The brain renin‐angiotensin system plays a crucial role in regulating body weight in diet‐induced obesity in rats

Abstract

Background and Purpose

Reduced weight gain after treatment with AT₁ receptor antagonists may involve a brain‐related mechanism. Here, we investigated the role of the brain renin‐angiotensin system on weight regulation and food behaviour, with or without additional treatment with telmisartan.

Methods

Transgenic rats with a brain‐specific deficiency in angiotensinogen (TGR(ASrAOGEN)) and the corresponding wild‐type, Sprague Dawley (SD) rats were fed (3 months) with a high‐calorie cafeteria diet (CD) or standard chow. SD and TGR(ASrAOGEN) rats on the CD diet were also treated with telmisartan (8 mg·kg⁻¹·d⁻¹, 3 months).

Results

Compared with SD rats, TGR(ASrAOGEN) rats (i) had lower weights during chow feeding, (ii) did not become obese during CD feeding, (iii) had normal baseline leptin plasma concentrations independent of the feeding regimen, whereas plasma leptin of SD rats was increased due to CD, (iv) showed a reduced energy intake, (v) had a higher, strain‐dependent energy expenditure, which is additionally enhanced during CD feeding, (vi) had enhanced mRNA levels of pro‐opiomelanocortin and (vii) showed improved glucose control. Weight gain and energy intake in rats fed the CD diet were markedly reduced by telmisartan in SD rats but only to a minor extent in TGR(ASrAOGEN) rats.

Conclusions

The brain renin‐angiotensin system affects body weight regulation, feeding behaviour and metabolic disorders. When angiotensin II levels are low in brain, rats are protected from developing diet‐induced obesity and obesity‐related metabolic impairments. We further suggest that telmisartan at least partly lowers body weight via a CNS‐driven mechanism.

Abbreviations

AngII

angiotensin II

AngI

angiotensin I

Ang(1‐7)

angiotensin‐(1‐7)

ARC

arcuate nucleus

BBB

blood‐brain barrier

BMI

body mass index

CART

cocaine‐ and amphetamine‐regulated transcript

CD

cafeteria diet

C_max

maximal concentration

EE

energy expenditure

GFAP

glial fibrillary acidic protein

HDL

high‐density lipoproteins

HOMA

Homeostatic model assessment index

HPA axis

hypothalamic‐pituitary‐adrenal axis

LRT

leptin resistance test

OGTT

oral glucose tolerance test

POMC

proopiomelanocortin

RAS

renin‐angiotensin system

RER

respiratory exchange rate

SBP

systolic blood pressure

SD

Sprague Dawley rat

TG

transgenic rat

T2DM

type 2 diabetes mellitus.

Tables of Links

TARGETS
GPCRs
AT1 receptor
AT2 receptor

LIGANDS
AngII, angiotensin II
Ang(1‐7), angiotensin‐(1‐7)
Telmisartan

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Introduction

A number of AT₁ receptor antagonists such as candesartan, olmesartan or telmisartan have repeatedly been demonstrated to reduce body weight in lean (Zorad et al., 2006; He et al., 2010; Müller‐Fielitz et al., 2011) or obese rodents (Schupp et al., 2005; He et al., 2010; Miesel et al., 2012; Müller‐Fielitz et al., 2012a; Vazquez‐Medina et al., 2013; Müller‐Fielitz et al., 2014; Müller‐Fielitz et al., 2015; Schuchard et al., 2015). In patients, a moderate weight loss was also observed during irbesartan therapy (Kintscher et al., 2007). In line with the findings after AT₁ receptor blockade, body weights of the double AT_1A/AT_1B receptor knockout (KO) mice were reduced (Gembardt et al., 2008), which strengthens the importance of these pharmacological insights into the effects of AT₁ receptor antagonists.

Considering the adipocyte‐brain crosstalk that is known to be involved in regulating energy intake, energy expenditure and body weight, several mechanisms have been found to participate in weight loss induced by AT₁ receptor blockade. These include peripheral‐associated pathways such as a reduced growth of adipocytes (Zorad et al., 2006; Müller‐Fielitz et al., 2012a), anti‐inflammatory effects (Cole et al., 2010; Guo et al., 2011) or the activation of PPAR‐δ (He et al., 2010). The fact that high doses of AT₁ receptor antagonists are required to induce anti‐obesity effects (Müller‐Fielitz et al., 2011; Müller‐Fielitz et al., 2014) may indicate that the underlying mechanism involves the CNS because the lipophilicity of these antagonists is low, which limits blood‐brain barrier (BBB) penetration (Michel et al., 2013). Thus, high doses of AT₁ receptor antagonists may be necessary in particular to affect energy intake and expenditure, while normal doses may help to control glucose and regulate blood pressure (Müller‐Fielitz et al., 2011; Müller‐Fielitz et al., 2014). The hypothesis of a brain‐related mechanism is supported by the observations that (i) orally administered AT₁ receptor antagonists were able to antagonize pressure responses to centrally administered angiotensin II (AngII), giving evidence for BBB penetration of the antagonists (Gohlke et al., 2001; Gohlke et al., 2002); (ii) hypothalamic orexigenic peptides were suppressed during treatment with AT₁ receptor antagonists (Müller‐Fielitz et al., 2011; Noma et al., 2011; Müller‐Fielitz et al., 2015); (iii) the stress‐induced stimulation of the hypothalamus‐pituitary‐adrenal (HPA) axis was attenuated in rats by AT₁ receptor antagonists, which was further associated with reduced intake of cafeteria diet (CD) (Miesel et al., 2012); and (iv) the leptin sensitivity was restored during treatment with AT₁ receptor antagonists (Müller‐Fielitz et al., 2014; Müller‐Fielitz et al., 2015).

Because both peripheral and centrally‐driven mechanisms may have anti‐obesity effects during treatment with AT₁ receptor antagonists, we aimed in this study to gather more evidence showing that an attenuated AT₁ receptor‐dependent signalling mediates weight reduction in a CNS‐dependent manner. Because of the lack of brain‐specific double AT_1A/AT_1B receptor KO mice, we used transgenic rats with low brain angiotensinogen (TGR(ASrAOGEN)) as an experimental model of brain‐specific, reduced Ang II signalling (Schinke et al., 1999). Because the TGR(AsrAOGEN) rats were generated on the Sprague Dawley (SD) rat genetic background and all breeding since has been performed on this background, we considered SD rats to be the correct controls. In TGR(ASrAOGEN) rats, the concentration of angiotensinogen protein was markedly reduced, compared with controls in medulla, pons, thalamus, cerebellum and particularly in the hypothalamus, an area that is well known to be involved in energy homeostasis, while plasma concentrations of angiotensinogen and plasma renin activity were similar. In terms of body weight, transgenic mice with a global angiotensinogen deficiency gained less weight, independently of feeding with normal chow or a high‐fat diet (Massiera et al., 2001b). Body weights in the TGR(ASrAOGEN) strain werer lower than in wild‐type (WT) controls (Kasper et al., 2005; Müller et al., 2010). As a limitation of the TGR(ASrAOGEN) strain, the marked down‐regulation of angiotensinogen in the brain may indeed mimic AT₁ receptor blockade but this deficiency also reduced formation of Ang(1‐7), which was recently demonstrated to be involved in regulating body weight (Schuchard et al., 2015).

