A role for PAK1 mediated phosphorylation of ß-catenin Ser552 in the regulation of insulin secretion.

The presence of adherens junctions and the associated protein ß-catenin are requirements for the development of glucose-stimulated insulin secretion (GSIS) in ß-cells. Evidence indicates that modulation of ß-catenin function in response to changes in glucose levels can modulate the levels of insulin secretion from ß-cells but the role of ß-catenin phosphorylation in this process has not been established. We find that a Ser552Ala version of ß-catenin attenuates glucose-stimulated insulin secretion indicating a functional role for Ser552 phosphorylation of ß-catenin in insulin secretion. This is associated with alterations F/G actin ratio but not the transcriptional activity of ß-catenin. Both glucose and GLP-1 stimulated phosphorylation of the serine 552 residue on ß-catenin. We investigated the possibility that an EPAC-PAK1 pathway might be involved in this phosphorylation event. We find that reduction in PAK1 levels using siRNA attenuates both glucose and GLP-1 stimulated phosphorylation of ß-catenin Ser552 and the effects of these on insulin secretion in ß-cell models. Furthermore, both the EPAC inhibitor ESI-09 and the PAK1 inhibitor IPA3 do the same in both ß-cell models and mouse islets. Together this identifies phosphorylation of ß-catenin at Ser552 as part of a cell signalling mechanism linking nutrient and hormonal regulation of ß-catenin to modulation of insulin secretory capacity of ß-cells and indicates this phosphorylation event is regulated downstream of EPAC and PAK1 in ß-cells.

α-catenin isoforms are regulated by glucose and involved in regulating insulin secretion in rat clonal ß-cell models.

The recent finding that ß-catenin levels play an important rate-limiting role in processes regulating insulin secretion lead us to investigate whether its binding partner α-catenin also plays a role in this process. We find that levels of both α-E-catenin and α-N-catenin are rapidly up-regulated as levels of glucose are increased in rat clonal ß-cell models INS-1E and INS-832/3. Lowering in levels of either α-catenin isoform using siRNA resulted in significant increases in glucose stimulated insulin secretion (GSIS) and this effect was attenuated when ß-catenin levels were lowered indicating these proteins have opposing effects on insulin release. This effect of α-catenin knockdown on GSIS was not due to increases in insulin expression but was associated with increases in calcium influx into cells. Moreover, simultaneous depletion of α-E catenin and α-N catenin decreased the actin polymerisation to a similar degree as latrunculin treatment and inhibition of ARP 2/3 mediated actin branching with CK666 attenuated the α-catenin depletion effect on GSIS. This suggests α-catenin mediated actin remodelling may be involved in the regulation of insulin secretion. Together this indicates that α-catenin and ß-catenin can play opposing roles in regulating insulin secretion, with some degree of functional redundancy in roles of α-E-catenin and α-N-catenin. The finding that, at least in ß-cell models, the levels of each can be regulated in the longer term by glucose also provides a potential mechanism by which sustained changes in glucose levels might impact on the magnitude of GSIS.

Evidence that RXFP4 is located in enterochromaffin cells and can regulate production and release of serotonin.

RXFP4 is a G protein-coupled receptor (GPCR) in the relaxin family. It has recently been recognised that this receptor and its cognate ligand INSL5 may have a role in the regulation of food intake, gut motility, and other functions relevant to metabolic health and disease. Recent data from reporter-mice showed co-location of Rxfp4 and serotonin (5-HT) in the lower gut. We used human single-cell RNA sequence data (scRNASeq) to show that RXFP4 is in a subset of gut enterochromaffin cells that produce 5-HT in humans. We also used RNAScope to show co-location of Rxfp4 mRNA and 5-HT in mouse colon, confirming prior findings. To understand whether RXFP4 might regulate serotonin production, we developed a cell model using Colo320, a human gut-derived immortalised cell line that produces and releases serotonin. Overexpression of RXFP4 in these cells resulted in a constitutive decrease in cAMP levels in both the basal state and in cells treated with forskolin. Treatment of cells with two RXFP4 agonists, INSL5 derived peptide INSL5-A13 and small molecule compound-4, further reduced cAMP levels. This was paralleled by a reduction in expression of mRNA for TPH1, the enzyme controlling the rate limiting step in the production of serotonin. Overexpression of RXFP4 also attenuated the cAMP-induced release of serotonin from Colo320 cells. Together this demonstrates that serotonin producing enterochromaffin cells are the major site of RXFP4 expression in the gut and that RXFP4 can have inhibitory functional impacts on cAMP production as well as TPH1 expression and serotonin release.

Preptin Deficiency Does Not Protect against High-Fat Diet-Induced Metabolic Dysfunction or Bone Loss in Mice.

