Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene

Adrenomedullin (AM) is a potent depressor peptide whose vascular action is suggested to involve nitric oxide (NO) release. To explore the role of endogenous AM in vascular and renal function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists AM(22-52) and CGRP(8-37) on the renal perfusion pressure (RPP) of kidneys isolated from AM transgenic (TG)/heterozygote knockout (KO) mice and wild-type littermates (WT). Furthermore, we evaluated the renal function and histology 24 hours after bilateral renal artery clamp for 45 minutes in TG, KO, and WT mice. Baseline RPP was significantly lower in TG than in KO and WT mice (KO 93.4+/-4.6, WT 85.8+/-4.2, TG 72.4+/-2.4 mm Hg [mean+/-SE], P<0.01). ACh and AM caused a dose-related reduction in RPP, but the degree of vasodilatation was smaller in TG than that in KO and WT (%DeltaRPP 10(-7) mol/L ACh: KO -48.1+/-3.9%, WT -57.5+/-5.6%, TG -22.8+/-4.8%, P<0.01), whereas N(G)-nitro-L-arginine methyl ester (L-NAME) caused greater vasoconstriction in TG (%DeltaRPP 10(-4) mol/L: KO 33.1+/-3.3%, WT 55.5+/-7.2%, TG 152.6+/-21.2%, P<0.01). Both AM antagonists increased RPP in TG to a greater extent compared with KO and WT mice (%DeltaRPP 10(-6) mol/L CGRP(8-37): KO 12.8+/-2.6%, WT 19.4+/-3.6%, TG 41.8+/-8.7%, P<0.01). In mice with ischemic kidneys, serum levels of urea nitrogen and renal damage scores showed smaller values in TG and greater values in KO mice (urea nitrogen: KO 104+/-5>WT 98+/-15>TG 38+/-7 mg/dL, P<0.05 each). Renal NO synthase activity was also greater in TG mice. However, the differences in serum urea nitrogen and renal damage scores among the 3 groups of mice were not observed in mice pretreated with L-NAME. In conclusion, AM antagonists increased renal vascular tone in WT as well as in TG, suggesting that endogenous AM plays a role in the physiological regulation of the vascular tone. AM is likely to protect renal tissues from ischemia/reperfusion injury through its NO releasing activity.     

aP2-Cre-mediated inactivation of acetyl-CoA carboxylase 1 causes growth retardation and reduced lipid accumulation in adipose tissues

Adipose tissue is one of the major sites for fatty acid synthesis and lipid storage. We generated adipose (fat)-specific ACC1 knockout (FACC1KO) mice using the aP2-Cre/loxP system. FACC1KO mice showed prenatal growth retardation; after weaning, however, their weight gain was comparable to that of wild-type (WT) mice on a normal diet. Under lipogenic conditions of fasting/re-feeding a fat-free diet, lipid accumulation in adipose tissues of FACC1KO mice was significantly decreased; this is consistent with a 50–66% reduction in the ACC activity in these tissues compared with that of WT mice. Surprisingly, FACC1KO mice manifested skeletal growth retardation phenotype accompanied by decreased chondrocyte proliferation in the growth plate and lower trabecular bone density. In addition, there was about a 30% decrease in serum insulin-like growth factor I (IGF1), and while the serum leptin level was decreased by about 50%, it did not counteract the osteopenic effects of IGF1 on the bone. Fatty acid analyses of mutant bone lipids revealed relatively higher levels of C18:2n-6 and C18:3n-3 and lower levels of their elongation C20 homologs than that of WT cohorts, leading to lower levels of C20 homologs and bone development. Moreover, aP2-Cre-mediated ACC1 inactivation in bone tissue led to a decreased number of osteoblasts but not of osteoclasts. The downregulation of ACC1 on osteoblastogenesis may be the cause for the osteopenia phenotype of FACC1KO bone homeostasis.     

Assessment of renal functional phenotype in mice lacking gp91PHOX subunit of NAD(P)H oxidase