We designed our study by particularly addressing the following hypotheses: that the development of diet‐induced obesity could be prevented in TGR(ASrAOGEN) rats and that the decreased weight gain following treatment with AT₁ receptor antagonists would be at least partly diminished in TGR(ASrAOGEN) rats. To investigate this, CD‐fed TGR(ASrAOGEN) rats were treated with telmisartan for 3 months. Some of these results have already been presented at the Annual Meetings of the German Society of Experimental and Clinical Pharmacology and Toxicology 2014 and 2015 (Piehl et al., 2014; Winkler et al., 2015).

Methods

Animals

All animal care and experimental procedures were in accordance with the NIH guidelines for the care and use of laboratory animals and were approved by the animal ethics committee of the local regulatory authority (Ministerium für Energiewende, Landwirtschaft, Umwelt und ländliche Räume des Bundeslandes Schleswig‐Holstein). The results of all studies involving animals are reported in accordance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). A total of 120 animals were used in the experiments described here. Male TGR(ASrAOGEN)L680 rats (designated in the following as TGR(ASrAOGEN), Max Delbrück Center for Molecular Medicine, Berlin, Germany) and SD rats (Saint‐Berthevin, Cedex, France) were used in the experiments. The animals were kept at room temperature with a 12 h/12 h dark (14:00–2:00 h)/light (2:00–14:00 h) cycle.

Feeding and treatment protocols

Protocol 1

Group sizes of n = 13 were estimated a priori by a power analysis and considering a difference of approximately 100 g between chow and CD feeding as the primary effect size, an α‐value of 0.008 and a power size of 0.815. At the age of 4 weeks, rats arrived in the laboratory and were kept in pairs. After a 2 week habituation period, a total of 26 TGR(ASrAOGEN) and 26 SD rats were randomized to one of the two groups per strain. One group of TGR(ASrAOGEN) and SD rats, respectively, was fed solely with standard chow (Maintenance 1320; Altromin, Lage, Germany). This feeding regimen is designated as ‘control’ throughout this text. A second group of TGR(ASrAOGEN) and SD rats had free access throughout the entire duration of the study to standard chow plus one of six various commercial chocolate/cookie bars, these being switched daily in a regular manner. This feeding regimen is designated as ‘CD’ throughout this text (Miesel et al., 2010). Blood samples were taken before and at weeks 4, 8 and 12 (all nonfasting) and at week 9 and 11 (fasting for 18 h) from a tail nick. Animals were phenotyped regarding fat mass (week 8) by using MRI techniques (Miesel et al., 2010), energy expenditure (week 11) by using the PhenoMaster/InfraMot System™ (TSE, Bad Homburg, Germany) (Müller‐Fielitz et al., 2014; Schuchard et al., 2015) and insulin sensitivity (oral glucose tolerance test (OGTT)], week 12 (Miesel et al., 2010). In week 8, abdominal girth and body length (not including tail length) were determined in a blinded manner to calculate the body mass index (BMI). After 12 weeks, rats were killed, and organs were removed. After preparing the femur, its length was measured using a vernier calliper. During the study and functional phenotyping, some rats unexpectedly died, and some animals had to be excluded from further study. Thus, this part of the study ultimately had the following group sizes: triglycerides (TG)_chow n = 12, SD_chow n = 10; TG_CD n = 12, SD_CD n = 13.

Protocol 2

Based on an a priori power analysis, the group size was estimated to be n = 17 (60 g difference in body weight as the primary effect size, α‐value: 0.008, power 0.78). For reasons of breeding and availability of animals, a total of 38 TGR(ASrAOGEN) and 30 SD rats arrived at the age of 4 weeks rats in the laboratory and were kept in pairs. After a 2 week habituation period, TGR(ASrAOGEN) and SD rats were randomized to one of the two groups per strain. To address the question of whether the weight loss induced by AT₁ receptor antagonists was mediated via a CNS pathway, one group of SD and TGR(ASrAOGEN) rats was treated with telmisartan (Boehringer Ingelheim Pharmaceuticals, Inc., Ingelheim, Germany) for the same time as they were fed the CD diet (8 mg·kg⁻¹, once per day by gavage, for 14 weeks), whereas a second group of CD‐fed SD and TGR(ASrAOGEN) rats received vehicle instead of telmisartan. Blood samples (non‐fasting, before, after weeks 4, 8 and 12 and fasting for 18 h at weeks 9 and 11) were taken. All animals were monitored for body weight, energy intake and energy expenditure (week 11), leptin sensitivity (week 12) (Müller‐Fielitz et al., 2015), glycemic control (OGTT at week 13) and fat mass distribution (week 13). At week 14, rats were killed, and organs were removed. Adipocytes were isolated for the glucose uptake assay according to (Santos et al., 2010, 2008). During the study and functional phenotyping, some rats unexpectedly died, and some animals had to be excluded from further study. Thus, this part of the study ultimately had the following group sizes: TG_VEH n = 17, SD_VEH n = 15; TG_TEL n = 19, SD_TEL n = 15.

Biochemical analysis

To determine AngII, blood (2 mL) was collected into an inhibitor solution containing 15.4 mM EDTA and 20 μM bestatin (final concentration). Plasma concentrations of insulin, adiponectin and glucagon (all from Millipore, St. Charles, MO, USA) or AngII (IBL, Hamburg, Germany) were determined by radioimmunoassay using commercial kits. Plasma concentrations of leptin were measured by commercial radioimmunoassay (Millipore) as well as elisa (R&D Systems, Inc., Minneapolis, MN, USA). TG and high‐density lipoproteins (HDL) were quantified in the plasma of fasting animals using a Roche/Hitachi Modular P Chemistry Analyzer (Mannheim, Germany) (Müller‐Fielitz et al., 2014, 2015; Schuchard et al., 2015). Blood glucose was determined using glucose sensors (Ascensia® ELITE; Bayer, Leverkusen, Germany). RNA was isolated from the hypothalamus, cDNA was synthesized and mRNA levels were quantified by using real‐time quantitative polymerase chain reaction.

Histology of liver and fat tissue

Formaldehyde‐fixed, paraffin‐embedded sections of liver and epididymal fat pads were stained with haematoxylin and eosin (H&E) according to the standard protocol of GILL. A pathologist unaware of the animal grouping evaluated the slides and scored each liver tissue specimen for hepatocytes with steatosis (Schuchard et al., 2015) and each fat sample for macrophage infiltration. Size of adipocytes was evaluated by counting 10 cells per animal in a blinded manner using labsens® version 1.1 2010 supported by Olympus (Hamburg, Germany)

Data and statistical analysis

These studies comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Means and upper and lower quartiles are depicted as boxplots. Whiskers indicate variability between the 10th and 90th percentiles. In line graphs and tables, data are expressed as means ± SEM.

As described previously, rats were fed either with chow or with chow plus chocolate/cookie bars. Because of the different calorie values of chow (11.7 kJ·g⁻¹) and chocolate/cookie bars (20.8 kJ·g⁻¹), we individually calculated the energy intake (in kJ) of each rat to correctly assess food intake on the basis of the consumed amounts of chow and chocolate/cookie bars.