Preptin is derived from the cleavage of the E-peptide of pro-insulin-like growth factor (IGF)-II and is an insulin secretagogue. Observational studies have linked elevated circulating preptin to metabolic dysfunction in humans; however, a causal role for preptin in metabolic dysfunction has not been established. Additionally, preptin can promote osteoblast proliferation and differentiation, suggesting a link with skeletal health. We previously described a global preptin knockout (KO) model. In this study, we sought to uncover the impact of preptin KO in mice on the response to a moderately high-fat diet (HFD) and low-fat diet (LFD). HFD groups had higher weight and fat mass gain, lower trabecular and cortical bone volume and fracture load, and higher liver triglycerides. In males, preptin deficiency led to lower blood glucose than wild-type (WT) mice under LFD conditions. This was accompanied by differences in bone microarchitecture, including lower trabecular bone volume fraction, trabecular number, and lower cortical thickness. These differences were absent in female mice, although KO females had a HFD-driven increase in fat mass and liver triglycerides that was absent in WT mice. Female WT mice had increased glucose-stimulated insulin secretion under HFD conditions that was absent in female KO mice. Overall, preptin may have a detrimental impact on metabolism and a positive impact on bone health in male mice and may protect against liver fat storage in females while enabling islet compensation under HFD conditions. When we consider that serum preptin levels are elevated in humans of both sexes in pathological states in which insulin levels are elevated, the impact of preptin on comorbidity risk needs to be better understood. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Vesiculin derived from IGF-II drives increased islet cell mass in a mouse model of pre-diabetes.

Pancreatic islet-cell function and volume are both key determinants of the maintenance of metabolic health. Insulin resistance and islet-cell dysfunction often occur in the earlier stages of type 2 diabetes (T2D) progression. The ability of the islet cells to respond to insulin resistance by increasing hormone output accompanied by increased islet-cell volume is key to maintaining blood glucose control and preventing further disease progression. Eventual ß-cell loss is the main driver of full-blown T2D and insulin-dependency. Researchers are targeting T2D with approaches that include those aimed at enhancing the function of the patient's existing ß-cell population, or replacing islet ß-cells. Another approach is to look for agents that enhance the natural capacity of the ß-cell population to expand. Here we aimed to study the effects of a new putative ß-cell growth factor on a mouse model of pre-diabetes. We asked whether: 1) 4-week's treatment with vesiculin, a two-chain peptide derived by processing from IGF-II, had any measurable effect on pre-diabetic mice vs vehicle; and 2) whether the effects were the same in non-diabetic littermate controls. Although treatment with vesiculin did not alter blood glucose levels over this time period, there was a doubling of the Proliferating Cell Nuclear Antigen (PCNA) detectable in the islets of treated pre-diabetic but not control mice and this was accompanied by increased insulin- and glucagon-positive stained areas in the pancreatic islets.

Investigating IGF-II and IGF2R serum markers as predictors of body weight loss following an 8-week acute weight loss intervention: PREVIEW sub-study.

BACKGROUND: Weight reduction is effective in preventing T2D however, weight reduction and maintenance is difficult to achieve on a population scale. Serum insulin-like growth factor II (IGF-II) and IGF-II receptor (IGF2R) have been associated with diabetic status and body weight in prior studies and, in addition, IGF-II has been indicated as predictive of future weight change. We measured these serum markers in participants with obesity/overweight and prediabetes from the New Zealand arm of the PREVIEW lifestyle intervention randomised trial before and after an 8-week low energy diet (LED). METHODS: Total IGF-II (n = 223) and soluble IGF2R (n = 151) were measured using commercial ELISA kits on fasted serum samples taken prior to an 8-week LED and also from participants completing the LED. RESULTS: IGF-II levels were not correlated with baseline body weight although mean levels did significantly decrease following the LED. Change in IGF-II serum level was correlated to fasting glucose change (p = 0.04) but not to weight change. Baseline serum IGF2R was correlated with BMI (p = 0.007) and was significantly higher in Maori compared to European Caucasian participants independent of body weight (p = 0.0016). Following LED, IGF2R change was positively associated with weight change (p = 0.02) when corrected for ethnicity. Pre-LED levels of these serum markers were not predictive of the magnitude of weight loss over the 8 weeks. CONCLUSION: Neither marker was useful in predicting magnitude of short-term weight loss. IGF2R is positively associated with BMI and is higher in Maori compared to European Caucasian individuals.

For Better or Worse: The Potential for Dose Limiting the On-Target Toxicity of PI 3-Kinase Inhibitors.

The hyper-activation of the phosphoinositide (PI) 3-kinase signaling pathway is a hallmark of many cancers and overgrowth syndromes, and as a result, there has been intense interest in the development of drugs that target the various isoforms of PI 3-kinase. Given the key role PI 3-kinases play in many normal cell functions, there is significant potential for the disruption of essential cellular functions by PI 3-kinase inhibitors in normal tissues; so-called on-target drug toxicity. It is, therefore, no surprise that progress within the clinical development of PI 3-kinase inhibitors as single-agent anti-cancer therapies has been slowed by the difficulty of identifying a therapeutic window. The aim of this review is to place the cellular, tissue and whole-body effects of PI 3-kinase inhibition in the context of understanding the potential for dose limiting on-target toxicities and to introduce possible strategies to overcome these.