To determine the role of endogenous superoxide (O2-) in the kidney, we assessed renal hemodynamics and excretory function in gp91(PHOX) (a NAD(P)H oxidase subunit) gene knockout (KO) mice and compared these findings with those of wild-type (WT) strain C57BL/6 mice. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearances respectively in anesthetized mice (n=8 in each group). There were higher baseline RBF (4.3+/-0.4 versus 2.5+/-0.2 mL/min per gram; P<0.002) and lower renal vascular resistance (RVR) (16+/-1.4 versus 29+/-2.3 mm Hg/mL/min per gram; P<0.0001) in KO compared with WT without a significant difference in mean arterial pressure (MAP) (67+/-2 versus 71+/-2 mm Hg) and GFR (0.66+/-0.09 versus 0.73+/-0.05 mL/min per gram) between the strains. Intravenous infusion of angiotensin II (Ang II) (2 ng/min per gram of body weight) for 30 minutes caused a lesser degree of decreases in RBF (-8% versus -33%) and of increases in RVR (+73% versus +173%) in KO compared with WT. GFR was increased (43%) in KO but not in WT during Ang II infusion. Urinary excretion of nitrate/nitrite was higher in conscious KO (n=5) than in WT (n=5), indicating an increase in nitric oxide bioavailability that could be the cause of high RBF and low RVR in KO. These data indicate that gp91(PHOX), a subunit of NAD(P)H oxidase, plays a regulatory role in the maintenance of renal vascular tone. These results also suggest that the mechanism of Ang II-mediated renal vascular action involves concomitant generation of O2-.     

Androgen inactivation and steroid-converting enzyme expression in abdominal adipose tissue in men

We examined 5alpha-dihydrotestosterone (5alpha-DHT) inactivation and the expression of several steroid-converting enzymes with a focus on aldoketoreductases 1C (AKR1C), especially AKR1C2, in abdominal adipose tissue in men. AKR1C2 is mainly involved in the conversion of the potent androgen 5alpha-DHT to its inactive forms 5alpha-androstane-3alpha/beta,17beta-diol (3alpha/beta-diol). Subcutaneous (s.c.) and omental (Om) adipose tissue biopsies were obtained from 21 morbidly obese men undergoing biliopancreatic derivation surgery and 11 lean to obese men undergoing general abdominal surgery. AKR1C2 mRNA and 5alpha-DHT inactivation were detected in both s.c. and Om adipose tissue. After incubation of preadipocytes with 5alpha-DHT, both 3alpha-diol and 3beta-diol were produced through 3alpha/beta-ketosteroid reductase (3alpha/beta-HSD) activity. In preadipocyte cultures, 3alpha-reductase activity was significantly predominant over 3beta-reductase activity in cells from both the s.c. and Om compartments. Expression levels of AKR1C1, AKR1C3 and of the androgen receptor were significantly higher in s.c. versus Om adipose tissue while mRNA levels of 17beta-HSD-2 (hydroxysteroid dehydrogenase type 2) and 3(alpha-->beta)-hydroxysteroid epimerase were significantly higher in Om fat. 3Alpha/beta-HSD activity was mainly detected in the cytosolic fraction, suggesting that AKR1C may be responsible for this reaction. Experiments with isoform-specific AKR1C inhibitors in preadipocytes showed that AKR1C2 inhibition significantly decreased 3alpha-HSD and 3beta-HSD activities (3alpha-HSD: 30 +/- 24% of control for s.c. and 32 +/- 9% of control for Om, 3beta-HSD: 44 +/- 12% of control for s.c.). When cells were incubated with both AKR1C2 and AKR1C3 inhibitors, no significant additional inhibition was observed. 5Alpha-DHT inactivation was significantly higher in mature adipocytes compared with preadipocyte cultures in s.c. adipose tissue, as expressed per microgram total protein (755 +/- 830 versus 245 +/- 151 fmol 3alpha/beta-diol per microg protein over 24 h, P < 0.05 n = 10 cultures). 5Alpha-DHT inactivation measured in tissue homogenates was significantly higher in the s.c. depot compared with Om fat (117 +/- 39 versus 79 +/- 38 fmol 3alpha/beta-diol per microg prot over 24 h, P < 0.0001). On the other hand, Om 3alpha/beta-HSD activity was significantly higher in obese men (body mass index (BMI) >or= 30 kg/m2) compared with lean and overweight men (84 +/- 37 versus 52 +/- 30 fmol 3alpha/beta-diol per microg protein over 24 h, P < 0.03). No difference was found in s.c. 3alpha/beta-HSD activity between these groups. Positive correlations were found between s.c. 5alpha-DHT inactivation rate and circulating levels of the androgen metabolites androsterone-glucuronide (r = 0.41, P < 0.02) and 3alpha-diol-glucuronide (r = 0.38, P < 0.03) and with the adrenal precursor androstenedione (r = 0.42, P < 0.02). In conclusion, androgen inactivation was detected in abdominal adipose tissue in men, with higher 3alpha/beta-HSD activity in the s.c. versus Om depot. Higher Om 5alpha-DHT inactivation rates were found in obese compared with lean men. Further studies are required to elucidate whether local androgen inactivation in abdominal adipose tissue is involved in the modulation of adipocyte metabolism and regional fat distribution in men.     

beta(2)-adrenergic receptor overexpression exacerbates development of heart failure after aortic stenosis

