The islet tissue plasminogen activator/plasmin system is upregulated with human islet amyloid polypeptide aggregation and protects beta cells from aggregation-induced toxicity.

AIMS/HYPOTHESIS: Apart from its fibrinolytic activity, the tissue plasminogen activator (tPA)/plasmin system has been reported to cleave the peptide amyloid beta, attenuating brain amyloid deposition in Alzheimer's disease. As aggregation of human islet amyloid polypeptide (hIAPP) is toxic to beta cells, we sought to determine whether activation of the fibrinolytic system can also reduce islet amyloid deposition and its cytotoxic effects, which are both observed in type 2 diabetes. METHODS: The expression of Plat (encoding tPA) and plasmin activity were measured in isolated islets from amyloid-prone hIAPP transgenic mice or non-transgenic control islets expressing non-amyloidogenic mouse islet amyloid polypeptide cultured in the absence or presence of the amyloid inhibitor Congo Red. Plat expression was also determined in hIAPP-treated primary islet endothelial cells, bone marrow-derived macrophages (BMDM) and INS-1 cells, in order to determine the islet cell type(s) producing tPA in response to hIAPP aggregation. Cell-free thioflavin-T assays and MS were used to respectively monitor hIAPP aggregation kinetics and investigate plasmin cleavage of hIAPP. Cell viability was assessed in INS-1 beta cells treated with hIAPP with or without plasmin. Finally, to confirm the findings in human samples, PLAT expression was measured in freshly isolated islets from donors with and without type 2 diabetes. RESULTS: In isolated islets from transgenic mice, islet Plat expression and plasmin activity increased significantly with the process of amyloid deposition (p≤0.01, n=5); these effects were not observed in islets from non-transgenic mice and were blocked by Congo Red (p≤0.01, n=4). In response to hIAPP exposure, Plat expression increased in BMDM and INS-1 cells vs vehicle-treated cells (p≤0.05, n=4), but not in islet endothelial cells. Plasmin reduced hIAPP fibril formation in a dose-dependent manner in a cell-free system, and restored hIAPP-induced loss of cell viability in INS-1 beta cells (p≤0.01, n=5). Plasmin cleaved monomeric hIAPP, inducing a rapid decrease in the abundance of full-length hIAPP and the appearance of hIAPP 1-11 and 12-37 fragments. hIAPP 12-37, which contains the critical amyloidogenic region, was not toxic to INS-1 cells. Finally, PLAT expression was significantly increased by 2.4-fold in islets from donors with type 2 diabetes (n=4) vs islets from donors without type 2 diabetes (n=7) (p≤0.05). CONCLUSIONS/INTERPRETATION: The fibrinolytic system is upregulated in islets with hIAPP aggregation. Plasmin rapidly degrades hIAPP, limiting its aggregation into amyloid and thus protecting beta cells from hIAPP-induced toxicity. Thus, increasing islet plasmin activity might be a strategy to limit beta cell loss in type 2 diabetes.

[Don't neglect metabolic syndrome]. / Vignette diagnostique de l'étudiant. Ne pas négliger le syndrome métabolique.

The concept of «metabolic syndrome¼ was brought to the forefront in the early 2000s in international literature, but this interest seems to have faded somewhat in recent years. However, this constellation of cardiovascular risk factors should not be neglected. Taken individually, they hardly seem problematic, but when they are present within the same individual, they significantly increase the risk of cardiovascular morbidity and mortality. This clinical vignette aims to draw attention to the usefulness of the search for metabolic syndrome in clinical practic.

Le concept de «syndrome métabolique¼ a été mis en avant de la scène au début des années 2000 dans la littérature internationale, mais cet intérêt semble s'être quelque peu estompé au cours des dernières années. Il convient cependant de ne pas négliger cette constellation de facteurs de risque cardiovasculaire qui, pris individuellement, ne paraissent guère problématiques, mais qui, lorsqu'ils co-existent chez une même personne, augmentent sensiblement le risque de morbi-mortalité. Cette vignette clinique a pour but d'attirer l'attention sur l'importance de la recherche d'un syndrome métabolique dans la pratique clinique.

