Angiotensin 1-7 as means to prevent the metabolic syndrome: lessons from the fructose-fed rat model

Abstract

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7-treated animals had lower body weight (-9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (-51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (-40%) and serum aldosterone (-48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7-treated rats. WAT from Ang 1-7-treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7-treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7-treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

FIG. 1.

Insulin sensitivity tests. A and B: Glucose tolerance test. Ang 1-7–treated rats (n = 6) had normal levels of basal serum glucose and insulin but a significantly attenuated increase in serum glucose response to acute (2 g/kg) intraperitoneal glucose challenge, compared with fructose-fed rats (n = 9, P < 0.05), as revealed by the AUC analysis (A). Glucose levels were measured 30, 60, and 90 min after injection and were significantly reduced by Ang 1-7 at 30, 60, and 90 min postinjection (P < 0.05) (B). Data are presented as the mean value ± SEM. C: Intraperitoneal insulin tolerance test. Ang 1-7 rats had increased insulin sensitivity with a more prominent and prolonged hypoglycemic effect of insulin. Changes in basal (t = 0) blood glucose levels after intraperitoneal insulin administration were measured every 30 min over a total period of 120 min. Ang 1-7 treatment led to a significant hypoglycemic reaction at 30 and 120 min post–insulin reduction (P < 0.05). Data are presented as the mean value ± SEM. *P < 0.05.

FIG. 2.

MRI analysis of total and subcutaneous fat. Total and subcutaneous (red arrow pointing to subcutaneous fat) fat was significantly reduced in the 6-month HFrD Ang 1-7–treated rats (n = 6) (A) compared with HFrD-untreated rats (n = 5) (B). Averages ± SEM of total and subcutaneous fat in all studied groups are presented in C and D. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

Histological sections of epididymal fat tissue. Tissue sections from Ang 1-7–treated rats (n = 6) (A and B) display many small adipocytes and fewer large adipocytes (a field “enriched” with large adipocytes is shown in B). The large adipocytes did not display inflammatory infiltration. Tissue from HFrD and HFrDSal rats (n = 9) (C–G) displays large adipocytes with inflammatory infiltration (C, E, and F), mostly of macrophages (CD68 staining) (G). um, μm.

FIG. 4.

Histological sections of the liver from animals in the prevention arm. Photomicrographs of portal (A–C) and central (D and E) regions of liver samples stained with hematoxylin and eosin (H & E). Portal areas of livers from HFrD Ang 1-7–treated rats (n = 6) (A, F, H, and J) and HFrDSal (C) and HFrD rats (n = 9) (B, G, I, and K) stained with oil red O (O-R O) (F and G), CD68 (H and I), and masson trichrome (MTC) (J and K). Original magnification ×40. Scale bar, 100 μm.

FIG. 5.

Expression of pP65NFκB, FAS, MAS, and activated ERK in the epididymal fat of rats treated with Ang 1-7 for 6 months (n = 6) and control rats (n = 9). Ang 1-7 treatment significantly reduced the levels of phosphorylated p65 (A) and of phosphorylated ERK (C). Elevated levels of FAS were evident in the Ang 1-7–treated group (B); however, no downregulation of the Ang 1-7 receptor, MAS, was detected (D). Bar graphs represent a quantification of the respective protein expression levels from at least three experiments. Data are presented as mean ± SEM. *P < 0.05.

FIG. 6.

Epididymal fat NADPH-stimulated superoxide production as measured by lucigenin-enhanced chemiluminescence. Superoxide production was significantly lower in HFrD rats treated with Ang 1-7 compared with HFrD-untreated rats [fructose untreated (n = 5), 165 ± 47% of control rats; fructose + Ang 1-7 (n = 5), 42 ± 8% of control rats (n = 3); P < 0.05 in comparison with control values]. Data are presented as the mean ± SE. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)

FIG. 7.

Immunofluorescence of 10-day myofiber cultures. Individual myofibers were isolated from HFrD rats that were not (A, A′, and A′′) or were treated with Ang 1-7 (B, B′, and B′′). At least nine cultures per rat (three HFrD and four HFrD Ang 1-7 rats) were reacted with DAPI to allow visualization of nuclei (A′ and B′) and sarcomeric myosin for specific identification of skeletal muscle cells (A′′ and B′′). Scale bar, 20 μm.