BACKGROUND: Beta-adrenergic signaling is downregulated in the failing heart, and the significance of such change remains unclear. METHODS AND RESULTS: To address the role of beta-adrenergic dysfunction in heart failure (HF), aortic stenosis (AS) was induced in wild-type (WT) and transgenic (TG) mice with cardiac targeted overexpression of beta(2)-adrenergic receptors (ARs), and animals were studied 9 weeks later. The extents of increase in systolic arterial pressure (P<0.01 versus controls), left ventricular (LV) hypertrophy (TG, 94+/-6 to 175+/-7 mg; WT, 110+/-6 to 168+/-10 mg; both P<0.01), and expression of ANP mRNA were similar between TG and WT mice with AS. TG mice had higher incidences of premature death and critical illness due to heart failure (75% versus 23%), pleural effusion (81% versus 45%), and left atrial thrombosis (81% versus 36%, all P<0.05). A more extensive focal fibrosis was found in the hypertrophied LV of TG mice (P<0.05). These findings indicate a more severe LV dysfunction in TG mice. In sham-operated mice, LV dP/dt(max) and heart rate were markedly higher in TG than WT mice (both P<0.01). dP/dt(max) was lower in both AS groups than in sham-operated controls, and this tended to be more pronounced in TG than WT mice (-32+/-5% versus -16+/-6%, P=0.059), although dP/dt(max) remained higher in TG than WT groups (P<0.05). CONCLUSIONS: Elevated cardiac beta-adrenergic activity by beta(2)-AR overexpression leads to functional deterioration after pressure overload.     

Lack of hexose-6-phosphate dehydrogenase impairs lipid mobilization from mouse adipose tissue

In adipose tissue, glucocorticoids regulate lipogenesis and lipolysis. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme located in the endoplasmic reticulum that provides a cofactor for the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), regulating the set point of its activity and allowing for tissue-specific activation of glucocorticoids. The aim of this study was to examine the adipose tissue biology of the H6PDH null (H6PDH/KO) mouse. Real-time PCR analysis confirmed similar mRNA levels of 11beta-HSD1 and glucocorticoid receptor-alpha in wild-type (WT) and H6PDH/KO mice in liver and gonadal fat depots. Microsomal 11beta-HSD1 protein levels shown by Western blot analysis corresponded well with mRNA expression in gonadal fat of WT and H6PDH/KO mice. Despite this, the enzyme directionality in these tissues changed from predominately oxoreductase in WT to exclusively dehydrogenase activity in the H6PDH/KO mice. In the fed state, H6PDH/KO mice had reduced adipose tissue mass, but histological examination revealed no difference in average adipocyte size between genotypes. mRNA expression levels of the key lipogenic enzymes, acetyl CoA carboxylase, adiponutrin, and stearoyl-coenzyme A desaturase-2, were decreased in H6PDH/KO mice, indicative of impaired lipogenesis. In addition, lipolysis rates were also impaired in the H6PDH/KO as determined by lack of mobilization of fat and no change in serum free fatty acid concentrations upon fasting. In conclusion, in the absence of H6PDH, the set point of 11beta-HSD1 enzyme activity is switched from predominantly oxoreductase to dehydrogenase activity in adipose tissue; as a consequence, this leads to impairment of fat storage and mobilization.     

The antidepressant-like effect of the melatonin receptor ligand luzindole in mice during forced swimming requires expression of MT2 but not MT1 melatonin receptors

We previously reported an antidepressant-like effect in C3H/HeN mice during the forced swimming test (FST) following treatment with the MT1/MT2 melatonin receptor ligand, luzindole. This study investigated the role melatonin receptors (MT1 and/or MT2) may play in the effect of luzindole in the FST using C3H/HeN mice with a genetic deletion of either MT1 (MT1KO) or MT2 (MT2KO) melatonin receptors. In the light phase (ZT 9-11), luzindole (30 mg/kg, i.p.) significantly decreased immobility during swimming in both wild type (WT) (135.6 +/- 25.3 s, n = 7) and MT(1)KO (132.6 +/- 13.3 s, n = 8) as compared with vehicle-treated mice (WT: 207.1 +/- 6.0 s, n = 7; MT1KO: 209.5 +/- 6.2 s, n = 8) (P < 0.001). In the dark phase (ZT 20-22), luzindole also decreased time of immobility in both WT (89.5 +/- 13.9 s, n = 8) and MT1KO (66.5 +/- 6.4 s, n = 8) mice as compared with the vehicle treated (WT: 193.8 +/- 3.5, n = 6; MT1KO: 176.6 +/- 6.2 s, n = 8) (P < 0.001). Genetic disruption of the MT1 gene did not alter the diurnal rhythm of serum melatonin in MT1KO mice (ZT 9-11: 1.3 +/- 0.6 pg/mL, n = 7; ZT 20-22: 10.3 +/- 1.1 pg/mL, n = 8) as compared with WT (ZT 9-11: 1.4 +/- 0.7 pg/mL; ZT 20-22: 10.6 pg/mL). Swimming did not alter the serum melatonin diurnal rhythm in WT and MT1KO mice. Decreases in immobility of WT and MT1KO mice by luzindole treatment were not affected by gender or age (3 months versus 8 months). In contrast, luzindole did not decrease immobility during the FST in MT2KO mice. We conclude that the antidepressant-like effect of luzindole may be mediated through blockade of MT2 rather than MT1 melatonin receptors.     

