Jürgen Janke

Weight Loss and the Renin-Angiotensin-Aldosterone System

The renin-angiotensin-aldosterone system has been causally implicated in obesity-associated hypertension. We studied the influence of obesity and weight reduction on the circulating and adipose tissue renin-angiotensin-aldosterone system in menopausal women. Blood samples were analyzed for angiotensinogen, renin, aldosterone, angiotensin-converting enzyme activity, and angiotensin II. In adipose tissue biopsy samples, we analyzed angiotensinogen, renin, renin-receptor, angiotensin-converting enzyme, and angiotensin II type-1 receptor gene expression. Obese women (n=19) had higher circulating angiotensinogen, renin, aldosterone, and angiotensin-converting enzyme than lean women (n=19), and lower angiotensinogen gene expression in adipose tissue. Seventeen women successfully participated in a weight reduction protocol over 13 weeks to reduce daily caloric intake by 600 kcal. Body weight was reduced by −5%, as were angiotensinogen levels by −27%, renin by −43%, aldosterone by −31%, angiotensin-converting enzyme activity by −12%, and angiotensinogen expression by −20% in adipose tissue (all P<0.05). The plasma angiotensinogen decrease was highly correlated with the waist circumference decline (r=0.74; P<0.001). Weight and renin-angiotensin-aldosterone system reductions were accompanied by a −7-mm Hg reduced systolic ambulatory blood pressure. These data suggest that a 5% reduction in body weight can lead to a meaningfully reduced renin-angiotensin-aldosterone system in plasma and adipose tissue, which may contribute to the reduced blood pressure.

Retinol-Binding Protein 4 in Human Obesity

Studies in mice suggest that adipocytes serve as glucose sensors and regulate systemic glucose metabolism through release of serum retinol-binding protein 4 (RBP4). This model has not been validated in humans. RBP4 was highly expressed in isolated mature human adipocytes and secreted by differentiating human adipocytes. In contrast to the animal data, RBP4 mRNA was downregulated in subcutaneous adipose tissue of obese women, and circulating RBP4 concentrations were similar in normal weight, overweight, and obese women (n = 74). RBP4 was positively correlated with GLUT4 expression in adipose tissue, independent of any obesity-associated variable. Five percent weight loss slightly decreased adipose RBP4 expression but did not influence circulating RBP4. In another set of experiments, we stratified patients (n = 14) by low or high basal fasting interstitial glucose concentrations, as determined by the microdialysis technique. Venous glucose concentrations were similar throughout oral glucose tolerance testing, and basal RBP4 expression in adipose tissue and serum RBP4 concentrations were similar in the groups with higher and lower interstitial glucose levels. Our findings point to profound differences between rodents and humans in the regulation of adipose or circulating RBP4 and challenge the notion that glucose uptake by adipocytes has a dominant role in the regulation of RBP4.

AT1 Receptor Agonistic Antibodies From Preeclamptic Patients Stimulate NADPH Oxidase

Background—We recently identified agonistic autoantibodies directed against the angiotensin AT1 receptor (AT1-AA) in the plasma of preeclamptic women. To elucidate their role further, we studied the effects of AT1-AA on reactive oxygen species (ROS), NADPH oxidase expression, and nuclear factor-&kgr;B (NF-&kgr;B) activation. Methods and Results—We investigated human vascular smooth muscle cells (VSMC) and trophoblasts, as well as placentas. AT1-AA were isolated from sera of preeclamptic women. Angiotensin II (Ang II) and AT1-AA increased ROS production and the NADPH oxidase components, p22, p47, and p67 phox in Western blotting. We next tested if AT1-AA lead to NF-&kgr;B activation in VSMC and trophoblasts. AT1-AA activated NF-&kgr;B. Inhibitor-&kgr;B&agr; (I-&kgr;B&agr;) expression was reduced in response to AT1-AA. AT1 receptor blockade with losartan, diphenylene iodonium, tiron, and antisense against p22 phox all reduced ROS production and NF-&kgr;B activation. VSMC from p47phox−/− mice showed markedly reduced ROS generation and NF-&kgr;B activation in response to Ang II and AT1-AA. The p22, p47, and p67 phox expression in placentas from preeclamptic patients was increased, compared with normal placentas. Furthermore, NF-&kgr;B was activated and I-&kgr;B&agr; reduced in placentas from preeclamptic women. Conclusions—NADPH oxidase is potentially an important source of ROS that may upregulate NF-&kgr;B in preeclampsia. We suggest that AT1-AA through activation of NADPH oxidase could contribute to ROS production and inflammatory responses in preeclampsia.

Hormonal regulation of the human adipose-tissue renin-angiotensin system: relationship to obesity and hypertension.

