Dietary capsaicin reduces rodent visceral fat weight. We tested the hypothesis that intact intestinal mucosal afferent nerve function is necessary for fat deposition in visceral adipose tissue sites. Rats were treated daily for 2 weeks with intragastric (chronic treatment) vehicle or capsaicin. Superior mesenteric artery blood flow and mesenteric and inguinal fat blood flow were measured before and after capsaicin was administered into the duodenum (acute treatment). Fat from all sites was dissected and weighed. Chronic capsaicin significantly attenuated acute capsaicin-induced mesenteric hyperemia but did not abolish the reflex wiping of the eye exposed to capsaicin, indicating that functional ablation was limited to the intestinal mucosal afferent nerves. The associated vasoconstriction in adipose tissue was inhibited at the visceral (mesenteric) site and maintained but attenuated at the subcutaneous (inguinal) site. The onset of vasoconstriction was instantaneous, indicating a reflex mechanism. There was a redistribution of fat from visceral to subcutaneous sites, reflected by a decrease and an increase in the percentage of body fat in the visceral and subcutaneous sites, respectively. These pilot studies reveal for the first time that normal intestinal mucosal afferent nerve function is necessary for the physiologic accumulation of fat in visceral adipose tissue sites.

We describe a method for simultaneous extraction of angiotensins I and II from human blood. Blood was diluted with 10 volumes of isotonic salIne containing ammonium ethylenediaminetetraacetate, and the angiotensin peptides were extracted by cation-exchange chromatography. The dilution effectively Inhibits in vitro production of angiotensin I, conversion of anglotensin Ito angiotensin II, and degradation of the peptides. Analytical recovery of exogenous anglotensin I or II added to whole blood In physiological concentrations was essentially complete. The extraction procedure removes blood and plasma components that interfere with radloimmunoassay of angiotensin, thus eliminating the need to correct for nonspecific interference. Circulating angiotensins I and II, measured In patients with essential hypertension before and after a week of diuretic administration, increased in parallel with plasma renin activity.

Objective The present study was designed to determine the effects of insulin on cytosolic angiotensin II production and proliferation in cultured rat vascular smooth muscle cells. Design and methods Vascular smooth muscle cells were incubated with insulin for 48 h. Cytosolic angiotensin I and II were determined by radioimmunoassays of purified cell homogenates. Angiotensin II was also detected by immunohistochemistry of intact cells. Cell proliferation was determined by pulse labeling with radiolabeled thymidine. Angiotensinogen mRNA expression was determined by slot-blot analysis. Results Insulin significantly increased cytosolic angiotensin II concentration in vascular smooth muscle cells. Lisinopril, omapatrilat and irbesartan inhibited this increase of angiotensin II, but had no effect on angiotensin I levels. Immunohistochemical staining confirmed the presence of angiotensin II in control and insulin-treated vascular smooth muscle cells. Insulin increased cell proliferation, and addition of lisinopril, omapatrilat or irbesartan inhibited this effect. Insulin also increased expression of angiotensinogen mRNA in cultured vascular smooth muscle cells, but PD98059, a mitogen-activated protein kinase inhibitor, prevented the rise in angiotensinogen expression. Conclusion These results support the concept that insulin stimulates angiotensin II production in cultured vascular smooth muscle cells through a mitogen-activated, protein kinase-dependent pathway that might be a factor in the progression of atherosclerosis. Agents that block the renin–angiotensin system have direct protective effects, reducing vascular angiotensin II and growth of vascular smooth muscle cells and are thus of cardiovascular benefit.

Previous studies from our laboratory have shown that insulin increases cytosolic angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMC). The present study was performed to assess the hypothesis that angiotensin II is internalized by the action of angiotensin type-1 receptors (AT1-receptors) in insulin-mediated VSMC. Rat aortic VSMC were isolated and cultured in DMEM/F12 medium with 10% fetal calf serum. When cells were 90% confluent, they were deprived of serum for 24 hours. Then the medium was again changed to fresh serum-free medium containing either insulin (1mU/ml) or insulin with captopril (1 mmol/l) or insulin with losartan (1 mmol/l). Control cells were incubated in serum-free medium, alone. After 48 hours incubation with insulin or insulin plus inhibitors, the cells were scraped using PBS solution containing peptidase inhibitors. The cells were homogenized and the cytosolic fraction obtained by ultracentrifugation. Cytosolic Ang II was measured by radioimmunoassay and protein content was measured by BCA method. The concentration of Ang II produced in control cells was 114 ± 4.1 pg/mg protein. Insulin increased cytosolic Ang II concentration to 134.3 ± 7.2 pg/mg protein (p<0.01, n=6 for each group). The angiotensin converting enzyme inhibitor captopril reduced the insulin-stimulated Ang II levels to 86 ± 1.8 pg/mg protein (p<0.01 from insulin alone, n=6). The AT1 receptor blocker losartan reduced the insulin-stimulated Ang II levels to 101 ± 5.9 pg/mg protein (p<0.01, n=6). Thus, insulin increases cytosolic Ang II concentration in VSMC, and both AT-1 receptor blockers and ACE inhibitors blunt the insulin-stimulated increase of Ang II. In conclusion, the concentration of cytosolic Ang II in insulin-mediated VSMC is dependent on both angiotensin converting enzyme and AT1 receptors, and the present results support a mechanism by which insulin-stimulated Ang I is converted to Ang II extracellularly, and is subsequently internalized after binding to AT1 receptors.