Activation of the inducible nitric oxide synthase pathway contributes to inflammation-induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis.

OBJECTIVE: Osteoporosis is a major clinical problem in chronic inflammatory diseases such as rheumatoid arthritis. The mechanism of bone loss in this condition remains unclear, but previous studies have indicated that depressed bone formation plays a causal role. Since cytokine-induced nitric oxide (NO) production has been shown to inhibit osteoblast growth and differentiation in vitro, this study was undertaken to investigate the role of the inducible NO synthase (iNOS) pathway in the pathogenesis of inflammation-mediated osteoporosis (IMO) by studying mice with targeted inactivation of the iNOS gene (iNOS knockout [iNOS KO] mice). METHODS: IMO was induced in wild-type (WT) and iNOS KO mice by subcutaneous injections of magnesium silicate. The skeletal response was assessed at the tibial metaphysis by measurements of bone mineral density (BMD), using peripheral quantitative computed tomography, by bone histomorphometry, and by measurements of bone cell apoptosis. RESULTS: NO production increased 2.5-fold (P < 0.005) in WT mice with IMO, but did not change significantly in iNOS KO mice. Total BMD values decreased by a mean +/- SEM of 14.4+/-2.0% in WT mice with IMO, compared with a decrease of 8.6+/-1.2% in iNOS KO mice with IMO (P < 0.01). Histomorphometric analysis confirmed that trabecular bone volume was lower in WT mice with IMO compared with iNOS KO mice with IMO (16.2+/-1.5% versus 23.4+/-2.6%; P < 0.05) and showed that IMO was associated with reduced bone formation and a 320% increase in osteoblast apoptosis (P < 0.005) in WT mice. In contrast, iNOS KO mice with IMO showed less inhibition of bone formation than WT mice and showed no significant increase in osteoblast apoptosis. CONCLUSION: Inducible NOS-mediated osteoblast apoptosis and depressed bone formation play important roles in the pathogenesis of IMO.     

The α-glucosidase inhibitor acarbose prevents obesity and simple steatosis in sequestosome 1/A170/p62 deficient mice

Aim:  Sequestosome 1 (SQSTM1)/A170/p62 plays an important role in membrane-receptor mediated signal transduction and autophagic protein degradation. Although the mechanism involved is not clear, sqstm1 gene knockout (KO) mice develop mature-onset obesity and insulin resistance, leading to type II diabetes. KO mice show accumulation of fat in white adipose tissue and the liver when fed a standard diet. Acarbose is an α-glucosidase inhibitor that improves insulin sensitivity and decreases postprandial hyperglycemia, and it is used to treat type 2 diabetes. We examined whether or not dietary acarbose prevented obesity and simple steatosis in KO mice.
Methods:  Wild-type (WT) and KO mice were fed a standard diet with or without acarbose (0.8% w/w) from 15–25 weeks of age. The body weight and the fat content of adipose tissue and the liver were measured, and changes of lipid metabolism in these tissues were assessed from gene expression.
Results:  Acarbose treatment suppressed weight gain and the development of hepatic steatosis in KO mice. Acarbose treatment up-regulated hepatic expression of the pparα , ucp-2, and abca1 genes, as well as srebp1c , pparα , and pparγ in adipose tissue. In WT mice, however, acarbose treatment had little influence on weight gain and gene expression.
Conclusions:  The results of this study suggest that long-term administration of acarbose is effective for prevention of obesity and simple steatosis in SQSTM1-KO mice.     