[Update of nutritional recommendations in the treatment of type 2 diabetes]. / Actualisation des recommandations nutritionnelles dans le traitement du diabète de type 2.

Dietary management of type 2 diabetes is essential to improve glycaemic control et reduce risk of diabetes complications. Key recommendations for people with diabetes are largely similar to those for the general population. Overweight or obese diabetic persons should be supported with evidence-based treatments to achieve and maintain weight loss. A wide range of carbohydrate intakes are acceptable and diets with a low glycaemic index or low glycaemic load may be recommended, provided their composition is consistent with overall diet recommendations for dietary fibers, sugars, saturated fats and proteins. It is also important to consume minimally processed plant foods, such as whole grains, vegetables, whole fruits, legumes, while reducing the consumption of red and processed meats, sodium and sugar-sweetened beverages.

La prise en charge nutritionnelle du diabète de type 2 est essentielle afin d'améliorer l'équilibre glycémique et réduire le risque de complications liées au diabète. Les principales recommandations diététiques pour les personnes diabétiques sont largement comparables à celles prodiguées dans la population générale. Les sujets diabétiques en surpoids ou obèses devraient bénéficier de prises en charge validées pour obtenir et maintenir une perte de poids. Un apport en glucides très variable est autorisé, avec des aliments à charge glycémique faible, et une composition adéquate en fibres, sucres, acides gras saturés et protéines. Il est important de réduire la consommation d'aliments transformés et de favoriser les céréales complètes, les produits végétaux (fruits et légumes), tout en limitant l'apport en viandes, sel et boissons sucrées.

Islet amyloid polypeptide aggregation exerts cytotoxic and proinflammatory effects on the islet vasculature in mice.

AIMS/HYPOTHESIS: The islet vasculature, including its constituent islet endothelial cells, is a key contributor to the microenvironment necessary for normal beta cell health and function. In type 2 diabetes, islet amyloid polypeptide (IAPP) aggregates, forming amyloid deposits that accumulate between beta cells and islet capillaries. This process is known to be toxic to beta cells but its impact on the islet vasculature has not previously been studied. Here, we report the first characterisation of the effects of IAPP aggregation on islet endothelial cells/capillaries using cell-based and animal models. METHODS: Primary and immortalised islet endothelial cells were treated with amyloidogenic human IAPP (hIAPP) alone or in the presence of the amyloid blocker Congo Red or the Toll-like receptor (TLR) 2/4 antagonist OxPAPc. Cell viability was determined0 along with mRNA and protein levels of inflammatory markers. Islet capillary abundance, morphology and pericyte coverage were determined in pancreases from transgenic mice with beta cell expression of hIAPP using conventional and confocal microscopy. RESULTS: Aggregated hIAPP decreased endothelial cell viability in immortalised and primary islet endothelial cells (by 78% and 60%, respectively) and significantly increased expression of inflammatory markers Il6, Vcam1 and Edn1 mRNA relative to vehicle treatment in both cell types (p<0.05; n=4). Both cytotoxicity and the proinflammatory response were ameliorated by Congo Red (p<0.05; n=4); whereas TLR2/4-inhibition blocked inflammatory gene expression (p<0.05; n=6) without improving viability. Islets from high-fat-diet-fed amyloid-laden hIAPP transgenic mice also exhibited significantly increased expression of most markers of endothelial inflammation (p<0.05; n=5) along with decreased capillary density compared with non-transgenic littermates fed the same diet (p<0.01). Moreover, a 16% increase in capillary diameter was observed in amyloid-adjacent capillaries (p<0.01), accompanied by a doubling in pericyte structures positive for neuron-glial antigen 2 (p<0.001). CONCLUSIONS/INTERPRETATION: Islet endothelial cells are susceptible to hIAPP-induced cytotoxicity and exhibit a TLR2/4-dependent proinflammatory response to aggregated hIAPP. Additionally, we observed amyloid-selective effects that decreased islet capillary density, accompanied by increased capillary diameter and increased pericyte number. Together, these data demonstrate that the islet vasculature is a target of the cytotoxic and proinflammatory effects of aggregated hIAPP that likely contribute to the detrimental effects of hIAPP aggregation on beta cell function and survival in type 2 diabetes.