Food efficiency ratio (FER) was calculated according to the equation ‘FER = body weight gain · total energy⁻¹’. Homeostatic model assessment (HOMA) index was calculated considering plasma levels of fasting animals according to the formula ‘insulin (μU·mL⁻¹) · glucose (mg·dL⁻¹)·405⁻¹’. The amount of fat was semi‐automatically quantified in retroperitoneal fat pads and in subcutaneous fat on the basis of the transverse T1‐weighted turbo spin‐echo images by using the freeware MRIcro version 1.4 build 1 (http://downloads.fyxm.net/MRIcro‐117936.html) and the Vitom for Windows software (Essen, Deutschland). Only intensity signals of 70 grey scales were considered to ensure that fat was being analysed (Vogt et al., 2007). In order to quantify the total effect over the observation period in response to OGTT for changes in plasma concentrations of glucose, the areas under the curve were calculated for each individual animal on the basis of their delta values. C_max values were also calculated considering delta values. All data were checked for outliers using the graphpad® outlier calculator and tested for Gaussian distribution. A two‐way analysis of variance (ANOVA) was performed to examine the effects of two variables. P‐values given in the figures originated from two‐way ANOVA by testing strain differences. P‐values given in legends originated from two‐way ANOVA by testing diet differences (protocol 1; P ^diet _{2‐way ANOVA}) or drug differences (protocol 2; P ^drug _{2‐way ANOVA}). Bonferroni's post hoc test for multiple comparisons test was only performed if F achieved P < 0.05 and there was no significant variance inhomogeneity. Differences were considered to be statistically significant at P < 0.05.

Results

Results for TGR(ASrAOGEN) before any feeding or treating regimen

At the age of 6 weeks, body weight of TGR(ASrAOGEN) was lower than in wild‐type controls in both protocols (Tables 1, 2; Figures 1A and 4A). Plasma leptin levels did not differ between the two strains (Figure 1B), while adiponectin was markedly higher in TGR(ASrAOGEN) than in SD rats (Figure S1) Fasting glucose of TGR(ASrAOGEN) and SD rats at the age of 6 weeks was similar (3.35 ± 0.17 vs. 3.33 ± 0.18 mmol·L⁻¹).

Table 1.

Growth of SD and TGR(ASrAOGEN) rats after long‐term feeding with high‐calorie cafeteria diet

	SDCON	SDCD	TGCON	TGCD	Two‐way ANOVA P‐values
	(n = 10)	(n = 13)	(n = 12)	(n = 12)	Diet	Strain
Body weight at age 6 weeks	247 ± 9	255 ± 7	177 ± 5	178 ± 5	0.534	<0.0001
Body weight gain (g)	315 ± 12	434 ± 14 *	221 ± 5	232 ± 5	<0.0001	<0.0001
BMI (kg · m−2)	7.11 ± 0.11	7.75 ± 0.09 *	6.09 ± 0.06	6.09 ± 0.08	0.0005	<0.0001
Subcutaneous fat volume (mL)	12.5 ± 1.4	28.9 ± 2.9 *	13.7 ± 0.5	12.1 ± 0.7	0.0001	<0.0001
Visceral fat volume (mL)	28.9 ± 2.3	66.8 ± 5.1 *	19.3 ± 1.0	21.0 ± 0.8	<0.0001	<0.0001
Body length (cm)	27.0 ± 0.2	28.3 ± 0.3 *	24.5 ± 0.2	24.6 ± 0.2	0.002	<0.0001
Femur length (mm)	40.3 ± 0.2	40.9 ± 0.2	39.2 ± 0.3	39.7 ± 0.2	0.012	<0.0001
Girth (cm)	21.6 ± 0.2	23.7 ± 0.3 *	18.9 ± 0.2	18.8 ± 0.2	<0.0001	<0.0001
Left ventricle (g)	0.98 ± 0.02	1.15 ± 0.02 *	1.01 ± 0.02	1.09 ± 0.01 *	<0.0001	0.6396
Liver (g)	16.3 ± 0.6	18.6 ± 0.7 *	11.2 ± 0.3	10.9 ± 0.2	0.066	<0.0001
Kidneys (g)	1.65 ± 0.05	1.66 ± 0.04	1.27 ± 0.02	1.20 ± 0.02	0.310	<0.0001
Adrenal glands (mg)	25.9 ± 1.1	25.9 ± 1.1	25.4 ± 0.4	25.7 ± 0.5	0.485	0.300

Data shown are means ± SEM.

^* P < 0.05, significantly different from corresponding chow‐fed rats after Bonferroni's post hoc test.

Table 2.

Growth of CD‐fed SD and TGR(ASrAOGEN) rats after treatment with telmisartan (8 mg·kg⁻¹).

	SDVEH	SDTEL	TGVEH	TGTEL	Two‐way ANOVA P‐values
	(n = 15)	(n = 15)	(n = 17)	(n = 19)	Drug	Strain
Body weight at age 6	248 ± 7	251 ± 9	184 ± 7	185 ± 5	0.7770	<0.0001
Body weight gain (g)	414 ± 22	309 ± 11 *	212 ± 5	178 ± 5	<0.0001	<0.0001
BMI (kg · m−2)	7.98 ± 0.23	7.16 ± 0.15 *	6.10 ± 0.07	5.75 ± 0.09	0.0001	<0.0001
Subcutaneous fat volume (mL)	47.5 ± 7.3	32.2 ± 2.9 *	16.3 ± 1.1	11.6 ± 0.7	0.0039	0.0039
Visceral fat volume (mL)	86.3 ± 11.3	58.8 ± 3.6 *	31.4 ± 1.7	21.9 ± 0.8	0.0004	<0.0001
Femur length (mm)	40.9 ± 0.3	40.1 ± 0.4 *	38.8 ± 0.2	38.4 ± 0.2	0.0075	<0.0001
Body length (vm)	28.4 ± 0.2	27.9 ± 0.2	25.5 ± 0.1	24.9 ± 0.2	0.0009	<0.0001
Girth (cm)	22.3 ± 0.7	20.3 ± 0.3 *	18.1 ± 0.2	16.9 ± 0.3	0.0006	<0.0001
Left ventricle (g)	1.19 ± 0.04	0.94 ± 0.04 *	1.04 ± 0.01	0.77 ± 0.02 *	<0.0001	<0.0001
Liver (g)	18.6 ± 0.9	17.2 ± 1.0	11.8 ± 0.3	10.4 ± 0.2	0.0312	<0.0001
Kidneys (g)	1.76 ± 0.5	1.78 ± 0.06	1.11 ± 0.02	1.13 ± 0.02	0.7207	<0.0001
Adrenal glands (mg)	28.3 ± 1.6	26.5 ± 0.8	25.4 ± 0.6	23.7 ± 0.2	0.0280	0.0004

Data shown are means ± SEM. Control rats received vehicle.

^* P < 0.05, significantly different from corresponding vehicle‐treated rats after Bonferroni's post hoc test.

Figure 1.

Growth and food intake of TGR(ASrAOGEN) and SD rats after control or CD feeding (TG_chow n = 12, SD_chow n = 10, TG_CD n = 12, SD_CD n = 13; Protocol 1. (A) Gain in body weight during experiment. (B) Plasma leptin are enhanced by CD feeding in SD (P ^diet _{2‐way ANOVA} < 0.0001), but not in TGR(ASrAOGEN) rats. Plasma concentrations at the end of the study of triglycerides (C) (P ^diet _{2‐way ANOVA} < 0.0001) and HDL (D) (P ^diet _{2‐way ANOVA} = 0.0001). (E) Energy intake after control or CD feeding during the study (P ^diet _{2‐way ANOVA} for both SD and TG rats is 0.0001). (F) Food efficiency ratio (FER; P ^diet _{2‐way ANOVA} = 0.0012), (G) hypothalamic POMC expression (P ^diet _{2‐way ANOVA} = 0.024). (H) water intake per week (P ^diet _{2‐way ANOVA} < 0.0001). Means ± SEM are shown in line graphs; means and upper and lower quartiles as boxplots. Whiskers indicate variability between the 10th and 90th percentile; *P < 0.05 after Bonferroni's post hoc test.

Figure 4.

