Miriam O. Ribeiro

Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension.

Recent studies have indicated that acute inhibition of nitric oxide biosynthesis in the rat promotes arterial hypertension and renal vasoconstriction. We evaluated the renal and systemic effects of 4-6 weeks of nitric oxide blockade in Munich-Wistar rats receiving the nitric oxide inhibitor nitro-L-arginine orally. Age-matched untreated rats were used as controls. In an additional seven rats, nitric oxide blockade was carried out in conjunction with oral administration of the novel angiotensin II antagonist losartan potassium. Tail-cuff pressure rose progressively in nitro-L-arginine-treated rats, reaching 164 +/- 6 mm Hg at 4-6 weeks, compared with 108 +/- 3 mm Hg in controls. In rats concomitantly receiving losartan, tail-cuff pressure reached 125 +/- 6 mm Hg, still elevated compared with rats receiving losartan alone (98 +/- 3 mm Hg). Nitro-L-arginine-treated rats presented marked renal vasoconstriction and hypoperfusion, as well as a 30% fall in glomerular filtration rate and a 39% increase in filtration fraction. Treatment with Losartan normalized glomerular filtration rate, but not filtration fraction or renal vascular resistance. Plasma renin activity was elevated after nitro-L-arginine treatment. Renal histological examination revealed widespread arteriolar narrowing, focal arteriolar obliteration, and segmental fibrinoid necrosis in the glomeruli. In a separate group of rats, nitro-L-arginine administered for 1 week induced hypertension that was partially reversed by acute L-arginine, but not D-arginine or L-glycine, infusions. We conclude that chronic nitric oxide blockade may constitute a new model of severe arterial hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal effects of acute and chronic nitric oxide inhibition in experimental diabetes

We investigated whether nitric oxide (NO) contributes to glomerular hyperfiltration in experimental diabetes. Thirty-five adult male Munich-Wistar streptozocin-diabetic rats and 39 nondiabetic controls were distributed among 4 groups: C, normal control; C + L-NAME, controls receiving the NO inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), 40 mg/dl in drinking water; DM, diabetic rats; DM + L-NAME, diabetic rats receiving L-NAME, 15 mg/dl in drinking water. After 1 month of treatment, the DM + L-NAME group exhibited renal vasoconstriction and lacked hyperfiltration. Acute administration of L-NAME, 2.5 mg/kg, depressed the glomerular filtration rate and promoted renal vasoconstriction to a much greater extent in the DM than in the C group. Acute administration of endothelin 1 (600 ng/kg, bolus) or angiotensin II (25 micrograms/kg/min, continuous infusion) exerted similar hemodynamic effects in the C and DM groups, suggesting that the enhanced response of DM to L-NAME reflected specific sensitivity to NO inhibition. Urinary excretion of nitrites and nitrates was fourfold higher in DM compared to C. These results support the notion that augmented NO production may contribute to renal hyperfiltration and hyperperfusion in diabetes.

β1 Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple β-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the β(1) isoform in energy homeostasis. First, the 30  min i.v. infusion of norepinephrine (NE) or the β(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the β(1) gene (KO of β(1) adrenergic receptor (β(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, β(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, β(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the β(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

Effects of thyroid hormone analogs on lipid metabolism and thermogenesis.

Thyroid hormone affects in a myriad of biological processes such as development, growth, and metabolic control. Triiodothyronine (T3) is the biologically active form of thyroid hormone that acts through nuclear receptors, TRalpha and TRbeta, regulating gene expression. Given that the distribution of these receptors is heterogeneous amongst the different tissues, it is not surprising that some physiological effects of T3 are isoform specific. For example, while TRalpha is the dominant receptor in the brain and skeletal system and mediates most of the synergism between T3 and the sympathetic signaling pathway in the heart, TRbeta is abundant in liver and is probably the isoform that mediates most of the T3 effects on lipid metabolism. Thus, it makes sense to develop compounds that selectively act on either one of the TRs, allowing for the activation of specific T3-dependent pathways. This article reviews the recent progress made in this area, focusing on the physiological effects of compounds that lower serum cholesterol and decrease fat mass, as they spare skeletal muscle and bone masses, as well as the heart. The available studies indicate that achieving selective activation of different TR-mediated pathways is a promising strategy for treating lipid disorders and obesity.

A TRβ-selective agonist confers resistance to diet-induced obesity

Thyroid hormone receptor beta (TRbeta also listed as THRB on the MGI Database)-selective agonists activate brown adipose tissue (BAT) thermogenesis, while only minimally affecting cardiac activity or lean body mass. Here, we tested the hypothesis that daily administration of the TRbeta agonist GC-24 prevents the metabolic alterations associated with a hypercaloric diet. Rats were placed on a high-fat diet and after a month exhibited increased body weight (BW) and adiposity, fasting hyperglycemia and glucose intolerance, increased plasma levels of triglycerides, cholesterol, nonesterified fatty acids and interleukin-6. While GC-24 administration to these animals did not affect food ingestion or modified the progression of BW gain, it did increase energy expenditure, eliminating the increase in adiposity without causing cardiac hypertrophy. Fasting hyperglycemia remained unchanged, but treatment with GC-24 improved glucose tolerance by increasing insulin sensitivity, and also normalized plasma triglyceride levels. Plasma cholesterol levels were only partially normalized and liver cholesterol content remained high in the GC-24-treated animals. Gene expression in liver, skeletal muscle, and white adipose tissue was only minimally affected by treatment with GC-24, with the main target being BAT. In conclusion, during high-fat feeding treatment with the TRbeta-selective agonist, GC-24 only partially improves metabolic control probably as a result of accelerating the resting metabolic rate.