Substance P (SP)-neurokinin-1 receptor (NK-1R) alters adipose tissue responses to high-fat diet and insulin action

Peripheral administration of a specific neurokinin-1 receptor (NK-1R) antagonist to mice leads to reduced weight gain and circulating levels of insulin and leptin after high-fat diet (HFD). Here, we assessed the contribution of substance P (SP) and NK-1R in diet-induced obesity using NK-1R deficient [knockout (KO)] mice and extended our previous findings to show the effects of SP-NK-1R interactions on adipose tissue-associated insulin signaling and glucose metabolic responses. NK-1R KO and wild-type (WT) littermates were fed a HFD for 3 wk, and obesity-associated responses were determined. Compared with WT, NK-1 KO mice show reduced weight gain and circulating levels of leptin and insulin in response to HFD. Adiponectin receptor mRNA levels are higher in mesenteric fat and liver in NK-1 KO animals compared with WT, after HFD. Mesenteric fat from NK-1R KO mice fed with HFD has reduced stress-activated protein kinase/c-Jun N-terminal kinase and protein kinase C activation compared with WT mice. After glucose challenge, NK-1R KO mice remove glucose from the circulation more efficiently than WT and pair-fed controls, suggesting an additional peripheral effect of NK-1R-mediated signaling on glucose metabolism. Glucose uptake experiments in isolated rat adipocytes showed that SP directly inhibits insulin-mediated glucose uptake. Our results further establish a role for SP-NK-1R interactions in adipose tissue responses, specifically as they relate to obesity-associated pathologies such as glucose intolerance and insulin resistance. Our results highlight this pathway as an important therapeutic approach for type 2 diabetes.     

The hypertriglyceridemic clamp technique. Studies using long-chain and structured triglyceride emulsions in healthy subjects

In a randomized crossover study, plasma kinetics of 2 different types of fat emulsions were studied in 8 healthy volunteers by using a hypertriglyceridemic clamp technique. The method involves the stabilization of serum triglyceride (TG) concentration during 180 minutes at a predetermined level (4 mmol/L) by adjustment of TG infusion rate by repeated online measurements of serum TG concentration. The fat emulsions under study were a long-chain fatty acid triglyceride (LCT) emulsion (Intralipid 20%, Fresenius Kabi, Sweden) and a structured triglyceride (STG) emulsion (Structolipid 20%, Fresenius Kabi) where medium- and long-chain fatty acids have been interesterified within a TG molecule. The hypertriglyceridemic clamp was found to have acceptable reproducibility when tested in 3 healthy individuals on 2 different occasions, as similar steady-state TG levels were obtained by infusing similar amounts of fat. The average (+/-SEM) TG concentration during the 180-minute clamp was similar for STGs and LCTs (4.0 +/- 0.1 vs 3.9 +/- 0.1 mmol/L; not significant), but the amount of fat that had to be infused was significantly higher during STG than during LCT clamping (0.31 +/- 0.04 vs 0.21 +/- 0.02 g TG per minute; P < .05). Higher serum levels of free fatty acids (1.80 +/- 0.13 vs 0.96 +/- 0.09 mmol/L; P < .05), free glycerol (1.30 +/- 0.07 vs 0.76 +/- 0.08 mmol/L; P < .001), and beta-OH butyrate (1.61 +/- 0.44 vs 1.17 +/- 0.23 mmol/L; not significant) were obtained at the end of the clamp during infusion of STGs compared with LCTs. During infusion of STGs the medium-chain fatty acids octanoic (C:8) and decanoic acid (C:10) constituted approximately half of circulating fatty acids that correspond to the compositional ratio of the emulsion. Plasma lipoprotein lipase (LPL) concentration was higher during STG than during LCT clamping (6.06 +/- 0.62 vs 3.15 +/- 0.40 mU/mL; P < .05), and there was a positive correlation between the mean LPL concentration and the amount of infused TG during the steady-state period (r = 0.58; P < .05). In conclusion, the hypertriglyceridemic clamp method was found to give reproducible results and could be considered for comparison of metabolic clearance properties of different types of fat emulsions. The capacity of healthy subjects to eliminate STGs from blood was greater than for LCTs. An increased LPL activity induced by the higher TG infusion rate may have contributed to the increased metabolic clearance of STGs.     

Inhibitory effect on arterial injury-induced neointimal formation by adoptive B-cell transfer in Rag-1 knockout mice

We investigated the effect of B-cell reconstitution in immune-deficient Rag-1 knockout (KO) mice subjected to arterial injury. After 21 days, injury induced a 4- to 5-fold increase in neointimal formation in Rag-1 KO mice fed normal chow compared with wild-type (WT) mice (0.020+/-0.0160 [n=8] versus 0.0049+/-0.0022 [n=8] mm(2), respectively; P<0.05) and in western-type diet-fed Rag-1 KO mice compared with WT mice (0.0312+/-0.0174 [n=7] versus 0.0050+/-0.0028 [n=6] mm(2), respectively; P<0.05). To investigate the role of B cells in response to injury, Rag-1 KO mice were reconstituted with B cells derived from the spleens of WT mice, with donors and recipients on the same diet. Reconstitution of Rag-1 KO mice with B cells from WT mice (both fed normal chow) reduced neointimal formation compared with the effect in unreconstituted Rag-1 KO mice (0.0076+/-0.0039 [n=9] versus 0.020+/-0.0160 [n=8] mm(2), respectively; P<0.05). Reconstitution of Rag-1 KO mice with B cells from WT mice (both fed a western diet) reduced neointimal formation compared the effect in Rag-1 KO mice (0.0087+/-0.0037 [n=8] versus 0.0312+/-0.0174 [n=7] mm(2), respectively; P<0.05). Injured carotid arteries from reconstituted Rag-1 KO mice had detectable IgM and IgG, indicating viable transfer of B cells. The results suggest that B cells modulate the response to arterial injury.     