Growth and food intake of TGR(ASrAOGEN) and SD rats after CD feeding and treatment with telmisartan (TEL, 8 mg·kg⁻¹·d⁻¹, 3 months) or vehicle. (A) Body weight during experiment (P ^drug _{2‐way ANOVA} = 0.028 for TG_VEH versus TG_TEL, P ^drug _{2‐way ANOVA} = 0.010 for SD_VEH versus SD_TEL). Plasma concentrations of leptin (B) (P^drug _{2‐way ANOVA} = 0.003), triglycerides (C) P ^drug _{2‐way ANOVA} = 0.0003) and HDL (D) (P^drug _{2‐way ANOVA} = 0.026) at the end of the study. (E) Energy intake after control or CD feeding during the study (P ^drug _{2‐way ANOVA} = 0.0034 for TG_VEH versus TG_TEL, P ^drug _{2‐way ANOVA} = 0.018 for SD_VEH versus SD_TEL). (F) Food efficiency ratio (FER; P ^drug _{2‐way ANOVA} = 0.005). (G) Hypothalamic POMC expression, (H) mean water intake per week. Means ± SEM are shown in line graphs; means and upper and lower quartiles as boxplots. Whiskers indicate variability between the 10th and 90th percentile. Group sizes (n) are shown in the figures. Data lost in leptin TG and HDL was due to limited blood volume; data lost in POMC was due to technical complications. *P < 0.05 versus appropriate vehicle‐treated rats after Bonferroni's post hoc test.

Influence of CD feeding on TGR(ASrAOGEN) with a brain‐specific angiotensinogen deficiency

Weight regulation and energy balance

Gain in body weight differed in a strain‐dependent manner. Because femurs, body lengths and kidneys were smaller in TGR(ASrAOGEN) than in SD rats (Table 1), growth of TGR(ASrAOGEN) rats is thought to be less than that of SD rats. However, SD rats clearly became obese under CD feeding because BMI, girth and fat mass also increased (Table 1, Figures 1A, S2A). Hence, plasma levels of leptin, TG and also adiponectin increased, while HDL decreased (Table 1, Figures 1B–D, S1C). In contrast, TGR(ASrAOGEN) did not become obese despite CD feeding (Table 1, Figures 1A, S2A). Plasma levels of lipids and leptin remained unaltered (Figure 1B–D), while adiponectin also increased (Figure S1C). Plasma AngII levels of chow‐fed SD rats were 9.1 ± 0.8 pmol·L⁻¹ and were not influenced by CD feeding or by strain (data not shown). Weights of livers were higher in SD than in TGR(ASrAOGEN) rats. CD feeding further increased liver weights selectively in SD rats while left ventricular weight as enhanced in both strains (Table 1). The level of steatosis was also higher in these rats (Figure S3A). The energy intake was higher when rats were fed with CD, which was more distinct in SD than in the TGR(ASrAOGEN) strain (ANOVA P‐values indicating strain differences for diet <0.0001 and time <0.0001) (Figures 1E, 2E, S4A). However, CD feeding only increased energy intake in both strains during the dark period (Figure 2E). In TGR(ASrAOGEN) rats, the food efficiency ratio was lower than in SD rats, and furthermore, this parameter was reduced when TGR(ASrAOGEN) rats were fed with CD (Figure 1I).

Figure 2.

Energy expenditure (A) (P ^diet _{2‐way ANOVA} for EE_dark = 0.0013), respiratory ratio (B) (P ^diet _{2‐way ANOVA} = 0.025 , P ^drug _{2‐way ANOVA} < 0.0001), locomotion (C) (P ^diet _{2‐way ANOVA} for locomotion_dark = 0.006) and energy intake (D) (P ^diet _{2‐way ANOVA} for energy intake_dark < 0.0001) considering light and dark periods during indirect calorimetry measurements of TGR(ASrAOGEN) and SD rats after control or CD feeding (TG_chow n = 12, SD_chow n = 10; TG_CD n = 12, SD_CD n = 13). Animals were housed for 3 days in calorimetry cages, but only the data from the third day are shown. Means and upper and lower quartiles are shown as boxplots. Whiskers indicate variability between the 10th and 90th percentile. *P < 0.05 versus appropriate control‐fed rats after Bonferroni's post hoc test.

In CD‐fed, but not chow‐fed SD rats, final plasma leptin concentrations positively correlated with food intake, which indicates leptin resistance (Figure S5A). The hypothalamic mRNA levels of the anorexigenic peptide pro‐opiomelanocortin (POMC) were increased only in the TGR(ASrAOGEN) rats (Figure 1G). Water intake was lower in TGR(ASrAOGEN) rats only but was reduced by CD feeding in both strains (Figure 1H). mRNA levels of leptin receptor in hypothalamus of chow‐fed SD rats were 1.0 ± 0.7 × 10⁴ copies·ng RNA⁻¹ and not influenced by strain or diet (data not shown). Hypothalamic mRNA levels of glial fibrillary acidic protein (GFAP) differed strain dependently (SD_CON: 18.7 ± 1.6 × 10⁵, SD_CD: 19.7 ± 1.1 × 10⁵, TG_CON: 14.3 ± 0.6 × 10⁵, TG_CD: 15.4 ± 0.7 × 10⁵ copies·ng RNA⁻¹, P = 0.0002). mRNA levels of AT_1B receptor were reduced in the hypothalami of TGR(ASrAOGEN) rats, but those of AT_1A and AT₂ receptors did not differ between strains (Figure S6A). The energy expenditure (EE) varied between the strains and increased particularly in CD‐fed TGR(ASrAOGEN) rats during the dark period (Figures 2A, S4A; P ^diet _{2‐way ANOVA} for EE_dark is 0.0013). Moreover, the respiratory exchange rate (RER) was strain‐dependently lower in TGR(ASrAOGEN) rats and selectively reduced during CD feeding within the light period (Figures 2B, S4A; P ^diet _{2‐way ANOVA} for RR_light is <0.0001). There was less locomotion in the TGR(ASrAOGEN) strain, but it increased in particular during the dark period when they were fed with CD (Figures 2C, S4A; P ^diet _{2‐way ANOVA} for locomotion_dark is 0.006).

Glucose control

Because of CD feeding, baseline levels of both glucose and insulin and the HOMA index were selectively increased in SD, but not in TGR(ASrAOGEN) rats (Figure 3A–D). The glucose response in OGTT was higher in TGR(ASrAOGEN) rats (Figure 3E–G). In contrast, the insulin response in OGTT was clearly increased in CD‐fed SD rats (Figure 3H–J), indicating insulin resistance, which was also suggested from the baseline levels.

Figure 3.

CD feeding impaired glucose utilization in SD, but not in TGR(ASrAOGEN) rats (TG_chow n = 12, SD_chow n = 10, TG_CD n = 12, SD_CD n = 13). (A) Baseline fasting glucose levels (P ^diet _{2‐way ANOVA} = 0.0069), (B) baseline fasting insulin levels (P ^diet _{2‐way ANOVA} = 0.008), (C) HOMA index (P ^diet _{2‐way ANOVA} = 0.003), (D) baseline fasting glucagon levels (P ^diet _{2‐way ANOVA} = 0.003), (E) plasma glucose levels in response to an oral glucose tolerance test (1 g glucose·kg_bw ⁻¹), AUC (F) (P ^diet _{2‐way ANOVA} = 0.017) and C_max (G) values of plasma glucose time curves. (H) Plasma insulin levels in OGTT, AUC (I) P ^diet _{2‐way ANOVA} < 0.0001) and C_max (J) (P ^diet _{2‐way ANOVA} = 0.018) values of plasma glucose time curves. Means ± SEM are shown in line graphs; means and upper and lower quartiles as boxplots. Whiskers indicate variability between the 10th and 90th percentile. *P < 0.05 versus appropriate control‐fed rats after Bonferroni's post hoc test. AUC, area under control.