Neprilysin inhibition improves intravenous but not oral glucose-mediated insulin secretion via GLP-1R signaling in mice with ß-cell dysfunction.

Type 2 diabetes is associated with the upregulation of neprilysin, a peptidase capable of cleaving glucoregulatory peptides such as glucagon-like peptide-1 (GLP-1). In humans, use of the neprilysin inhibitor sacubitril in combination with an angiotensin II receptor blocker was associated with increased plasma GLP-1 levels and improved glycemic control. Whether neprilysin inhibition per se is mediating these effects remains unknown. We sought to determine whether pharmacological neprilysin inhibition on its own confers beneficial effects on glycemic status and ß-cell function in a mouse model of reduced insulin secretion, and whether any such effects are dependent on GLP-1 receptor (GLP-1R) signaling. High-fat-fed male wild-type (Glp1r+/+) and GLP-1R knockout (Glp1r-/-) mice were treated with low-dose streptozotocin (STZ) to recapitulate type 2 diabetes-associated ß-cell dysfunction, or vehicle as control. Mice were continued on high-fat diet alone or supplemented with the neprilysin inhibitor sacubitril for 8 wk. At the end of the study period, ß-cell function was assessed by oral or intravenous glucose-tolerance test. Fasting and fed glucose were significantly lower in wild-type mice treated with sacubitril, although active GLP-1 levels and insulin secretion during oral glucose challenge were unchanged. In contrast, insulin secretion in response to intravenous glucose was significantly enhanced in sacubitril-treated wild-type mice, and this effect was blunted in Glp1r-/- mice. Similarly, sacubitril enhanced insulin secretion in vitro in islets from STZ-treated Glp1r+/+ but not Glp1r-/- mice. Together, our data suggest the insulinotropic effects of pharmacological neprilysin inhibition in a mouse model of ß-cell dysfunction are mediated via intra-islet GLP-1R signaling.NEW & NOTEWORTHY The neprilysin inhibitor, sacubitril, improves glycemic status in a mouse model of reduced insulin secretion. Sacubitril enhances intravenous but not oral glucose-mediated insulin secretion. The increased glucose-mediated insulin secretion is GLP-1 receptor-dependent. Neprilysin inhibition does not raise postprandial circulating active GLP-1 levels.

[Potential role of sacubitril/valsartan combination in type 2 diabetes]. / Intérêt de la combinaison sacubitril/valsartan dans le diabète de type 2.

Sacubitril/valsartan, a first-in-class angiotensin receptor-neprilysin inhibitor (ARNI), is indicated for the treatment of heart failure with reduced ejection fraction. It has demonstrated benefits in terms of cardiovascular morbidity and mortality reduction in this population. Recently, this drug association has also been shown to improve glycemic control and insulin sensitivity in patients with obesity and/or type 2 diabetes. Furthermore, some studies suggest a protective role of this new drug class in diabetic nephropathy. Altogether, these data raise the question about the potential place of ARNI in prevention and treatment of type 2 diabetes, a condition closely associated with heart failure.

Le sacubitril/valsartan, premier médicament de la classe des ARNI (Angiotensin Receptor-Neprilysin Inhibitor), est indiqué dans le traitement de l'insuffisance cardiaque à fraction d'éjection réduite. Il a démontré des bénéfices en termes de réduction de morbimortalité cardiovasculaire dans cette population. Récemment, il a également été rapporté que cette association thérapeutique améliore le contrôle glycémique et la sensibilité à l'insuline des patients avec une obésité et/ou un diabète de type 2. De plus, certaines études suggèrent également un rôle protecteur de cette nouvelle classe médicamenteuse dans la néphropathie diabétique. Ces données soulèvent la question de la place éventuelle des ARNI dans la prévention et le traitement du diabète de type 2, une pathologie étroitement associée à l'insuffisance cardiaque.

