandin E2 Receptor, EP3, Is Induced in Diabetic Islets and Negatively Regulates Glucose- and Hormone-Stimulated Insulin Secretion

BTBR mice develop severe diabetes in response to genetically induced obesity due to a failure of the β-cells to compensate for peripheral insulin resistance. In analyzing BTBR islet gene expression patterns, we observed that Pgter3, the gene for the prostaglandin E receptor 3 (EP3), was upregulated with diabetes. The EP3 receptor is stimulated by prostaglandin E2 (PGE2) and couples to G-proteins of the Gi subfamily to decrease intracellular cAMP, blunting glucose-stimulated insulin secretion (GSIS). Also upregulated were several genes involved in the synthesis of PGE2. We hypothesized that increased signaling through EP3 might be coincident with the development of diabetes and contribute to β-cell dysfunction. We confirmed that the PGE2-to-EP3 signaling pathway was active in islets from confirmed diabetic BTBR mice and human cadaveric donors, with increased EP3 expression, PGE2 production, and function of EP3 agonists and antagonists to modulate cAMP production and GSIS. We also analyzed the impact of EP3 receptor activation on signaling through the glucagon-like peptide (GLP)-1 receptor. We demonstrated that EP3 agonists antagonize GLP-1 signaling, decreasing the maximal effect that GLP-1 can elicit on cAMP production and GSIS. Taken together, our results identify EP3 as a new therapeutic target for β-cell dysfunction in T2D.

Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system

Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA‐approved drug rapamycin has been shown to promote lifespan and delay age‐related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long‐term prophylactic use of rapamycin as a therapy for age‐related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR‐containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA‐approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA‐approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs.

Phenotypic Characterization of MIP-CreERT1Lphi Mice With Transgene-Driven Islet Expression of Human Growth Hormone

There is growing concern over confounding artifacts associated with β-cell–specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible β-cell–specific transgenic (MIP-CreERT1Lphi) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT1Lphi mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for β-cell research.

Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of β-cell mass.

Background: Gαz can block insulin secretion, but no one had looked at its role in glucose intolerance. Results: Mice that do not express Gαz mice do not develop glucose intolerance when fed a high fat diet. Conclusion: A Gαz signaling pathway contributes to the pathophysiology of glucose intolerance. Significance: This is the first work describing the specific role(s) of Gαz in glucose intolerance. Insufficient plasma insulin levels caused by deficits in both pancreatic β-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed β-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating β-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric Gz protein, Gαz, impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gαz-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gαz is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gαz are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in β-cell proliferation is correlated with significantly increased β-cell mass. Further, an endogenous Gαz signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gαz to both major aspects of β-cell decompensation: insufficient β-cell function and mass.

A single-islet microplate assay to measure mouse and human islet insulin secretion

One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.

Synergy Between Gαz Deficiency and GLP-1 Analog Treatment in Preserving Functional β-Cell Mass in Experimental Diabetes.

A defining characteristic of type 1 diabetes mellitus (T1DM) pathophysiology is pancreatic β-cell death and dysfunction, resulting in insufficient insulin secretion to properly control blood glucose levels. Treatments that promote β-cell replication and survival, thus reversing the loss of β-cell mass, while also preserving β-cell function, could lead to a real cure for T1DM. The α-subunit of the heterotrimeric Gz protein, Gαz, is a tonic negative regulator of adenylate cyclase and downstream cAMP production. cAMP is one of a few identified signaling molecules that can simultaneously have a positive impact on pancreatic islet β-cell proliferation, survival, and function. The purpose of our study was to determine whether mice lacking Gαz might be protected, at least partially, from β-cell loss and dysfunction after streptozotocin treatment. We also aimed to determine whether Gαz might act in concert with an activator of the cAMP-stimulatory glucagon-like peptide 1 receptor, exendin-4 (Ex4). Without Ex4 treatment, Gαz-null mice still developed hyperglycemia, albeit delayed. The same finding held true for wild-type mice treated with Ex4. With Ex4 treatment, Gαz-null mice were protected from developing severe hyperglycemia. Immunohistological studies performed on pancreas sections and in vitro apoptosis, cytotoxicity, and survival assays demonstrated a clear effect of Gαz signaling on pancreatic β-cell replication and death; β-cell function was also improved in Gαz-null islets. These data support our hypothesis that a combination of therapies targeting both stimulatory and inhibitory pathways will be more effective than either alone at protecting, preserving, and possibly regenerating β-cell mass and function in T1DM.