Objective Adipose tissue secretes vasoactive substances which may contribute to the development of obesity-related hypertension. The aim of this work was to study the expression of renin–angiotensin system genes in adipose tissue of obese hypertensive subjects and the hormonal regulation of these genes. Design Differential expression of renin–angiotensin system genes in subcutaneous abdominal adipocytes of 12 lean normotensive, eight obese normotensive, and 10 obese hypertensive women was determined in a cross-sectional study. In vitro hormonal regulation of these genes was studied in primary human adipocytes obtained by breast reduction from healthy women. Methods In the clinical study, 24-h ambulatory blood pressure measurement and anthropometry were used to characterize the volunteers, and adipocytes were obtained by subcutaneous needle biopsy. The in vitro regulation of renin–angiotensin system genes by hydrocortisone, insulin, thyroxin, estradiol and angiotensin II on primary cultured human mammary adipocytes was studied by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results While expression of the angiotensinogen gene was significantly lower in adipocytes from both obese groups, the renin, angiotensin-converting enzyme and angiotensin II type 1 receptor genes were significantly upregulated in obese hypertensives. Hydrocortisone increased angiotensin II type 1 receptor gene and protein expression in a time- and dose-dependent manner in human adipocytes, but had no significant influence on other renin–angiotensin system genes. Expression of these genes was not significantly affected by any of the other tested hormones. Conclusions Renin–angiotensin system genes are differentially regulated in human obesity and hypertension. The role of the adipose-tissue renin–angiotensin system in the development of obesity-associated hypertension or metabolic disease clearly warrants further study.

Angiotensin type 1 receptor antagonists induce human in-vitro adipogenesis through peroxisome proliferator-activated receptor-gamma activation.

Objective In clonal animal cells, certain angiotensin receptor blockers (ARB) activate the peroxisome proliferator-activated receptor-γ (PPARγ). The aim of this work was to validate that observation in human cells and humans. Methods We investigated the induction of in-vitro adipogenesis and the activation of PPARγ-target genes, adiponectin and lipoprotein lipase, by ARB in human preadipocytes. We also studied PPARγ response-element-driven luciferase reporter gene activation in human adipocytes. Finally, we treated 14 obese men for 10 days with placebo crossed over with 150 mg/day irbesartan. Subcutaneous fat was analyzed for mRNA expression of adiponectin and lipoprotein lipase. Results Telmisartan and irbesartan, and to a lesser degree losartan, induced adipogenesis and activated PPARγ-target genes. This stimulation of PPARγ-target genes was prevented by the PPARγ antagonist GW9662. Eprosartan had no effect. Paradoxically, all ARB activated the luciferase reporter gene. PPARγ activity increased approximately two-fold with pioglitazone and 1.5-fold with the ARB in all assays. In the cross-over clinical study, irbesartan lowered blood pressure but had no effect on adiponectin or lipoprotein lipase mRNA expression. Conclusions Our data are the first to show that ARB induce adipogenesis and PPARγ-target gene expression in human adipocytes. Pharmacokinetic differences may contribute to the heterogeneous effects on metabolism and preadipocyte differentiation. In humans, larger doses of ARB, longer treatments, or both may be required to activate PPARγ in adipose cells.

Retinol saturase promotes adipogenesis and is downregulated in obesity

Adipocyte differentiation is controlled by many transcription factors, but few known downstream targets of these factors are necessary for adipogenesis. Here we report that retinol saturase (RetSat), which is an enzyme implicated in the generation of dihydroretinoid metabolites, is induced during adipogenesis and is directly regulated by the transcription factor peroxisome proliferator activated receptor γ (PPARγ). Ablation of RetSat dramatically inhibited adipogenesis but, surprisingly, this block was not overcome by the putative product of RetSat enzymatic activity. On the other hand, ectopic RetSat with an intact, but not a mutated, FAD/NAD dinucleotide-binding motif increased endogenous PPARγ transcriptional activity and promoted adipogenesis. Indeed, RetSat was not required for adipogenesis when cells were provided with exogenous PPARγ ligands. In adipose tissue, RetSat is expressed in adipocytes but is unexpectedly downregulated in obesity, most likely owing to infiltration of macrophages that we demonstrate to repress RetSat expression. Thiazolidinedione treatment reversed low RetSat expression in adipose tissue of obese mice. Thus, RetSat plays an important role in the biology of adipocytes, where it favors normal differentiation, yet is reduced in the obese state. RetSat is thus a novel target for therapeutic intervention in metabolic disease.

Association of CRP genetic variants with blood concentrations of C-reactive protein and colorectal cancer risk.