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting (∼70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase (∼3.0°C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.

Nifedipine Prevents Renal Injury in Rats With Chronic Nitric Oxide Inhibition

Chronic nitric oxide inhibition promotes hypertension, renal dysfunction, and renal injury by unclear mechanisms. We examined the effects in this model of concomitant treatment with the calcium channel blocker nifedipine. Six adult male Munich-Wistar rats received 0.025% nifedipine in chow. Six untreated rats served as controls. Fifteen days later, renal function was evaluated in anesthetized rats before and after a bolus injection of the nitric oxide inhibitor N omega-nitro-L-arginine methyl ester at 3 mg/kg IV. Renal vasoconstriction and systemic hypertension induced by the inhibitor were similar in untreated and nifedipine-treated rats. In a second protocol, eight rats received the nitric oxide inhibitor in their drinking water at 2.6 mmol/L. Eight additional rats also received nifedipine as above. At day 15, rats given the nitric oxide inhibitor exhibited systemic hypertension and renal vasoconstriction. Simultaneous nifedipine lowered blood pressure slightly without ameliorating renal hemodynamics. Tail-cuff pressure rose continuously in rats receiving the nitric oxide blocker, reaching 171 +/- 7 mm Hg at 30 days, but remained at 143 +/- 3 mm Hg in rats also given nifedipine. At this stage, rats treated with the nitric oxide inhibitor exhibited extremely variable plasma renin activity, tuft collapse in 10.1 +/- 2.2% of the glomeruli, and renal interstitial fibrosis. Simultaneous nifedipine treatment normalized the dispersion of plasma renin levels, while preventing renal morphological abnormalities. These results suggest that in the chronic nitric oxide inhibition model, sustained operation of voltage-sensitive calcium channels is not essential for renal vasoconstriction but contributes to systemic hypertension and plays a pivotal role in the development of renal structural injury.

Scope and limitations of iodothyronine deiodinases in hypothyroidism

The coordinated expression and activity of the iodothyronine deiodinases regulate thyroid hormone levels in hypothyroidism. Once heralded as the pathway underpinning adequate thyroid-hormone replacement therapy with levothyroxine, the role of these enzymes has come into question as they have been implicated in both an inability to normalize serum levels of tri-iodothyronine (T3) and the incomplete resolution of hypothyroid symptoms. These observations, some of which were validated in animal models of levothyroxine monotherapy, challenge the paradigm that tissue levels of T3 and thyroid-hormone signalling can be fully restored by administration of levothyroxine alone. The low serum levels of T3 observed among patients receiving levothyroxine monotherapy occur as a consequence of type 2 iodothyronine deiodinase (DIO2) in the hypothalamus being fairly insensitive to ubiquitination. In addition, residual symptoms of hypothyroidism have been linked to a prevalent polymorphism in the DIO2 gene that might be a risk factor for neurodegenerative disease. Here, we discuss how these novel findings underscore the clinical importance of iodothyronine deiodinases in hypothyroidism and how an improved understanding of these enzymes might translate to therapeutic advances in the care of millions of patients with this condition.

Mice lacking brain-type creatine kinase activity show defective thermoregulation

The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and UbCKmit) are expressed during the prepubescent and adult period of mammalian life. These creatine kinase (CK) isoforms are present in neural cell types throughout the central and peripheral nervous system and in smooth muscle containing tissues, where they have an important role in cellular energy homeostasis. Here, we report on the coupling of CK activity to body temperature rhythm and adaptive thermoregulation in mice. With both brain-type CK isoforms being absent, the body temperature reproducibly drops ~1.0 degrees C below normal during every morning (inactive) period in the daily cycle. Facultative non-shivering thermogenesis is also impaired, since CK--/-- mice develop severe hypothermia during 24 h cold exposure. A relationship with fat metabolism was suggested because comparison of CK--/-- mice with wildtype controls revealed decreased weight gain associated with less white and brown fat accumulation and smaller brown adipocytes. Also, circulating levels of glucose, triglycerides and leptin are reduced. Extensive physiological testing and uncoupling protein1 analysis showed, however, that the thermogenic problems are not due to abnormal responsiveness of brown adipocytes, since noradrenaline infusion produced a normal increase of body temperature. Moreover, we demonstrate that the cyclic drop in morning temperature is also not related to altered rhythmicity with reduced locomotion, diminished food intake or increased torpor sensitivity. Although several integral functions appear altered when CK is absent in the brain, combined findings point into the direction of inefficient neuronal transmission as the dominant factor in the thermoregulatory defect.

Positive impact of omega-3 fatty acid supplementation in a dog with drug-resistant epilepsy: a case study.

Epilepsy is the most common neurological disorder in both dogs and humans. Although the pharmacological options for treatment of epilepsies have increased, it has been reported that two-thirds of dogs with epilepsy are refractory to antiepileptic drug therapy. To our knowledge, there are no experimental studies in the literature that show an effect of omega-3 supplementation on epilepsy in dogs. Our case study describes the effectiveness of daily intake of a moderate amount of fish oil in a case of canine epilepsy.