We have previously reported that exposure of vascular smooth muscle cells (VSMCs) to insulin results in stimulation of angiotensinogen gene expression. The objective of this investigation was to determine whether, after insulin stimulation, the angiotensin II (Ang II) generating cascade takes place intracellularly. We also evaluated the effect of insulin-induced production of AngII on the VSMCs growth, and the action of the angiotensin converting enzyme inhibitor (ACEI), lisinopril (Lis), or the vasopeptidase inhibitor (VPI), omapatrilat (OP), on the insulin-induced growth. Cultured VSMCs, from rat thoracic aorta, were deprived of serum for 24h. Insulin with OP or Lis was added. After 48 hours incubation, Ang II concentration in the cell homogenate was quantified by radioimmunoassay, a method highly specific and sensitive for Ang II. Proliferation was estimated by determination of the rate of DNA synthesis as assayed by pulse labeled 3H-methyl thymidine (3H-TdR) uptake, adjusted to the protein concentration of each sample. Exposure of VSMCs to 1000μU/ml insulin for 48 hours resulted in a 37.4% increases in Ang II (p<0.01). OP (10-7M) or Lis (10-7M), prevented this insulin-mediated increase of Ang II. Insulin increased the rate of 3H-TdR uptake by 31.3+/- 6.7% (p<0.01) compared to control. In comparison, OP and LIS reduced this action to basal values not significantly different from control. In conclusion, our results demonstrate that in VSMCs: Insulin increases intracellular Ang II production, indicating the presence of an intracellular renin-angiotensin system; Both inhibitors suppress insulin-induced intracellular production of Ang II, suggesting an intracellular action of these drugs. This may clarify the action of ACEI irrespective of the plasma renin-angiotensin system level; and ACEI and VPI inhibit the insulin-mediated growth of VSMCs to the same extent.

With aging, blood pressure and norepinephrine (NE) levels gradually increase, and the duration and quality of sleep decrease. This raises the possibility that nighttime enhanced sympathetic nervous sytem activity may affect blood pressure and sleep patterns in aging men. We studied 12 normotensive aging men and 13 age and weight matched essential hypertensives (ages 55–70), as well as 9 young normotensive and 7 hypertensive subjects (ages 22–46 yrs) on a 100 mEq Na/80 mEq K diet. Supine blood pressure and hormone levels were determined at bi-hourly intervals between 09.00–21.00 h and every 30 min between 21.00–09.00 h. Elderly hypertensives and normotensives exhibited circadian rhythms of blood pressure, with the highest levels recorded in the late morning hours and the lowest between 01.00–04.00 h. Contrary to the findings in early essential hypertension, older hypertensives had lower mean 24 h levels of NE (377 ± 9 vs 455 ± 9 pg/ml; p < 0.001) than normotensives. The nighttime decrease in NE levels encountered in young individuals was not observed in aging subjects. Elderly hypertensives had lower mean 24 h PRA (0.92 ± 0.03 vs. 1.41 ± 0.06 ng/ml/h; p < 0.01). Circadian rhythm for PRA was barely detectable in older hypertensives. The circadian patterns of aldosterone, prolactin (PRL), and cortisol in older hypertensive and older normotensives were similar to those observed in younger hypertensives and normotensives. Since the secretory pattern of both sleep-independent hormones (cortisol, aldosterone) and sleep responsive hormones (PRL) is unchanged, the disrupted circadian rhythm of NE and PRA in aging hypertensives represent specific abnormalities.