Early beta cell dysfunction vs insulin hypersecretion as the primary event in the pathogenesis of dysglycaemia.

Obesity and insulin resistance are associated with the development of type 2 diabetes. It is well accepted that beta cell dysfunction is required for hyperglycaemia to occur. The prevailing view is that, in the presence of insulin resistance, beta cell dysfunction that occurs early in the course of the disease process is the critical abnormality. An alternative model has been proposed in which primary beta cell overstimulation results in insulin hypersecretion that then leads to the development of obesity and insulin resistance, and ultimately to beta cell exhaustion. In this review, data from preclinical and clinical studies, including intervention studies, are discussed in the context of these models. The preponderance of the data supports the view that an early beta cell functional defect is the more likely mechanism underlying the pathogenesis of hyperglycaemia in the majority of individuals who develop type 2 diabetes. Graphical abstract.

Low concentration IL-1ß promotes islet amyloid formation by increasing hIAPP release from humanised mouse islets in vitro.

AIMS/HYPOTHESIS: Aggregation of the beta cell secretory product human islet amyloid polypeptide (hIAPP) results in islet amyloid deposition, a pathological feature of type 2 diabetes. Amyloid formation is associated with increased levels of islet IL-1ß as well as beta cell dysfunction and death, but the mechanisms that promote amyloid deposition in situ remain unclear. We hypothesised that physiologically relevant concentrations of IL-1ß stimulate beta cell islet amyloid polypeptide (IAPP) release and promote amyloid formation. METHODS: We used a humanised mouse model of endogenous beta cell hIAPP expression to examine whether low (pg/ml) concentrations of IL-1ß promote islet amyloid formation in vitro. Amyloid-forming islets were cultured for 48 h in the presence or absence of IL-1ß with or without an IL-1ß neutralising antibody. Islet morphology was assessed by immunohistochemistry and islet mRNA expression, hormone content and release were also quantified. Cell-free thioflavin T assays were used to monitor hIAPP aggregation kinetics in the presence and absence of IL-1ß. RESULTS: Treatment with a low concentration of IL-1ß (4 pg/ml) for 48 h increased islet amyloid prevalence (93.52 ± 3.89% vs 43.83 ± 9.67% amyloid-containing islets) and amyloid severity (4.45 ± 0.82% vs 2.16 ± 0.50% amyloid area/islet area) in hIAPP-expressing mouse islets in vitro. This effect of IL-1ß was reduced when hIAPP-expressing islets were co-treated with an IL-1ß neutralising antibody. Cell-free hIAPP aggregation assays showed no effect of IL-1ß on hIAPP aggregation in vitro. Low concentration IL-1ß did not increase markers of the unfolded protein response (Atf4, Ddit3) or alter proIAPP processing enzyme gene expression (Pcsk1, Pcsk2, Cpe) in hIAPP-expressing islets. However, release of IAPP and insulin were increased over 48 h in IL-1ß-treated vs control islets (IAPP 0.409 ± 0.082 vs 0.165 ± 0.051 pmol/5 islets; insulin 87.5 ± 8.81 vs 48.3 ± 17.3 pmol/5 islets), and this effect was blocked by co-treatment with IL-1ß neutralising antibody. CONCLUSIONS/INTERPRETATION: Under amyloidogenic conditions, physiologically relevant levels of IL-1ß promote islet amyloid formation by increasing beta cell release of IAPP. Neutralisation of this effect of IL-1ß may decrease the deleterious effects of islet amyloid formation on beta cell function and survival.

Neprilysin inhibition: a new therapeutic option for type 2 diabetes?