De novo lipogenesis in adipose tissue of lean and obese women: application of deuterated water and isotope ratio mass spectrometry

OBJECTIVE: To evaluate the feasibility of using deuterated water and isotope ratio mass spectrometry to measure de novo fatty acid synthesis in adipose tissue, and to compare this parameter in obese and lean women. SUBJECTS: Six lean and six obese premenopausal Caucasian women in the main study and three obese Pima Indians in a pilot study. MEASUREMENTS: Deuterated water was administered orally twice daily for 14 days to create stable deuterium enrichment in body water, during which series of blood samples were collected to measure body water deuterium enrichment and deuterium incorporation into plasma total Triacylglycerol (TG) fatty acids and total cholesterol. Subcutaneous fat at different sites were sampled at the beginning and the end of deuterium administration to measure deuterium incorporation into TG fatty acids. RESULTS: Fractional de novo synthesis rate of TG fatty acids in adipose tissue was 0. 014+/-0.005 and 0.014+/-0.007% in lean and obese Caucasian women, corresponding to 2+/-0.7 and 5.6+/-3.2 g (P=0.3) of fatty acids synthesized daily, respectively. Plasma TG fatty acids and cholesterol synthesis rates were comparable to those reported previously. A pilot study showed that de novo lipid synthesis in adipose tissue of obese Pima Indians was also quantitatively minor. CONCLUSION: Human adipose tissue, like the liver, does not make a major contribution to whole body lipogenesis under eucaloric conditions. A combination of deuterated water and isotope ratio mass spectrometry is a useful research tool for studying accumulation of de novo synthesized lipids in human adipose tissue.     

Changes in adipose tissue composition in malnourished patients before and after liver transplantation: A carbon-13 magnetic resonance spectroscopy and gas-liquid chromatography study

We investigated adipose tissue fatty acid composition in 22 moderately to severely malnourished patients with cirrhosis and in 22 healthy volunteers by in vivo carbon-13 magnetic resonance spectroscopy (MRS). Gas-liquid chromatography (GLC) of adipose tissue samples was also performed in 11 of the patients and in 4 volunteers. In vivo ¹³C magnetic resonance spectra were obtained from the subcutaneous adipose tissue before and after eight weeks following orthotopic liver transplantation (OLT). Adipose tissue biopsy samples were obtained for GLC analysis at the time of transplantation in the patients and at inguinal hernia repair in the 4 volunteers. No significant differences were found in the subcutaneous adipose tissue total-saturated, - polyunsaturated or -monounsaturated fatty acid composition between patients and healthy volunteers by in vivo ¹³C MRS. GLC analysis of adipose tissue samples confirmed that total levels of saturated, poly-, and monounsaturated fatty acids remained the same but revealed significant differences in levels of individual fatty acids, particularly n-3 fatty acids (total n-3, cirrhotics: .84% +/- .07% vs. controls: 1.36% +/- .13%, P < .01). Eight weeks following transplantation, recipients showed a considerable increase in body mass (pretransplantation: 59.3 +/- 3.2 vs. posttransplantation: 63.2 +/- 3 kg, P < .01). ¹³C MRS revealed a significant increase in saturated fatty acids (pretransplantation: 21.6 +/- 2.8 vs. posttransplantation: 25.5% +/- 1.2%, P < .05) and a significant decrease in unsaturated fatty acids. The application of noninvasive MRS techniques may be important to identify the differential uptake of fats, examining both specific fatty acids and different body fat compartments. In the future, this may be useful in optimizing the dietary management of severely malnourished patients with chronic liver disease before liver transplantation.(Hepatology 1997 Jan;25(1):178-83)     

The role of the RhoA/Rho-kinase signaling pathway in renal vascular reactivity in endothelial nitric oxide synthase null mice