Background Circulating insulin and insulin-like growth factor-I (IGF-I) levels are increased in patients with hypertension and insulin resistance. Since both hormones are known to have cell growth-promoting effects, they may contribute to the progression of vascular hypertrophy in patients with insulin resistance. Insulin-mediated activation of the vascular renin-angiotensin system (RAS) stimulates growth in cultured rat vascular smooth muscle cells (VSMC).Objective In order to evaluate the role of IGF-I-mediated activation of components of the tissue RAS, we examined the effect of IGF-I receptor stimulation on cell proliferation, and production of angiotensinogen in cultured VSMC.Study Design Aortic VSMC were derived from male Sprague-Dawley rats. IGF-I and insulin-mediated DNA synthesis were estimated by H-3-thymidine uptake (H-3-TdR) with or without the angiotensin I converting enzyme inhibitor, captopril. Moreover, angiotensinogen released by the cells to the culture medium was determined by radioimmunoassay with or without the anti-IGF-I receptor antibody alpha IR3 or captopril.Results Both IGF-I and insulin increased 3H-TdR uptake by cultured rat VSMC (P < 0.05). Captopril blocked IGF-I and insulin-mediated 3H-TdR uptake (-34.4 +/- 1.9% and -32.7 +/- 1.8%, P < 0.05, respectively). IGF-I increased the angiotensinogen level in the medium by 30.6 +/- 2.9% (P < 0.01). Insulin also stimulated angiotensinogen synthesis by 26.3 +/- 2.2% (P < 0.01). Captopril and alpha IR3 significantly suppressed angiotensinogen production stimulated by both IGF-I and insulin.Conclusions These results indicate that IGF-I as well as insulin stimulates angiotensinogen production and growth in VSMC. Thus, both hormones may independently play a role in progression of the vascular hypertrophy and atherosclerosis in patients with hypertension and insulin resistance through activation of the tissue PAS. J Hypertens 2000, 18:1051 -1056 (C) Lippincott Williams & Wilkins.

Objective Plasma renin is not elevated in recombinant human erythropoietin (rhEPO)-induced hypertension but angiotensin converting enzyme inhibitors reduce blood pressure in both human and animal studies. Since rhEPO elevates renin and angiotensinogen messenger RNAs in angiotensin II target tissues such as the aorta, we explored the actions of rhEPO on renin-angiotensin system-related gene transcription of cultured rat vascular smooth muscle cells.Design and methods To separate direct actions of rhEPO from those mediated secondarily by potential activation of the renin-angiotensin system, vascular smooth muscle cells were cultured with rhEPO and enalapril to inhibit the angiotensin converting enzyme and losartan to inhibit angiotensin II type 1 receptors,Results Vascular smooth muscle cells cultured with rhEPO (6-8 units/ml) demonstrated elevations (40-120%) in messenger RNAs of the renin-angiotensin system (renin, angiotensinogen, angiotensin receptor types 1 and 2) and increased levels of several messenger RNAs known to respond to angiotensin It (transforming growth factor-p, insulin-like growth factor-II, epidermal growth factor, c-fos and platelet-derived growth factor). In contrast, cells cultured in the presence of rhEPO and enalapril or losartan showed elevations of messenger RNA for only the two types of angiotensin II receptor. This increase was higher than that obtained when cells were cultured with rhEPO or either antagonist alone. The increase in specific binding of angiotensin II to cells cultured in the presence of rhEPO and enalapril or rhEPO and losartan paralleled the changes in receptor messenger RNA.Conclusions rhEPO exerts its primary action on vascular smooth muscle cells via an increase in angiotensin receptor messenger RNA, resulting in a parallel increase in angiotensin II receptor expression. We suggest that increased receptor expression secondarily mediates the expression of other renin-angiotensin system messenger RNAs, which leads to angiotensin Ii-responsive gene transcription. The elevation in angiotensin II receptors, as observed in response to rhEPO, may provide a mechanism by which other forms of renin-dependent hypertension are initiated. (C) Lippincott Williams & Wilkins.

To assess the mechanism of the effect of cigarette smoke on ulcer disease we employed a rat model in which cigarette smoke increases the size of acetic acid-induced gastric ulcer and decreases the hyperemia at the ulcer margin. We postulate that cigarette smoke increases angiotensin II (a vasoconstrictor) in ulcer tissue. Since direct measurement of angiotensin II in small tissue samples is problematic, we compared the messenger ribonucleic acid (mRNA) for its precursors (angiotensinogen and renin) in ulcer and normal gastric tissue. We also evaluated the effect of enalapril, which blocks the conversion of angiotensin I to angiotensin II on ulcer size. In the ulcer tissue, cigarette smoke produced a significant increase in mRNA for angiotensinogen but not for renin. Enalapril decreased the size of the gastric ulcer in rats exposed to cigarette smoke. The data support the possibility that in ulcer tissue cigarette smoke stimulates an angiotensin II-mediated mechanism, which may in part be responsible for the impairment of ulcer margin hyperemia and aggravation of ulcer size.