Glucoregulatory activity of vesiculin in insulin sensitive and resistant mice.

Pancreatic islet-derived peptide hormones play key roles in the maintenance of systemic energy homeostasis and glucose balance and defects in their regulation are strongly implicated in the pathogenesis of obesity and diabetes. Peptides have also been used as lead compounds for therapeutics targeting metabolic disease. It is therefore important to understand the activity and function of islet hormones in both their target tissues and the whole organism. Insulin-like growth factor II (IGF-II) is an insulin homolog secreted by the islet ß-cells. Vesiculin is a newly discovered peptide hormone, processed from IGF-II and secreted from islet ß-cells in response to glucose. We postulated that vesiculin might act to regulate systemic glucose metabolism. Here we report our original investigations of vesiculin's activity in relation to glucoregulation. Vesiculin and IGF-II displayed similar dose-response relationships for lowering blood glucose in insulin-responsive FVB/n mice. By contrast, the ability of IGF-II to lower blood glucose was blunted in insulin-resistant triprolyl human-amylin transgenic mice, whereas vesiculin's ability to lower blood glucose remained unaffected. We also confirmed the ability of vesiculin to bypass insulin resistance in a second mouse model. In vitro analysis of signalling by vesiculin and IGF-II indicates that, like IGF-II, vesiculin signals through the IR/ IGF1R. Overall, we show that removal of only four amino acids from IGF-II has generated a peptide hormone with different bioactivity relevant to blood-glucose regulation. Investigating the differences among vesiculin, IGF-II and insulin signalling and activity may provide new insights into insulin resistance and potentially inform the design of novel therapeutics.

Using mass spectrometry to detect, differentiate, and semiquantitate closely related peptide hormones in complex milieu: measurement of IGF-II and vesiculin.

The search for an islet ß-cell growth factor has been a key objective in recent diabetes research, because the ability to regenerate and/or protect the functioning ß-cell population in patients could result in a great advancement for diabetes treatment. IGF-I and IGF-II are known to play crucial roles in fetal growth and prenatal development, and there is growing evidence that IGF-II increases ß-cell proliferation and survival in vitro and in vivo. A search for the source of IGF-II-like immunoreactivity in isolated ß-cell secretory granules from the murine cell line ßTC6-F7 revealed a novel 2-chain IGF-II-derived peptide, which we named vesiculin and which has been shown to be a full insulin agonist. Here, we present a liquid chromatography-tandem mass spectrometry method that enables selective detection and semiquantitation of the highly related IGF-II and vesiculin molecules. We have used this method to measure these 2 peptides in conditioned media from 2 ß-cell lines, produced under increasing glucose concentrations. This technique detected both IGF-II and vesiculin in media conditioned by MIN6 and ßTC6-F7 cells at levels in the range of 0 to 6 µM (total insulin, 80-450 µM) and revealed a glucose-stimulated increase in insulin, IGF-II, and vesiculin. IGF-II was detected in adult human and neonatal mouse serum in high levels, but vesiculin was not present. The methodology we present herein has utility for detecting and differentiating active peptides that are highly related and of low abundance.

Bone marrow mononuclear cells reduce myocardial reperfusion injury by activating the PI3K/Akt survival pathway.

OBJECTIVE: Adult bone marrow mononuclear cells (BMMNCs) can restore cardiac function following myocardial necrosis. Protocols used to date have administered cells relatively late after ischaemia/reperfusion injury, but there is the opportunity with elective procedures to infuse cells shortly after restoration of blood flow, for example after angioplasty. Our aim was therefore to try and quantify protection from myocardial injury by early infusion of BMMNCs in a rat ischaemia reperfusion (I/R) model. METHODS AND RESULTS: Male Wistar rats underwent 25 min of ischaemia followed by 2 h reperfusion of the left anterior descending coronary artery. Ten million BMMNCs were injected i.v. at reperfusion. We found BMMNCs caused a significant reduction in infarct size at 2 h when assessed by staining the area at risk with p-nitro blue tetrazolium (42% reduction, P<0.01). Apoptosis and necrosis of isolated cardiomyocytes was significantly reduced in the area at risk. Functional assessment at 7 days using echocardiography and left ventricular catheterisation showed improved systolic and diastolic function in the BMMNC treatment group (LVEF: BMMNC 71 ± 3% vs. PBS 48 ± 4%, P<0.0001). In functional studies BMMNC injected animals showed increased activation of Akt, inhibition of GSK-3ß, amelioration of p38 MAP kinase phosphorylation and NF-κB activity compared to control myocardium. Inhibition of PI3K with LY294002 abolished all beneficial effects of BMMNC treatment. Proteomic analysis also demonstrated that BMMNC treatment induced alterations in proteins within known cardioprotective pathways, e.g., heat shock proteins, stress-70 protein as well as the chaperone protein 14-3-3 epsilon. CONCLUSIONS: Early BMMNC injection during reperfusion preserves the myocardium, with evidence of reduced apoptosis, necrosis, and activation of survival pathways.