Results of treating CD‐fed SD and TGR(ASrAOGEN) rats with telmisartan

Weight regulation and energy balance

In accordance with the first part of our study, body weights and plasma levels of leptin and triglycerides in the TGR(ASrAOGEN) rats were clearly lower (Table 2 and Figure 4). Obesity was markedly improved when SD rats were treated with telmisartan in parallel to CD feeding because weight gain, BMI, girth and fat mass were diminished (Table 2, Figures 4A, S2B). Liver weights were strain‐dependently lower in TGR(ASrAOGEN) than in SD rats. Telmisartan lowered left ventricular weight in both strains, too. An overall, strain‐independent telmisartan effect on liver weight was found (P = 0.0312, Table 2). Steatosis was lower in TGR(ASrAOGEN) rats but remained strain independently unaffected by telmisartan treatment (Figure S3B). Macrophage infiltration into fat tissue was reduced when CD‐fed SD rats were treated with telmisartan, but this was never observed in TGR(ASrAOGEN) rats (Figure S8). Moreover, leptin and triglycerides decreased, while HDL (Figure 4B–D) increased. In contrast, markedly fewer telmisartan effects on weight gain, girth, fat mass or lipid levels were observed in the TGR(ASrAOGEN) strain. (Table 2, Figure 5B–D). An overall effect of telmisartan treatment on adiponectin was detected for both strains (P = 0.0142, Figure S1D). The number of adipocytes per g fat mass was clearly higher in epidymial fat of TGR(ASrAOGEN) rats and particularly after telmisartan treatment, but the cell area was halved when compared with adipocytes from SD rats. Thus, size and amount of adipocytes correlated negatively (Figure S7). Size of adipocytes tended to be strain independently reduced when animals were treated with telmisartan (P = 0.08) (Figure S7D,F,G). During the course of telmisartan treatment, energy intake lessened in both SD and TGR(ASrAOGEN) rats (Figure 4E). The FER was again lower in TGR(ASrAOGEN) and reduced by telmisartan only in SD rats (Figure 4E) Hypothalamic mRNA levels of POMC were found not to be decreased by telmisartan (Figure 5G). Telmisartan did not affect water intake either (Figure 4H). mRNA levels of leptin receptor in hypothalamus were not influenced by strain or telmisartan (data not shown). In accordance with the first part of our study, hypothalamic mRNA levels of GFAP were lower in TGR(ASrAOGEN) (P < 0.0001) and, moreover, were reduced in both strains after telmisartan treatment (P = 0.03) (SD_VEH: 18.0 ± 1.2 × 10⁵, SD_TEL: 15.6 ± 0.9 × 10⁵, TG_CON: 12.1 ± 0.6 × 10⁵, TG_CD: 10.7 ± 0.7 × 10⁵ copies·ng RNA⁻¹, P = 0.0002). Levels of mRNA for AT_1B receptors were reduced in the hypothalami of TGR(ASrAOGEN) rats, while those of AT_1A and AT₂ receptors did not differ (Figure S6B). GFAP was reduced in TGR(ASrAOGEN) rats, while AT_1A and AT_1B receptors did not differ (Figure S6B). We also tested whether leptin sensitivity might be altered by telmisartan. In response to exogenous leptin applications, body weight decreased in SD rats when treated with telmisartan, which indicates that leptin sensitivity was maintained (Figure 5). Food intake only tended to be decreased in these rats. In TGR(ASrAOGEN) rats, telmisartan did not increase responses to leptin (Figure 5). Energy expenditure was also found in this part of the study to be higher in TGR(ASrAOGEN) than in SD rats without being further influenced by telmisartan in either strain (Figure 6A) although less locomotion was observed in this strain particularly during the light period (Figure 6C). However, treatment with telmisartan decreased RER in SD rats during the dark period and in TGR(ASrAOGEN) rats during the light phase (Figure 6B).

Figure 5.

Leptin sensitivity in CD‐fed TGR(ASrAOGEN) and SD rats after telmisartan (TEL) treatment was determined by repeated leptin injections over 2 days. The grey bars indicate the dark periods. (A/B) Gain in body weight after exogenous leptin was selectively attenuated by telmisartan in TGR(ASrAOGEN) compared with SD rats (P ^drug _{2‐way ANOVA} = 0.025). (C/D) The energy intake after exogenous leptin was lower in TGR(ASrAOGEN) but only tended to be reduced by telmisartan (P ^drug _{2‐way ANOVA} = 0.066). Means ± SEM are shown in line graphs; means and upper and lower quartiles as boxplots. Whiskers indicate variability between the 10th and 90th percentile. Group sizes: SD_VEH n = 12, SD_TEL n = 14, TG_VEH n = 12, TG_TEL n = 16; technical complications in leptin applications led to reduced group sizes. *P < 0.05 versus appropriate vehicle‐treated rats after Bonferroni's post hoc test.

Figure 6.

Energy expenditure (A), respiratory ratio (B) (P ^drug _{2‐way ANOVA} for RR_light = 0.009 and P ^drug _{2‐way ANOVA} for RR_dark = 0.005), locomotion (C) and energy intake (D) (P ^drug _{2‐way ANOVA} for energy intake_light = 0.035 and P ^drug _{2‐way ANOVA} for energy intake_dark = 0.08) considering light and dark periods during indirect calorimetry measurements. Animals were housed for 3 days in calorimetry cages but only the data from the third day are shown. Means and upper and lower quartiles are shown as boxplots. Whiskers indicate variability between the 10th and 90th percentile. Group sizes: SD_VEH n = 15, SD_TEL n = 15, TG_VEH n = 17, TG_TEL n = 19; technical complications in calorimetry led to reduced group sizes. *P < 0.05 versus appropriate vehicle‐treated rats after Bonferroni's post hoc test.

Glucose control

Strain differences with lower insulin levels in the TGR(ASrAOGEN) strain and attenuated glucose and insulin responses in OGTT were also seen in this part of our study (Figure 7). Telmisartan did not affect fasting glucose in either strain, but insulin was reduced in SD rats (Figure 7B). Telmisartan also lowered the glucose response in OGTT in SD rats, while the insulin response remained constant. In TGR(ASrAOGEN) rats, telmisartan clearly diminished the insulin response in OGTT, while the glucose response only tended to be reduced (Figure 7D–I). To further determine glucose control, uptake studies were performed on isolated adipocytes. Glucose uptake was clearly higher in adipocytes (originating from 1 g fat) of TGR(ASrAOGEN) rats, which was further enhanced in telmisartan‐treated TGR(ASrAOGEN) rats (Figure 8A). Considering the number of adipocytes in epidymial fat and the total epidymial fat mass, the glucose uptake per adipocyte and per total fat mass, respectively, was similar among all groups (Figure 8B–C).

Figure 7.