Neprilysin is a widely expressed peptidase with broad substrate specificity that preferentially hydrolyses oligopeptide substrates, many of which regulate the cardiovascular, nervous and immune systems. Emerging evidence suggests that neprilysin also hydrolyses peptides that play an important role in glucose metabolism. In recent studies in humans, a dual angiotensin receptor-neprilysin inhibitor (ARNi) improved glycaemic control and insulin sensitivity in individuals with type 2 diabetes and/or obesity. Moreover, preclinical studies have also reported that neprilysin inhibition, alone or in combination with renin-angiotensin system blockers, elicits beneficial effects on glucose homeostasis. Since neprilysin inhibitors have been approved for the treatment of heart failure, their repurposing for treating type 2 diabetes would provide a novel therapeutic strategy. In this review, we evaluate existing evidence from preclinical and clinical studies in which neprilysin is deleted/inhibited, we highlight potential mechanisms underlying the beneficial glycaemic effects of neprilysin inhibition, and discuss possible deleterious effects that may limit the efficacy and safety of neprilysin inhibitors in the clinic. We also review the favourable impact neprilysin inhibition can have on diabetic complications, in addition to glucose control. Finally, we conclude that neprilysin inhibitors may be a useful therapeutic option for treating type 2 diabetes; however, their combination with angiotensin II receptor blockers is needed to circumvent deleterious consequences of neprilysin inhibition alone.

Gut-specific Neprilysin Deletion Protects Against Fat-induced Insulin Secretory Dysfunction in Male Mice.

Neprilysin is a ubiquitous peptidase that can modulate glucose homeostasis by cleaving insulinotropic peptides. While global deletion of neprilysin protects mice against high-fat diet (HFD)-induced insulin secretory dysfunction, strategies to ablate neprilysin in a tissue-specific manner are favored to limit off-target effects. Since insulinotropic peptides are produced in the gut, we sought to determine whether gut-specific neprilysin deletion confers beneficial effects on insulin secretion similar to that of global neprilysin deletion in mice fed a HFD. Mice with conditional deletion of neprilysin in enterocytes (NEPGut-/-) were generated by crossing Vil-Cre and floxed neprilysin mice. Neprilysin activity was almost abolished throughout the gut in NEPGut-/- mice, and was similar in plasma, pancreas, and kidney in NEPGut-/- vs control mice. An oral glucose tolerance test was performed at baseline and following 14 weeks of HFD feeding, during which glucose tolerance and glucose-stimulated insulin secretion (GSIS) were assessed. Despite similar body weight gain at 14 weeks, NEPGut-/- displayed lower fasting plasma glucose levels, improved glucose tolerance, and increased GSIS compared to control mice. In conclusion, gut-specific neprilysin deletion recapitulates the enhanced GSIS seen with global neprilysin deletion in HFD-fed mice. Thus, strategies to inhibit neprilysin specifically in the gut may protect against fat-induced glucose intolerance and beta-cell dysfunction.

RIPK3 promotes islet amyloid-induced ß-cell loss and glucose intolerance in a humanized mouse model of type 2 diabetes.

