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

Abstract

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.

Keywords: angiotensin receptor-neprilysin inhibitor; insulin secretion; mouse; obesity; renin-angiotensin system; sacubitril; type 2 diabetes; valsartan.

Figure 1

Sacubitril and sacubitril/valsartan but not valsartan reduce plasma neprilysin activity. Plasma neprilysin activity after 8 weeks of treatment with control (CTL), sacubitril (SAC), valsartan (VAL) or sacubitril/valsartan (S/V) in high fat-fed mice that received vehicle (A; VEH) or streptozotocin (B; STZ) injections. n=11-14/group. *p<0.05.

Figure 2

Sacubitril and valsartan but not sacubitril/valsartan lower fed and fasting glucose in diabetic mice. Fed blood glucose levels over time in vehicle (A;VEH) and streptozotocin (B; STZ)- injected high fat-fed mice treated for 8 weeks with control (circles, CTL), sacubitril (inverted triangles, SAC), valsartan (triangles, VAL) or sacubitril/valsartan (squares, S/V). The arrow shows when VEH or STZ was injected. n=11-14/group. *p<0.05 SAC and VAL vs CTL, †p<0.05 SAC and VAL vs S/V. 16-hour fasted plasma glucose (C, D) and insulin (E, F) measured in VEH (C, E) and STZ (D, F) groups of mice at baseline (week -3.5) and at the end of the 8-week treatment period with CTL, SAC, VAL or S/V. n=11-14/group. *p<0.05.

Figure 3

Sacubitril and valsartan but not sacubitril/valsartan increase glucose-stimulated insulin secretion in diabetic mice. Plasma glucose (A, B) and insulin (C, D) during an IVGTT in vehicle [(A, C) VEH]- and streptozotocin [(B, D) STZ]-injected high fat-fed mice treated for 8 weeks with control (circles; CTL), sacubitril (inverted triangles; SAC), valsartan (triangles; VAL) or sacubitril/valsartan (squares; S/V). The insets in A and B represent the rate of glucose disappearance (Kg) computed from 10 to 45 minutes during the IVGTT. The insets in C and D represent the incremental areas under the curve (iAUC) for insulin from 0 to 45 minutes. First-phase [0-5’] and second-phase [5-45’] glucose-stimulated insulin secretion (E, F) during the IVGTT in VEH (E)- and STZ (F)- injected mice treated for 8 weeks with CTL, SAC, VAL or S/V. n=11-14/group. *p<0.05.

Figure 4

Sacubitril, valsartan and sacubitril/valsartan do not affect beta- and alpha-cell mass. Insulin content (A, B), glucagon content (C, D), beta-cell mass (E, F) and alpha-cell mass (G, H) at the end of the 8-week treatment period in vehicle (A, C, E, G; VEH) and streptozotocin (B, D, F, H; STZ) injected high fat-fed mice treated with control (CTL), sacubitril (SAC), valsartan (VAL) or sacubitril/valsartan (S/V). n=11-14/group. *p<0.05.

Figure 5

Sacubitril, valsartan and sacubitril/valsartan do not alter insulin sensitivity in both obese and diabetic mice. Blood glucose levels, as absolute values (A, B) and as % of baseline (C, D), during an intraperitoneal insulin tolerance test in high fat-fed mice injected with vehicle (A, C; VEH) or streptozotocin (B, D; STZ) and treated for 8 weeks with control (circles, CTL), sacubitril (inverted triangles, SAC), valsartan (triangles, VAL) or sacubitril/valsartan (squares, S/V). Inverse area under the % of baseline glucose curve accounting for adjustment for glucose baseline in VEH (E)- or STZ (F)-injected mice treated for 8 weeks with CTL, SAC, VAL or S/V. n=11-14/group.

Figure 6

Sacubitril, valsartan and sacubitril/valsartan reduce body weight gain in both obese and diabetic mice. Body weight over time in high fat-fed mice injected with vehicle (A; VEH) or streptozotocin (B; STZ), and then treated for 8 weeks with control (circles, CTL), sacubitril (inverted triangles, SAC), valsartan (triangles, VAL) or sacubitril/valsartan (squares, S/V). The arrow shows when VEH or STZ was injected. Cumulative body weight gain over the 8-week treatment period in high fat-fed mice injected with VEH (C) or STZ (D) and treated with CTL, SAC, VAL or S/V. n=11-14/group. *p<0.05 SAC, VAL and S/V vs. CTL, ^‡p<0.05 VAL vs. S/V, ^†p<0.05 SAC vs. S/V.

Figure 7

Sacubitrilat and valsartan alone but not combined, increase glucose-stimulated insulin secretion in angiotensin II-treated islets. Insulin secretion in response to 2.8 mmol/l (white bars) and 20 mmol/l (grey bars) glucose in isolated islets from lean C57BL6/J mice following incubation with vehicle (CTL, open circles), 100 nmol/l angiotensin II alone (Ang II, black circles), 100 nmol/l angiotensin II + 1 μmol/l sacubitrilat (SAC, black inverted triangles), 1 μmol/l valsartan (VAL, black triangles), or 1 μmol/l sacubitrilat and 1 μmol/l valsartan (S/V, black squares). Data are mean ± S.E.M of 5 independent experiments. *p<0.05.