Influence of telmisartan (TEL) on in vivo glucose control in CD‐fed SD rats and TGR(ASrAOGEN) rats (=TG). (A) Baseline fasting glucose levels, (B) baseline fasting insulin levels (P^drug _{2‐way ANOVA} = 0.006), (C) HOMA index (P ^drug _{2‐way ANOVA} = 0.053), (D) plasma glucose levels in response to an oral glucose tolerance test (1 g glucose·kg_bw ⁻¹), AUC (E) (P ^drug _{2‐way ANOVA} = 0.040) and C_max (F) values of plasma glucose time curves. (G) Plasma insulin levels in OGTT, AUC (H) P ^drug _{2‐way ANOVA} = 0.044) and C_max (I) P ^drug _{2‐way ANOVA} = 0.018) values of plasma glucose time curves. Means ± SEM are shown in line graphs; means and upper and lower quartiles as boxplots. Whiskers indicate variability between the 10th and 90th percentile. Group sizes: SD_VEH n = 13, SD_TEL n = 14, TG_VEH n = 17, TG_TEL n = 18; reduced group sizes were due to death of animals prior to OGTT or due to technical complications in glucose applications.*P < 0.05 versus appropriate vehicle‐treated rats after Bonferroni's post hoc test.

Figure 8.

Influence of telmisartan (TEL) on ex vivo glucose control in CD‐fed SD rats and TGR(ASrAOGEN) rats (=TG). Glucose uptake in epidymial fat related to 1 g tissue (A) P ^drug _{2‐way ANOVA} = 0.010), adipocyte (B) or total epidymial fat mass (C). Means and upper and lower quartiles are shown as boxplots. Whiskers indicate variability between the 10th and 90th percentile. Group sizes: SD_VEH n = 14, SD_TEL n = 12, TG_VEH n = 11, TG_TEL n = 13; because this ex vivo assay was not yet established at the beginning of the study, we were not able to include all animals. *P < 0.05 versus appropriate vehicle‐treated rats after Bonferroni's post hoc test.

Discussion

The main finding of our study is that selective inhibition of the brain RAS by genetic means prevents CD‐induced obesity, supporting a role of the brain RAS in metabolic regulation. In line with the first publication on TGR(ASrAOGEN) rats (Schinke et al., 1999), we also observed here that baseline levels of AngII were indeed similar between TGR(ASrAOGEN) and SD rats, but that drinking responses were markedly reduced in the transgenic animals, which confirms the biochemical and functional down‐regulation of brain angiotensinogen in the TGR(ASrAOGEN) strain. The AngII‐dependent dipsogenic action was related to an AT_1B receptor‐mediated mechanism (Davisson et al., 2000). In agreement with others, we also found that hypothalamic expression of AT_1A receptors exceeded that of AT_1B receptors (Jöhren et al., 1995; Davisson et al., 2000), but expression was lower in TGR(ASrAOGEN) rats, which did not reflect increased AngII binding as others have shown (Monti et al., 2001). Thus, we assume that hypothalamic levels of mRNA for AT_1B receptors only reflect expression of these receptors, to a minor degree. Although increased water intake in response to intravenous AngII administration was blocked by intracerebroventricular AT₁ receptor blockade (Hohle et al., 1995), drinking responses after telmisartan were not reduced, which was also found in another recent study (Schuchard et al., 2015).

Effects on body weight

We have found that body length, femur, livers and kidneys are smaller in the TGR(ASrAOGEN) strain, suggesting that growth in general is reduced in transgenic animals, which is in line with the findings of other authors (Kasper et al., 2005; Groban et al., 2012). However, the strain‐dependent decrease in BMI, girth and visceral fat mass also indicates that not only is animal growth impaired but also that development of obesity can be prevented particularly in TGR(ASrAOGEN) rats (especially when fed with CD), which was associated with maintenance of normal lipid profiles and glucose control. Less weight gain was also found in angiotensinogen‐deficient mice [showing no detectable plasma levels of angiotensinogen and angiotensin I (AngI) (Tanimoto et al., 1994)] compared with WT controls although their intake of a high‐fat diet was not lower (Massiera et al., 2001a, 2001b). In contrast, specific overexpression of angiotensinogen in adipose tissue induced obesity (Massiera et al., 2001a). Considering that angiotensinogen is the unique substrate of renin and the precursor of AngII after the initial product, AngI, is metabilised by ACE, it seems confusing that body weight increased when angiotensinogen was overexpressed and reduced when it is deficient because, on the one hand, AngII induced weight loss after peripheral (Cabassi et al., 2005; Song et al., 2005; Zhang et al., 2009; Müller‐Fielitz et al., 2012b; Müller‐Fielitz and Raasch, 2013; Tabony et al., 2014) and brain‐specific administration of AngII (de Kloet et al., 2011) and, on the other hand, plasma levels of angiotensinogen or AngII positively correlated with body weight in obesity (Engeli et al., 2005; Harte et al., 2005). Addressing this discrepancy, properly balanced angiotensinogen levels in adipocytes must be maintained for normal adipose tissue growth and weight gain. Indeed, overexpression of angiotensinogen in adipose tissue induced obesity, whereas restricted angiotensinogen expression in adipose tissue was more likely to reduce body weight (Massiera et al., 2001a).

Regarding the underlying mechanisms of preventing obesity, we have shown in this study that energy intake was lower throughout the entire observation period of 12 weeks in the TGR(ASrAOGEN) strain. In contrast, others had observed that body weight‐related food intake was increased in TGR(AsrAOGEN) rats despite weight loss (Kasper et al., 2005). This inconsistency was thought to be not simply due to a suppression of appetite and was interpreted as the result of a centrally mediated long‐term effect of AngII on overall body metabolism' (Kasper et al., 2005). Moreover, we found that FER is lower in transgenic rats and particularly after CD feeding, thus suggesting that food is less sufficiently converted to body weight but more prone to be utilized as energy. Indeed, energy expenditure was higher in the TGR(ASrAOGEN) strain particularly when fed with CD. Increased locomotor activity, serving as a parameter for energy expenditure, was also detected in angiotensinogen‐deficient mice to explain their reduced body weight although the chow intake was similar to wild‐type controls (Massiera et al., 2001b). RER was also lower in TGR(ASrOGEN) than in SD rats, thus indicating higher fat burning. This finding also corresponds to our observation that the fat mass is reduced in these rats.

Increased action of leptin in the arcuate nucleus (ARC) inhibits the neuropeptide Y /agouti‐related protein anabolic pathway and stimulates the proopiomelanocortin/cocaine‐ and amphetamine‐regulated transcript (POMC/CART) catabolic pathway. Downstream to the ARC, the paraventricular nucleus produces anorexigenic peptides such as corticotrophin‐releasing hormone, and the lateral hypothalamus and perifornical area secrete the orexigenic substances orexin‐A and melanin‐concentrating hormone. Here, we observed higher hypothalamic mRNA levels of POMC in TRG(ASrAOGEN) than in SD rats, which is in line with the lower energy intake in this strain. Up to now, we only have little evidence for a direct mechanism of how low brain angiotensinogen levels may increase POMC. However, we did observe that leptin sensitivity was better in transgenic than in control rats, which may be influenced by the lower triglyceride levels of TGR(ASrAOGEN)rats. On the one hand, triglycerides are an important cause of leptin resistance as mediated by impaired transport across the BBB, and on the other, decreasing triglycerides may potentiate the anorectic effect of leptin by enhancing leptin transport across the BBB (Banks et al., 2004). In vitro experiments on microvessel endothelial cells supporting the notion that AngII directly modulates BBB endothelial cell function by altering both transcellular and paracellular permeability (Fleegal‐DeMotta et al., 2009). Decreases in hypothalamic GFAP may also contribute to the weight loss of TGR(ASrAOGEN) rats, an effect which was also observed in angiotensinogen‐deficient mice (Kakinuma et al., 1998) because obesity was related to enhanced GFAP immunoreactivity or GFAP protein as an indicator for astrogliosis in the CNS (Hsuchou et al., 2009; Buckman et al., 2013; Tomassoni et al., 2013).