OBJECTIVE: Aggregation of human islet amyloid polypeptide (hIAPP), a ß-cell secretory product, leads to islet amyloid deposition, islet inflammation and ß-cell loss in type 2 diabetes (T2D), but the mechanisms that underlie this process are incompletely understood. Receptor interacting protein kinase 3 (RIPK3) is a pro-death signaling molecule that has recently been implicated in amyloid-associated brain pathology and ß-cell cytotoxicity. Here, we evaluated the role of RIPK3 in amyloid-induced ß-cell loss using a humanized mouse model of T2D that expresses hIAPP and is prone to islet amyloid formation. METHODS: We quantified amyloid deposition, cell death and caspase 3/7 activity in islets isolated from WT, Ripk3-/-, hIAPP and hIAPP; Ripk3-/- mice in real time, and evaluated hIAPP-stimulated inflammation in WT and Ripk3-/- bone marrow derived macrophages (BMDMs) in vitro. We also characterized the role of RIPK3 in glucose stimulated insulin secretion (GSIS) in vitro and in vivo. Finally, we examined the role of RIPK3 in high fat diet (HFD)-induced islet amyloid deposition, ß-cell loss and glucose homeostasis in vivo. RESULTS: We found that amyloid-prone hIAPP mouse islets exhibited increased cell death and caspase 3/7 activity compared to amyloid-free WT islets in vitro, and this was associated with increased RIPK3 expression. hIAPP; Ripk3-/- islets were protected from amyloid-induced cell death compared to hIAPP islets in vitro, although amyloid deposition and caspase 3/7 activity were not different between genotypes. We observed that macrophages are a source of Ripk3 expression in isolated islets, and that Ripk3-/- BMDMs were protected from hIAPP-stimulated inflammatory gene expression (Tnf, Il1b, Nos2). Following 52 weeks of HFD feeding, islet amyloid-prone hIAPP mice exhibited impaired glucose tolerance and decreased ß-cell area compared to WT mice in vivo, whereas hIAPP; Ripk3-/- mice were protected from these impairments. CONCLUSIONS: In conclusion, loss of RIPK3 protects from amyloid-induced inflammation and islet cell death in vitro and amyloid-induced ß-cell loss and glucose intolerance in vivo. We propose that therapies targeting RIPK3 may reduce islet inflammation and ß-cell loss and improve glucose homeostasis in the pathogenesis of T2D.

Adipose tissue macrophage infiltration and hepatocyte stress increase GDF-15 throughout development of obesity to MASH.

Plasma growth differentiation factor-15 (GDF-15) levels increase with obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) but the underlying mechanism remains poorly defined. Using male mouse models of obesity and MASLD, and biopsies from carefully-characterized patients regarding obesity, type 2 diabetes (T2D) and MASLD status, we identify adipose tissue (AT) as the key source of GDF-15 at onset of obesity and T2D, followed by liver during the progression towards metabolic dysfunction-associated steatohepatitis (MASH). Obesity and T2D increase GDF15 expression in AT through the accumulation of macrophages, which are the main immune cells expressing GDF15. Inactivation of Gdf15 in macrophages reduces plasma GDF-15 concentrations and exacerbates obesity in mice. During MASH development, Gdf15 expression additionally increases in hepatocytes through stress-induced TFEB and DDIT3 signaling. Together, these results demonstrate a dual contribution of AT and liver to GDF-15 production in metabolic diseases and identify potential therapeutic targets to raise endogenous GDF-15 levels.

Unsaturated fatty acids prevent activation of NLRP3 inflammasome in human monocytes/macrophages.

The NLRP3 inflammasome is involved in many obesity-associated diseases, such as type 2 diabetes, atherosclerosis, and gouty arthritis, through its ability to induce interleukin (IL)-1ß release. The molecular link between obesity and inflammasome activation is still unclear, but free fatty acids have been proposed as one triggering event. Here we reported opposite effects of saturated fatty acids (SFAs) compared with unsaturated fatty acids (UFAs) on NLRP3 inflammasome in human monocytes/macrophages. Palmitate and stearate, both SFAs, triggered IL-1ß secretion in a caspase-1/ASC/NLRP3-dependent pathway. Unlike SFAs, the UFAs oleate and linoleate did not lead to IL-1ß secretion. In addition, they totally prevented the IL-1ß release induced by SFAs and, with less efficiency, by a broad range of NLRP3 inducers, including nigericin, alum, and monosodium urate. UFAs did not affect the transcriptional effect of SFAs, suggesting a specific effect on the NLRP3 activation. These results provide a new anti-inflammatory mechanism of UFAs by preventing the activation of the NLRP3 inflammasome and, therefore, IL-1ß processing. By this way, UFAs might play a protective role in NLRP3-associated diseases.

Obesity phenotype is related to NLRP3 inflammasome activity and immunological profile of visceral adipose tissue.