The gastrin-releasing peptide analog bombesin preserves exocrine and endocrine pancreas morphology and function during parenteral nutrition

Stimulation of digestive organs by enteric peptides is lost during total parental nutrition (PN). Here we examine the role of the enteric peptide bombesin (BBS) in stimulation of the exocrine and endocrine pancreas during PN. BBS protects against exocrine pancreas atrophy and dysfunction caused by PN. BBS also augments circulating insulin levels, suggesting an endocrine pancreas phenotype. While no significant changes in gross endocrine pancreas morphology were observed, pancreatic islets isolated from BBS-treated PN mice showed a significantly enhanced insulin secretion response to the glucagon-like peptide-1 (GLP-1) agonist exendin-4, correlating with enhanced GLP-1 receptor expression. BBS itself had no effect on islet function, as reflected in low expression of BBS receptors in islet samples. Intestinal BBS receptor expression was enhanced in PN with BBS, and circulating active GLP-1 levels were significantly enhanced in BBS-treated PN mice. We hypothesized that BBS preserved islet function indirectly, through the enteroendocrine cell-pancreas axis. We confirmed the ability of BBS to directly stimulate intestinal enteroid cells to express the GLP-1 precursor preproglucagon. In conclusion, BBS preserves the exocrine and endocrine pancreas functions during PN; however, the endocrine stimulation is likely indirect, through the enteroendocrine cell-pancreas axis.

The Inhibitory G Protein α-Subunit, Gαz, Promotes Type 1 Diabetes-Like Pathophysiology in NOD Mice

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes β-cell death and inhibits β-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional β-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive β-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive β-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, β-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a β-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in β-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of β-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

ID: 21: ALTERNATIVE RAPAMYCIN TREATMENT REGIMENS MITIGATE THE IMPACT OF RAPAMYCIN ON GLUCOSE HOMEOSTASIS AND THE IMMUNE SYSTEM, AND EXTENDS LIFESPAN

Inhibition of the mechanistic Target Of Rapamycin (mTOR) signaling pathway by the FDA-approved drug rapamycin promotes lifespan in numerous model organisms and delays age-related disease in mice. However, the utilization of rapamycin as a therapy for age-related diseases will likely prove challenging due to the serious metabolic and immunological side effects of rapamycin in humans. While the beneficial effects of rapamycin are largely mediated by inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by inhibition of mTOR complex 2 (mTORC2), which is chronically sensitive to rapamycin. We hypothesized that a more relaxed rapamycin dosing schedule or the use of rapamycin analogs might specifically target mTORC1, reducing mTORC2 mediated side effects and still promoting longevity. Here, we identified an intermittent rapamycin treatment regimen – 2 mg/kg administered every five days – with reduced impact on glucose metabolism and the immune system compared to daily rapamycin treatment. Further, we show that the FDA-approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 signaling while having a reduced impact on glucose metabolism compared to rapamycin. Finally, we find that intermittent rapamycin administration extends the lifespan of mice even to a greater degree than continuous fed rapamycin. First, we tested in C57BL/6J mice, an inbred mouse line of which of which the lifespan can be extended by rapamycin, several intermittent dosing regimens and determined that IP administration of rapamycin once every 5 days was the most intensive dosing routing with no significant impact on glucose tolerance. We analyzed the impact of intermittent rapamycin administration on glucose homeostasis and determined that compared to daily treatment, intermittent rapamycin has reduced pyruvate intolerance (impaired gluconeogensis regualtion). Surprisingly, intermittent rapamycin resulted in similar fasting plasma insulin, insulin sensitivity, and ex vivo insulin secretion than vehicle treatment. We also determined that intermittent rapamycin has reduced impact on the immune system compared to daily rapamycin, as indicated by spleen T regulatory cells percent. We analyzed the effect of intermittent rapamycin on mTOR signaling and observed a sustained impact of this dosing routine on adipose, but not muscle, mTORC1 downstream marker S6 (S240/244) phosphorylation after 5 days of treatment. Remarkable, we observed no impact of intermittent rapamycin on the mTORC2 substrate Akt (S473) phosphorylation on either tissue. We compared the impact of daily treatment with equimolar doses of the FDA-approved rapamycin analogs everolimus and temsirolimus on glucose homeostasis. Despite both analogs had similar reduction on muscle mTORC1 signaling than rapamycin, they had improved glucose tolerance. In addition, everolimus had improved pyruvate tolerance, reduced testis weight loss, and reduced inhibition of mTORC2 signaling in muscle. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs, yet still extend lifespan. Therefore, we analyzed the impact of every 5 days rapamycin administration on longevity when treatment started at 20 months of age. Fascinatingly, intermittent rapamycin extended median and maximum lifespan compared to vehicle treated mice, with minimum impact on metabolism. Our work demonstrates that the anti-aging potential of rapamycin is separable from many of its negative side-effects, and suggests that carefully designed dosing regimens may permit the safer use of rapamycin and its analogs for the treatment of age-related diseases in humans.