AT₂ receptor‐deficient mice showed (i) increased body weight (Gross et al., 2000) and (ii) lower resting body temperature and locomotor activity (Hein et al., 1995; Ichiki et al., 1995). Considering these findings, it is permissible to speculate as to whether the imbalance between energy intake and energy expenditure boosting obesity in SD rats when AngII is present is related to AT₂ receptors. We did demonstrate the presence of AT₂ receptor mRNA in the hypothalamus, but the levels were not regulated by strain, diet or telmisartan (Figure S6). Loss of AT₂ receptor expression was found to be sufficient to rescue obesity induced by high‐fat feeding (Yvan‐Charvet et al., 2005) or adipose tissue angiotensinogen overexpression (Yvan‐Charvet et al., 2009), which does strengthen the necessary role of AT₂ receptors for the onset of obesity, but can only support our hypothesis that a CNS‐mediated mechanism is involved in regulating body weight in diet‐induced rat obesity to a minor degree. Nevertheless, specifically activating AT₂ receptors with the AT₂ receptor agonist C21, attenuated rebound weight gain after food deprivation and modulated dopamine signalling, while deletion of AT₂ receptors enhanced rebound weight via excessive food intake by increasing dopamine levels in the striatum (Nakaoka et al., 2015). These findings more strongly support the idea that a brain‐related and AT₂ receptor‐dependent mechanism may contribute to diet‐induced obesity.

By treating TGR(ASrAOGEN) rats with telmisartan, we then aimed to answer the question of whether the prevention of weight gain by AT₁ receptor antagonists is likely to be related to a mechanism involving the CNS. As shown here, treating SD rats with telmisartan hampered development of obesity and decreased energy intake. These effects were also observed in TGR(ASrAOGN) rats. Hence, mechanisms involving AT₂ receptors, as discussed previously, or the Ang(1‐7)/Mas receptor pathway, as demonstrated recently (Schuchard et al., 2015) do not appear to be likely in transgenic rats because AngII cannot be up‐regulated and redirected to AT₂ receptors or Ang(1‐7) up‐regulated, which then acts via Mas, because of the angiotensinogen deficiency in these rats. Nonetheless, we were able to demonstrate a robust up‐regulation of POMC mRNA in TGR(ASrAOGEN) rats in parallel to the decreased food intake. However, findings on POMC do seem to be rather counterintuitive because telmisartan had no effect on hypothalamic mRNA levels in SD or TGR(ASrAOGEN) rats. In our recent paper, we did not find POMC to be up‐regulated after telmisartan (Schuchard et al., 2015), which in fact confirms the finding of the present study but unfortunately does not provide a satisfactory answer as to why (an‐)orexigenic peptides were not regulated by telmisartan despite alterations in food intake. Treating SD rats with telmisartan additionally normalized plasma levels of leptin, TG and HDL, restored leptin sensitivity and decreased energy intake and RER during the dark period, all of which is in agreement with our previous findings (Miesel et al., 2012; Müller‐Fielitz et al., 2012a; Müller‐Fielitz et al., 2014; Müller‐Fielitz et al., 2015). In contrast, TGR(ASrAOGEN) rats were influenced less by telmisartan, which strengthens the hypothesis of a brain‐dependent pathway in weight regulation. A small but significant effect of telmisartan on weight gain even in TGR(ASrAOGEN) rats suggests that peripheral pathways seem to be involved in addition to the central mechanism. In this regard, weight reduction may be related to reduced growth of adipocytes (Zorad et al., 2006; Müller‐Fielitz et al., 2012a) because effects of telmisartan treatment on adipocyte size were seen in this study when data were analysed with a a one‐tailed statistical test (P = 0.043). Obesity is widely accepted to be associated with chronic low‐grade inflammation in peripheral organs, accounting for comorbidities such as insulin resistance and cardiovascular diseases. Hence, anti‐inflammatory effects of AT₁ receptor antagonists may contribute to weight loss after treatment with these agents(Cole et al., 2010; Guo et al., 2011). Here, we found evidence that telmisartan reduced macrophage infiltration in fat tissue in SD rats, which, however, was not observed in the TGR(ASrAOGEN) strain. Obesity additionally promotes inflammation in the CNS because proinflammatory cytokines (De Souza et al., 2005) and GFAP immunoreactivity or GFAP protein (Hsuchou et al., 2009; Buckman et al., 2013; Tomassoni et al., 2013) increased in brain. Hypothalamic mRNA levels of GFAP were reduced by telmisartan, which is in line with findings for losartan. Losartan normalized GFAP‐positive hippocampal areas, which were increased compared with wild‐type mice in an Alzheimer's disease transgenic mice model (Ongali et al., 2014).

Effects on glucose control

Because obesity is hallmark characteristic feature of the metabolic syndrome and predisposes patients to developing type 2 diabetes mellitus (T2DM) (Sell et al., 2012), we investigated as a secondary parameter whether insulin resistance can be hampered in the TGR(ASrAOGEN) strain, in spite of feeding rats with CD and whether telmisartan treatment would have an additional effect on glucose control in this model. The adipose tissue is an important endocrine organ: here, leptin is released that inhibits insulin secretion from pancreatic beta cells (Emilsson et al., 1997), and adiponectin has the opposite effect (Ghoshal and Bhattacharyya, 2015). We observed in our study that plasma adiponectin was higher in TGR(ASrAOGEN) rats before the feeding period and increased during CD feeding in both strains, whereas baseline leptin was similar between TGR(ASrAOGEN) and SD rats, and selectively increased when SD rats were fed with CD. However, baseline insulin and insulin response in OGTT were similar in chow‐fed TGR(ASrAOGEN) and SD rats, which is in line with the findings of others (Massiera et al., 2001b; Kasper et al., 2005) and indicates that insulin is not necessarily regulated under normative conditions in the TGR(ASrAOGEN) strain even when adiponectin is increased. However, when animals were fed with CD, SD rats became obese and insulin resistant and had higher adiponectin and leptin levels; none of which was seen in TGR(ASrAOGEN) rats except for the increase in adiponectin. This allowed us to conclude that the better glucose control, as seen in TGR(ASrAOGEN) rats, may not simply be related to alterations in the leptin‐dependent and/or adiponectin‐dependent insulin release. We further asked whether glucose transport in adipocytes might be different between TGR(ASrAOGEN) and SD rats because glucose uptake in adipose tissue is suggested to be down‐regulated by leptin (Muller et al., 1997) and enhanced by adiponectin (Vu et al., 2007). When related to similar amounts of fat tissue, it seems likely that glucose uptake into adipocytes is higher in TGR(ASrAOGEN) than in SD rats, which may support the in vivo findings on adiponectin (Vu et al., 2007). However, due to the reduced cell size and increased number of adipocytes, glucose uptake per single adipocyte did not differ between the stains, which gives rise to doubt about whether this mechanism really does help to improve the control of glucose levels in TGR(ASrAOGEN) rats. The expansion of the fat mass in obesity goes hand in hand with infiltration of proinflammatory immune cells into adipose tissue, causing chronic, low‐grade inflammation, and with non‐alcoholic steatohepatitis. These effects were demonstrated to contribute to development of T2DM (Sell et al., 2012; Birkenfeld and Shulman, 2014). The relevance of both pathomechanisms was shown in SD rats because obesity and T2DM after CD feeding coincidently occurred with both macrophage infiltration as a sign of adipose tissue inflammation and with hepatic steatosis, all of which was not observed in TGR(ASrOGEN) rats. This strengthens the notion that the improved glucose control of these rat strains may be linked to their feature not to develop inflammation.

