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.

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.

Death receptors mediate the adverse effects of febrile-range hyperthermia on the outcome of lipopolysaccharide-induced lung injury.

We have shown that febrile-range hyperthermia enhances lung injury and mortality in mice exposed to inhaled LPS and is associated with increased TNF-α receptor activity, suppression of NF-κB activity in vitro, and increased apoptosis of alveolar epithelial cells in vivo. We hypothesized that hyperthermia enhances lung injury and mortality in vivo by a mechanism dependent on TNF receptor signaling. To test this, we exposed mice lacking the TNF-receptor family members TNFR1/R2 or Fas (TNFR1/R2(-/-) and lpr) to inhaled LPS with or without febrile-range hyperthermia. For comparison, we studied mice lacking IL-1 receptor activity (IL-1R(-/-)) to determine the role of inflammation on the effect of hyperthermia in vivo. TNFR1/R2(-/-) and lpr mice were protected from augmented alveolar permeability and mortality associated with hyperthermia, whereas IL-1R(-/-) mice were susceptible to augmented alveolar permeability but protected from mortality associated with hyperthermia. Hyperthermia decreased pulmonary concentrations of TNF-α and keratinocyte-derived chemokine after LPS in C57BL/6 mice and did not affect pulmonary inflammation but enhanced circulating markers of oxidative injury and nitric oxide metabolites. The data suggest that hyperthermia enhances lung injury by a mechanism that requires death receptor activity and is not directly associated with changes in inflammation mediated by hyperthermia. In addition, hyperthermia appears to enhance mortality by generating a systemic inflammatory response and not by a mechanism directly associated with respiratory failure. Finally, we observed that exposure to febrile-range hyperthermia converts a modest, survivable model of lung injury into a fatal syndrome associated with oxidative and nitrosative stress, similar to the systemic inflammatory response syndrome.

Febrile-range hyperthermia augments lipopolysaccharide-induced lung injury by a mechanism of enhanced alveolar epithelial apoptosis.

Fever is common in critically ill patients and is associated with worse clinical outcomes, including increased intensive care unit mortality. In animal models, febrile-range hyperthermia (FRH) worsens acute lung injury, but the mechanisms by which this occurs remain uncertain. We hypothesized that FRH augments the response of the alveolar epithelium to TNF-alpha receptor family signaling. We found that FRH augmented LPS-induced lung injury and increased LPS-induced mortality in mice. At 24 h, animals exposed to hyperthermia and LPS had significant increases in alveolar permeability without changes in inflammatory cells in bronchoalveolar lavage fluid or lung tissue as compared with animals exposed to LPS alone. The increase in alveolar permeability was associated with an increase in alveolar epithelial apoptosis and was attenuated by caspase inhibition with zVAD.fmk. At 48 h, the animals exposed to hyperthermia and LPS had an enhanced lung inflammatory response. In murine lung epithelial cell lines (MLE-15, LA-4) and in primary type II alveolar epithelial cells, FRH enhanced apoptosis in response to TNF-alpha but not Fas ligand. The increase in apoptosis was caspase-8 dependent and associated with suppression of NF-kappaB activity. The FRH-associated NF-kappaB suppression was not associated with persistence of IkappaB-alpha, suggesting that FRH-mediated suppression of NF-kappaB occurs by means other than alteration of IkappaB-alpha kinetics. These data show for the first time that FRH promotes lung injury in part by increasing lung epithelial apoptosis. The enhanced apoptotic response might relate to FRH-mediated suppression of NF-kappaB activity in the alveolar epithelium with a resultant increase in susceptibility to TNF-alpha-mediated cell death.