BACKGROUND: Smooth muscle contraction is regulated by the small GTPase RhoA and its target, Rho-kinase and recent evidence indicates that nitric oxide (NO) causes vasodilation through inhibition of the RhoA/Rho-kinase (ROCK) signaling pathway. AIM: This study tested the hypothesis that the enhanced renal vascular tone and systemic hypertension in endothelial nitric oxide synthase (eNOS) null mice is due to disinhibition of the ROCK signaling pathway. METHODS: Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and the isolated Krebs-perfused kidney preparation was used to evaluate renal vascular responses in C57BL/6 (wild type, WT) and eNOS knockout (KO) mice treated with Y-27632, a ROCK inhibitor. RESULTS: Compared with the WT mice, Rho kinase activity was higher in eNOS KO mice (37 +/- 8%, P < 0.05) as was SBP (33 +/- 4%, P < 0.05), basal renal perfusion pressure (31 +/- 4%, P < 0.05) and renal vascular resistance (35 +/- 4%, P < 0.05). Y-27632 abolished these differences. Vasoconstriction elicited by angiotensin II (Ang II) or phenylephrine (PE), G-protein-coupled receptor (GPCR) agonists, but not that elicited by arachidonic acid or KCl, was greater in eNOS KO mice. Y-27632 eliminated the amplified vasoconstriction elicited by Ang II or phenylephrine but to a greater extent in eNOS KO mice. Similarly, responses elicited by guanosine 5'-gamma-thiotriphosphate (GTPgammaS), a non-hydrolyzable GTP analog, or sodium tetrafluoride (NaF4), an activator of G-proteins, was greater in eNOS KO mice, 53 +/- 14 and 50 +/- 3%, respectively. Y-27632 normalized the difference. Y-27632 also elicited a dose-dependent renal vasodilation that was greater in eNOS KO mice. CONCLUSIONS: These results show that the ROCK signaling pathway is amplified in the eNOS KO mouse kidney and that the enhanced renal vascular tone and selective increase in reactivity to GPCR agonists supports a role for ROCK in the hypertension and vascular dysfunction in the eNOS KO mice.     

Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice

Oxygen-derived free radicals are involved in the vascular response to angiotensin II (Ang II), but the role of NADPH oxidase, its subunit proteins, and their vascular localization remain controversial. Our purpose was to address the role of NADPH oxidase in the blood pressure (BP), aortic hypertrophic, and oxidant responses to Ang II by taking advantage of knockout (KO) mice that are genetically deficient in gp91(phox), an NADPH oxidase subunit protein. The baseline BP was significantly lower in KO mice than in wild-type (WT) (92+/-2 [KO] versus 101+/-1 [WT] mm Hg, P<0.01), but infusion of Ang II for 6 days caused similar increases in BP in the 2 strains (33+/-4 [KO] versus 38+/-2 [WT] mm Hg, P>0.4). Ang II increased aortic superoxide anion production 2-fold in the aorta of WT mice but did not do so in KO mice. Aortic medial area increased in WT (0.12+/-0.02 to 0.17+/-0.02 mm(2), P<0.05), but did not do so in KO mice (0.10+/-0.01 to 0.11+/-0.01 mm(2), P>0.05). Histochemistry and polymerase chain reaction demonstrated gp91(phox) localized in endothelium and adventitia of WT mice. Levels of reactive oxidant species as indicated by 3-nitrotyrosine immunoreactivity increased in these regions in WT but not in KO mouse aorta in response to Ang II. These results indicate an essential role in vivo of gp91(phox) and NADPH oxidase-derived superoxide anion in the regulation of basal BP and a pressure-independent vascular hypertrophic and oxidant stress response to Ang II.     

Mice lacking the thyroid hormone receptor-alpha gene spend more energy in thermogenesis, burn more fat, and are less sensitive to high-fat diet-induced obesity

Unable to activate brown adipose tissue (BAT) thermogenesis, alphaT3-receptor-deficient mice (Thra-0/0) are cold intolerant. Our objective was to investigate the impact on energy economy and mechanisms of the alternate facultative thermogenesis developed. Energy expenditure (oxygen and food consumption) is elevated in Thra-0/0 mice reared at room temperature. Such difference disappears at thermoneutrality (30 C) and expands as ambient temperature becomes colder (P < 0.001). Despite eating more, Thra-0/0 are leaner than wild-type (WT) mice (P < 0.01), whereas these, whether on chow or high-fat diet, gained more weight (g/d: 0.12 +/- 0.002 vs. 0.08 +/- 0.002 and 0.25 +/- 0.005 vs. 0.17 +/- 0.005, respectively) and adiposity than Thra-0/0 mice (P < 0.001). The respiratory quotient was lower in Thra-0/0 than WT mice (P < 0.001), after feeding or fasted, on chow or high-fat diet, indicating a preference for fat as fuel, which was associated with increased lipoprotein lipase (LPL) expression in skeletal muscle of Thra-0/0 mice but with no differences in gene expression in white adipose tissue. Type-2 deiodinase (D2) was increased in BAT and aerobic muscle of Thra-0/0 mice. This and liver D1 were increased by a high-fat diet in both genotypes, as also were serum T3 and T3/T4 ratio, but more in Thra-0/0 than WT mice (P < 0.001). Remarkably, when studied at thermoneutrality, genotype differences in weight and adiposity gain, respiratory quotient, D2, and LPL disappeared. Thus, disruption of BAT thermogenesis in Thra-0/0 mice activates an alternate facultative thermogenesis that is more energy demanding and associated with reduced fuel efficiency, leanness, increased capacity to oxidize fat, and relative resistance to diet-induced obesity, in all of which muscle LPL and deiodinases play a key role.     