AIMS/HYPOTHESIS: Obesity is a heterogeneous condition comprising both individuals who remain metabolically healthy (MHO) and those who develop metabolic disorders (metabolically unhealthy, MUO). Adipose tissue is also heterogeneous in that its visceral component is more frequently associated with metabolic dysfunction than its subcutaneous component. The development of metabolic disorders is partly mediated by the NLR family pyrin domain containing-3 (NLRP3) inflammasome, which increases the secretion of inflammatory cytokines via activation of caspase-1. We compared the immunological profile and NLRP3 activity in adipose tissue between MUO and MHO individuals. METHODS: MHO and MUO phenotypes were defined, respectively, as the absence and the presence of the metabolic syndrome. Cellular composition and intrinsic inflammasome activity were investigated by flow cytometry, quantitative RT-PCR and tissue culture studies in subcutaneous and visceral adipose tissue from 23 MUO, 21 MHO and nine lean individuals. RESULTS: We found significant differences between the three study groups, including an increased secretion of IL-1ß, increased expression of IL1B and NLRP3, increased number of adipose tissue macrophages and decreased number of regulatory T cells in the visceral adipose tissue of MUO patients compared with MHO and lean participants. In macrophages derived from visceral adipose tissue, both caspase-1 activity and IL-1ß levels were higher in MUO patients than in MHO patients. Furthermore, caspase-1 activity was higher in CD11c(+)CD206(+) adipose tissue macrophages than in CD11c(-)CD206(+) cells. CONCLUSIONS/INTERPRETATION: The MUO phenotype seems to be associated with an increased activation of the NLPR3 inflammasome in macrophages infiltrating visceral adipose tissue, and a less favourable inflammatory profile compared with the MHO phenotype.

Acute Inhibition of Intestinal Neprilysin Enhances Insulin Secretion via GLP-1 Receptor Signaling in Male Mice.

The peptidase neprilysin modulates glucose homeostasis by cleaving and inactivating insulinotropic peptides, including some produced in the intestine such as glucagon-like peptide-1 (GLP-1). Under diabetic conditions, systemic or islet-selective inhibition of neprilysin enhances beta-cell function through GLP-1 receptor (GLP-1R) signaling. While neprilysin is expressed in intestine, its local contribution to modulation of beta-cell function remains unknown. We sought to determine whether acute selective pharmacological inhibition of intestinal neprilysin enhanced glucose-stimulated insulin secretion under physiological conditions, and whether this effect was mediated through GLP-1R. Lean chow-fed Glp1r+/+ and Glp1r-/- mice received a single oral low dose of the neprilysin inhibitor thiorphan or vehicle. To confirm selective intestinal neprilysin inhibition, neprilysin activity in plasma and intestine (ileum and colon) was assessed 40 minutes after thiorphan or vehicle administration. In a separate cohort of mice, an oral glucose tolerance test was performed 30 minutes after thiorphan or vehicle administration to assess glucose-stimulated insulin secretion. Systemic active GLP-1 levels were measured in plasma collected 10 minutes after glucose administration. In both Glp1r+/+ and Glp1r-/- mice, thiorphan inhibited neprilysin activity in ileum and colon without altering plasma neprilysin activity or active GLP-1 levels. Further, thiorphan significantly increased insulin secretion in Glp1r+/+ mice, whereas it did not change insulin secretion in Glp1r-/- mice. In conclusion, under physiological conditions, acute pharmacological inhibition of intestinal neprilysin increases glucose-stimulated insulin secretion in a GLP-1R-dependent manner. Since intestinal neprilysin modulates beta-cell function, strategies to inhibit its activity specifically in the intestine may improve beta-cell dysfunction in type 2 diabetes.

Neprilysin deficiency reduces hepatic gluconeogenesis in high fat-fed mice.