High blood concentrations of C‐reactive protein (CRP) have been associated with elevated risk of colorectal cancer in several prospective studies including the European Prospective Investigation into Cancer and Nutrition (EPIC), but it is unknown whether these observations reflect a causal relationship. We aimed to investigate whether CRP genetic variants associated with lifelong higher CRP concentrations translate into higher colorectal cancer risk. We conducted a prospective nested case–control study within EPIC including 727 cases diagnosed between 1992 and 2003 and 727 matched controls selected according to an incidence‐density sampling protocol. Baseline CRP concentrations were measured in plasma samples by a high sensitivity assay. Tagging single nucleotide polymorphisms (SNPs) in the CRP gene (rs1205, rs1800947, rs1130864, rs2808630, rs3093077) were identified via HapMap. The causal effect of CRP on colorectal cancer risk was examined in a Mendelian Randomization approach utilizing multiple CRP genetic variants as instrumental variables. The SNPs rs1205, rs1800947, rs1130864 and rs3093077 were significantly associated with CRP concentrations and were incorporated in a CRP allele score which was associated with 13% higher CRP concentrations per allele count (95% confidence interval 8–19%). Using the CRP‐score as instrumental variable, genetically twofold higher CRP concentrations were associated with higher risk of colorectal cancer (odds ratio 1.74, 95% confidence interval 1.06–2.85). Similar observations were made using alternative definitions of instrumental variables. Our findings give support to the hypothesis that elevated circulating CRP may play a direct role in the etiology of colorectal cancer.

Influence of dietary fat intake on the endocannabinoid system in lean and obese subjects

Endocannabinoid system (ECS) activation promotes obesity‐associated metabolic disease. Increased dietary fat intake increases blood endocannabinoids and alters adipose and skeletal muscle ECS gene expression in human.

Pressure Natriuresis in AT2 Receptor–Deficient Mice with L-NAME Hypertension

AT(2) receptor-disrupted (AT(2) -/-) mice provide a unique opportunity to investigate the cardiovascular and BP-related effects of NO depletion. This study compared the pressure-diuresis-natriuresis relationship in (AT(2) -/-) and wild-type (AT(2) +/+) mice after treating the animals with L-NAME (130 mg/kg body wt per day) for 1 wk. L-NAME increased mean arterial pressure (MAP) more in AT(2) -/- than in AT(2) +/+ mice (118 +/- 2 versus 108 +/- 4 mmHg). This difference occurred even though L-NAME-treated AT(2) +/+ mice had a greater sodium excretion than AT(2) -/- mice (10.9 +/- 0.5 versus 8.0 +/- 1.0 micro mol/h). The pressure-natriuresis relationship in conscious AT(2) -/- mice was shifted rightward compared with controls. RBF was decreased in AT(2) -/- compared with AT(2) +/+ mice. L-NAME decreased RBF in these mice further from 4.08 +/- 0.43 to 2.79 +/- 0.15 ml/min per g of kidney wt. GFR was not significantly different between AT(2) +/+ and AT(2) -/- mice (1.09 +/- 0.08 versus 1.21 +/- 0.09 ml/min per g of kidney wt). L-NAME reduced GFR in AT(2) -/- to 0.87 +/- 0.07 ml/min per g of kidney wt. Fractional sodium (FE(Na)) and water (FE(H2O)) curves were shifted more strongly to the right by L-NAME in AT(2) -/- mice than in AT(2) +/+ mice. AT(1) receptor blocker treatment lowered BP in both L-NAME-treated strains to basal values. It is concluded that the AT(1) receptor plays a key role in the impaired renal sodium and water excretion induced by NO synthesis blockade. Changes in RBF, GFR, and tubular sodium and water reabsorption are involved and may be also responsible for the greater BP increase in L-NAME-treated AT(2) -/- mice.

Cardiac hypertrophy and fibrosis in chronic L-NAME-treated AT2 receptor-deficient mice.

Background The role of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in cardiac hypertrophy and fibrosis is incompletely understood. The availability of AT2 receptor-deficient mice (AT2 −/y) makes it possible to study the effects of AT1 receptors without the confounding influence of AT2 receptor activity. Objective To test the hypothesis that the AT2 receptor affords protection from left ventricular hypertrophy and fibrosis in chronic hypertension induced by Nω-nitro-l-arginine methyl ester (l-NAME). Design Four groups of mice were studied over a period of 3 weeks: AT2 −/y mice with and without l-NAME, and AT2 +/y mice with and without l-NAME. Methods Blood pressure and heart rate were monitored by telemetry in groups of AT2 +/y and AT2 −/y mice for 4 weeks. l-NAME groups received the compound in drinking water for the last 3 weeks. We determined left ventricular AT1 receptor expression, cardiac hypertrophy and fibrosis, with and without l-NAME treatment. We used a miniaturized conductance-manometer system to measure pressure–volume loops at the time when the animals were killed. Results AT2 −/y mice treated with l-NAME showed worse left ventricular hypertrophy, more perivascular fibrosis and greater concentrations of brain natriuretic peptide than did AT2 +/y mice treated with l-NAME. The end-systolic pressure–volume relationship, an index of left ventricular contractility, was decreased in AT2 −/y mice treated with l-NAME. Conclusions The AT2 receptor is not essential for development of l-NAME-induced cardiac hypertrophy, fibrosis and concomitant changes in left ventricular performance. In contrast, the AT2 receptor offers a protective effect.