In agreement with numerous experimental and clinical findings, we also observed here that telmisartan ameliorates glucose utilization, particularly in CD‐fed SD. This has been broadly discussed to be related to the potency of telmisartan to stimulate PPARγ (Kintscher et al., 2008). We only have poor evidence that this PPARγ‐dependent mechanism contributes to improved glucose homeostasis of CD‐fed SD rats because adiponectin, which is one of the target genes for PPARγ, was not up‐regulated in these rats, which is in line with findings also showing that improved insulin sensitivity after treatment with AT₁ receptor antagonists occurs independently of PPARγ regulation (Müller‐Fielitz et al., 2012a). Thus, we assume that the improved glucose utilization after AT₁ receptor blockade is related more to the anti‐inflammatory properties of these antagonists, which was demonstrated in this study by lower macrophage infiltration into adipose tissue and which was suggested to further protect islet function (Cole et al., 2010). Moreover, improved glucose homeostasis after AT₁ receptor blockade may also be attributed to normalized reactivity of the stress‐axis because AngII increased stress responses and concurrently worsened glucose homeostasis in obese rats (Müller et al., 2007; Müller‐Fielitz and Raasch, 2013), whereas telmisartan lowered both corticosterone and glucose levels after stress (Raasch et al., 2006; Miesel et al., 2012). However, we did not generate any data in the present study supporting this probable mechanism.

In summary, we clearly show in this study that rats with a selective brain angiotensinogen deficiency are protected from developing diet‐induced obesity and that the reduced weight gain after treatment with AT₁ receptor antagonists can at least partly be attributed to a brain‐related mechanism.

Author contributions

M.W., J.S., I.S., F.M.V., J.B., C.T., M.B. and W.R. performed the research; W.R., M.W. and M.B. designed the research study. W.R. and M.W. analysed the data, and W.R., M.W. and M.B. wrote the paper.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organizations engaged with supporting research.

Supporting information

Figure S1 Plasma adiponectin levels of SD and TGR(AsOGEN) (=TG) rats before (A) or after (C, P^diet _{2‐way ANOVA} < 0.0001) CD feeding. Plasma adiponectin of CD‐fed SD rats and TGR(AsOGEN) rats before (B) or after (D, P^drug _{2‐way ANOVA} = 0.014) TEL treatment. Means and upper and lower quartiles are shown as boxplots. Whiskers indicate variability between the 10^th and 90^th percentile. Group sizes (given in the figures) are lower than indicated in Methods since we did not obtain blood from all animals before start. *P < 0.05 vs. appropriate control‐fed rats after Bonferroni's post hoc test.

Figure S2 Typical MRI images obtained by transverse T1‐weighted turbo spin‐echo MRI of (A) SD and TGR(AsOGEN) (=TG) rats, which were fed with chow or CD or of (B) CD‐fed SD and TGR(AsOGEN) (=TG) rats, which were treated with telmisartan (TEL) or vehicle (VEH). The abundance of visceral (F) and subcutaneous (G) fat deposits was quantified by computer‐assisted planimetry and the results are shown in Tables 1, 2.

Figure S3 Histological findings of livers from control or CD‐fed SD and TGR(AsOGEN)(=TG) rats (upper panel; P^diet _{2‐way ANOVA} < 0.003) and of CD‐fed animals with respect to TEL treatment (lower panel). Tissue specimens were evaluated in a blinded manner based on and scored for hepatocytes with steatosis. Means and upper and lower quartiles are shown as boxplots. Whiskers indicate variability between the 10^th and 90^th percentile. Group sizes (given in the figures) are lower than indicated in Methods because of unexpected deaths of few rats during the study and technical problems, * P < 0.05 vs. appropriate control‐fed rats after Bonferroni's post hoc test.

Figure S4 Energy expenditure (EE), respiratory ratio (RER), locomotion, and energy intake during indirect calorimetry measurements of (A) SD and TGR(AsOGEN) (=TG) rats, which were fed with chow or CD or of (B) CD‐fed SD rats and TGR(AsOGEN) (=TG) rats which were treated with telmisartan (TEL) or vehicle (VEH). Animals were housed for 3 days in calorimetry cages, but only the data from the 3^RD day are shown. Mean values during light and dark periods for EE, RER, and locomotion as well as total energy intake are shown in Figures 2 and 7, respectively considering light and dark periods.

Figure S5 A: Correlation between plasma leptin concentrations and energy intake at week 12 of SD rats and TGR(AsOGEN) (=TG) rats when fed with standard chow or CD; B: Correlation between plasma leptin and energy intake at week 12 of SD rats and TGR(AsOGEN) rats when fed with CD and simultaneously treated with telmisartan (TEL) or vehicle (VEH).

Figure S6 mRNA levels of different subtypes of AT receptors in hypothalami of rats from protocol 1 (SD_chow n = 10, SD_CD n = 13, TG_chow n = 12, TG_CD n = 12) and protocol 2 (SD_VEH n = 12, SD_TEL n = 12, TG_VEH n = 16, TG_TEL n = 15). Means and upper and lower quartiles are shown as boxplots. Whiskers indicate variability between the 10^th and 90^th percentile. Group sizes (given in the figures) are lower than indicated in Methods because of unexpected deaths of few rats during the study and technical problems.

Figure S7 Epidymial fat mass and characteristics of isolated adipocytes. A: Total fat mass CD‐fed SD rats and TGR(AsOGEN) rats with respect to telmisartan (TEL) treatment (P^drug _{2‐way ANOVA} = 0.005); B: Cell number per gram fat was microscopically quantified (P^drug _{2‐way ANOVA} < 0.0001); C: extrapolated cell number in total epidymial fat. D: The cell area of adipocytes was determined in slices after HE staining by counting 10 cells per animal in a blinded manner (P^drug _{2‐way ANOVA} = 0.008). E: Correlation between number and size of adipocytes. F: Microscopic view of adipocytes using the Fuchs‐Rosenthal chamber for quantification (the area of a small square is 0.0625 mm²). G: Microscopic view of fat slices after HE staining for quantifying the area of adipocytes. Means and upper and lower quartiles are shown as boxplots. Whiskers indicate variability between the 10^th and 90^th percentile. Group sizes: SD_VEH n = 14, SD_TEL n = 12, TG_VEH n = 11, TG_TEL n = 13; since this ex vivo assay was not yet established at the beginning of the study we were not able to include all animals. * P < 0.05 vs. appropriate vehicle‐treated rats after Bonferroni's post hoc test.

Figure S8 Histological findings of fat tissue from CD‐fed SD and TGR(AsOGEN) (=TG) rats, after telmisartan (TEL) treatment (lower panel). Tissue specimens were evaluated in a blinded manner based on and scored for animals in which macrophage infiltration into fat tissue was observed; Group sizes: SD_VEH n = 14, SD_TEL n = 13, TG_VEH n = 15, TG_TEL n = 15; * P < 0.05 vs. appropriate control‐fed rats (Fisher's exact test).

Supporting info item

Winkler, M. , Schuchard, J. , Stölting, I. , Vogt, F. M. , Barkhausen, J. , Thorns, C. , Bader, M. , and Raasch, W. (2016) The brain renin‐angiotensin system plays a crucial role in regulating body weight in diet‐induced obesity in rats. British Journal of Pharmacology, 173: 1602–1617. doi: 10.1111/bph.13461.