Neprilysin is a peptidase that cleaves glucoregulatory peptides, including glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK). Some studies suggest that its inhibition in diabetes and/or obesity improves glycemia, and that this is associated with enhanced insulin secretion, glucose tolerance and insulin sensitivity. Whether reduced neprilysin activity also improves hepatic glucose metabolism has not been explored. We sought to determine whether genetic deletion of neprilysin suppresses hepatic glucose production (HGP) in high fat-fed mice. Nep+/+ and Nep-/- mice were fed high fat diet for 16 weeks, and then underwent a pyruvate tolerance test (PTT) to assess hepatic gluconeogenesis. Since glycogen breakdown in liver can also yield glucose, we assessed liver glycogen content in fasted and fed mice. In Nep-/- mice, glucose excursion during the PTT was reduced when compared to Nep+/+ mice. Further, liver glycogen levels were significantly greater in fasted but not fed Nep-/- versus Nep+/+ mice. Since gut-derived factors modulate HGP, we tested whether gut-selective inhibition of neprilysin could recapitulate the suppression of hepatic gluconeogenesis observed with whole-body inhibition, and this was indeed the case. Finally, the gut-derived neprilysin substrates, GLP-1 and CCK, are well-known to suppress HGP. Having previously demonstrated elevated plasma GLP-1 levels in Nep-/- mice, we now measured plasma CCK bioactivity and reveal an increase in Nep-/- versus Nep+/+ mice, suggesting GLP-1 and/or CCK may play a role in reducing HGP under conditions of neprilysin deficiency. In sum, neprilysin modulates hepatic gluconeogenesis and strategies to inhibit its activity may reduce HGP in type 2 diabetes and obesity.

Insulinotropic Effects of Neprilysin and/or Angiotensin Receptor Inhibition in Mice.

Treatment of heart failure with the angiotensin receptor-neprilysin inhibitor sacubitril/valsartan improved glycemic control in individuals with type 2 diabetes. The relative contribution of neprilysin inhibition versus angiotensin II receptor antagonism to this glycemic benefit remains unknown. Thus, we sought to determine the relative effects of the neprilysin inhibitor sacubitril versus the angiotensin II receptor blocker valsartan on beta-cell function and glucose homeostasis in a mouse model of reduced first-phase insulin secretion, and whether any beneficial effects are additive/synergistic when combined in sacubitril/valsartan. High fat-fed C57BL/6J mice treated with low-dose streptozotocin (or vehicle) were followed for eight weeks on high fat diet alone or supplemented with sacubitril, valsartan or sacubitril/valsartan. Body weight and fed glucose levels were assessed weekly. At the end of the treatment period, insulin release in response to intravenous glucose, insulin sensitivity, and beta-cell mass were determined. Sacubitril and valsartan, but not sacubitril/valsartan, lowered fasting and fed glucose levels and increased insulin release in diabetic mice. None of the drugs altered insulin sensitivity or beta-cell mass, but all reduced body weight gain. Effects of the drugs on insulin release were reproduced in angiotensin II-treated islets from lean C57BL/6J mice, suggesting the insulin response to each of the drugs is due to a direct effect on islets and mechanisms therein. In summary, sacubitril and valsartan each exert beneficial insulinotropic, glycemic and weight-reducing effects in obese and/or diabetic mice when administered alone; however, when combined, mechanisms within the islet contribute to their inability to enhance insulin release.

The ß Cell in Diabetes: Integrating Biomarkers With Functional Measures.

The pathogenesis of hyperglycemia observed in most forms of diabetes is intimately tied to the islet ß cell. Impairments in propeptide processing and secretory function, along with the loss of these vital cells, is demonstrable not only in those in whom the diagnosis is established but typically also in individuals who are at increased risk of developing the disease. Biomarkers are used to inform on the state of a biological process, pathological condition, or response to an intervention and are increasingly being used for predicting, diagnosing, and prognosticating disease. They are also proving to be of use in the different forms of diabetes in both research and clinical settings. This review focuses on the ß cell, addressing the potential utility of genetic markers, circulating molecules, immune cell phenotyping, and imaging approaches as biomarkers of cellular function and loss of this critical cell. Further, we consider how these biomarkers complement the more long-established, dynamic, and often complex measurements of ß-cell secretory function that themselves could be considered biomarkers.