Targeted deletion of matrix metalloproteinase 2 ameliorates myocardial remodeling in mice with chronic pressure overload

Matrix metalloproteinases (MMPs) play an important role in the extracellular matrix remodeling. Experimental and clinical studies have demonstrated that MMP 2 and 9 are upregulated in the dilated failing hearts and involved in the development and progression of myocardial remodeling. However, little is known about the role of MMPs in mediating adverse myocardial remodeling in response to chronic pressure overload (PO). We, thus, hypothesized that selective disruption of the MMP 2 gene could ameliorate PO-induced cardiac hypertrophy and dysfunction in mice. PO hypertrophy was induced by transverse aortic constriction (TAC) in male MMP 2 knockout (KO) mice (n=10) and sibling wild-type (WT) mice (n=9). At 6 weeks, myocardial MMP 2 zymographic activity was 2.4-fold increased in WT+TAC, and this increase was not observed in KO+TAC, with no significant alterations in other MMPs (MMP 1, 3, 8, and 9) or tissue inhibitors of MMPs (1, 2, 3, and 4). TAC resulted in a significant increase in left ventricular (LV) weight and LV end-diastolic pressure (EDP) with preserved systolic function. KO+TAC mice exerted significantly lower LV weight/body weight (4.2+/-0.2 versus 5.0+/-0.2 mg/g; P<0.01), lung weight/body weight (4.9+/-0.2 versus 6.2+/-0.4 mg/g; P<0.01), and LV end-diastolic pressure (4+/-1 versus 10+/-2 mm Hg; P<0.05) than WT+TAC mice despite comparable aortic pressure. KO+TAC mice had less myocyte hypertrophy (cross-sectional area; 322+/-14 versus 392+/-14 microm2; P<0.01) and interstitial fibrosis (collagen volume fraction; 3.3+/-0.5 versus 8.2+/-1.0%; P<0.01) than WT+TAC mice. MMP 2 plays an important role in PO-induced LV hypertrophy and dysfunction. The inhibition of MMP 2 activation may, therefore, be a useful therapeutic strategy to manage hypertensive heart disease.     

Differences in adipose tissue metabolism between postmenopausal and perimenopausal women

Changes in adipose tissue metabolism may contribute to the changes in body fat distribution seen during the menopause transition. We compared in vitro abdominal and gluteal sc adipose tissue metabolism [basal and stimulated lipolysis and activity of adipose tissue lipoprotein lipase (AT-LPL)] in postmenopausal and perimenopausal women (n = 12/group), matched for race, body mass index (29.5 +/- 3.8 kg/m(2); mean +/- SD), and percentage body fat (42 +/- 6%). The postmenopausal women were older (54 +/- 3 vs. 48 +/- 3 yr; P < 0.01) and had higher FSH (55.5 +/- 26.4 vs. 16.6 +/- 22.5 IU/ml; P < 0.01) and lower estradiol (33.8 +/- 14.9 vs. 97.4 +/- 61.7 pmol/liter; P < 0.05) concentrations than the perimenopausal women. Despite similar fat cell size and beta-adrenergic receptor and postreceptor (dibutyryl-cAMP)-stimulated lipolysis, basal lipolysis was 77% lower in gluteal adipose cells from postmenopausal compared with perimenopausal women (P < 0.05). Within each group, AT-LPL activity in the gluteal region was significantly higher than in the abdominal region (P < 0.05). In addition, AT-LPL activity was significantly higher in the postmenopausal compared with perimenopausal women in both gluteal (4.9 +/- 3.6 vs. 2.0 +/- 1.4 nmol free fatty acid/g.min; P < 0.05) and abdominal (3.2 +/- 2.6 vs. 1.3 +/- 0.9 nmol free fatty acid/g.min; P < 0.05) adipose cells. The results of this study suggest that menopause status is associated with differences in adipose tissue metabolism in both the abdominal and gluteal fat depots. The lower lipolysis and higher AT-LPL activity in postmenopausal women may predispose them to gain body fat after menopause.