Dietary sulfur amino acid restriction upregulates DICER to confer beneficial effects.

OBJECTIVE: Dietary restriction (DR) improves health and prolongs lifespan in part by upregulating type III endoribonuclease DICER in adipose tissue. In this study, we aimed to specifically test which missing dietary component was responsible for DICER upregulation. METHODS: We performed a nutrient screen in mouse preadipocytes and validated the results in vivo using different kinds of dietary interventions in wild type or genetically modified mice and worms, also testing the requirement of DICER on the effects of the diets. RESULTS: We found that sulfur amino acid restriction (i.e., methionine or cysteine) is sufficient to increase Dicer mRNA expression in preadipocytes. Consistently, while DR increases DICER expression in adipose tissue of mice, this effect is blunted by supplementation of the diet with methionine, cysteine, or casein, but not with a lipid or carbohydrate source. Accordingly, dietary methionine or protein restriction mirrors the effects of DR. These changes are associated with alterations in serum adiponectin. We also found that DICER controls and is controlled by adiponectin. In mice, DICER plays a role in methionine restriction-induced upregulation of Ucp1 in adipose tissue. In C. elegans, DR and a model of methionine restriction also promote DICER expression in the intestine (an analog of the adipose tissue) and prolong lifespan in a DICER-dependent manner. CONCLUSIONS: We propose an evolutionary conserved mechanism in which dietary sulfur amino acid restriction upregulates DICER levels in adipose tissue leading to beneficial health effects.

Adipocyte PU.1 knockout promotes insulin sensitivity in HFD-fed obese mice.

Insulin resistance is a key feature of obesity and type 2 diabetes. PU.1 is a master transcription factor predominantly expressed in macrophages but after HFD feeding PU.1 expression is also significantly increased in adipocytes. We generated adipocyte specific PU.1 knockout mice using adiponectin cre to investigate the role of PU.1 in adipocyte biology, insulin and glucose homeostasis. In HFD-fed obese mice systemic glucose tolerance and insulin sensitivity were improved in PU.1 AKO mice and clamp studies indicated improvements in both adipose and liver insulin sensitivity. At the level of adipose tissue, macrophage infiltration and inflammation was decreased and glucose uptake was increased in PU.1 AKO mice compared with controls. While PU.1 deletion in adipocytes did not affect the gene expression of PPARg itself, we observed increased expression of PPARg target genes in eWAT from HFD fed PU.1 AKO mice compared with controls. Furthermore, we observed decreased phosphorylation at serine 273 in PU.1 AKO mice compared with fl/fl controls, indicating that PPARg is more active when PU.1 expression is reduced in adipocytes. Therefore, in obesity the increased expression of PU.1 in adipocytes modifies the adipocyte PPARg cistrome resulting in impaired glucose tolerance and insulin sensitivity.

Loss of mitochondrial energetics is associated with poor recovery of muscle function but not mass following disuse atrophy.

Skeletal muscle atrophy is a clinically important outcome of disuse because of injury, immobilization, or bed rest. Disuse atrophy is accompanied by mitochondrial dysfunction, which likely contributes to activation of the muscle atrophy program. However, the linkage of muscle mass and mitochondrial energetics during disuse atrophy and its recovery is incompletely understood. Transcriptomic analysis of muscle biopsies from healthy older adults subject to complete bed rest revealed marked inhibition of mitochondrial energy metabolic pathways. To determine the temporal sequence of muscle atrophy and changes in intramyocellular lipid and mitochondrial energetics, we conducted a time course of hind limb unloading-induced atrophy in adult mice. Mitochondrial respiration and calcium retention capacity were diminished, whereas H2O2 emission was increased within 3 days of unloading before significant muscle atrophy. These changes were associated with a decrease in total cardiolipin and profound changes in remodeled cardiolipin species. Hind limb unloading performed in muscle-specific peroxisome proliferator-activated receptor-γ coactivator-1α/ß knockout mice, a model of mitochondrial dysfunction, did not affect muscle atrophy but impacted muscle function. These data suggest early mitochondrial remodeling affects muscle function but not mass during disuse atrophy. Early alterations in mitochondrial energetics and lipid remodeling may represent novel targets to prevent muscle functional impairment caused by disuse and to enhance recovery from periods of muscle atrophy.

Sarcolipin Signaling Promotes Mitochondrial Biogenesis and Oxidative Metabolism in Skeletal Muscle.

The major objective of this study was to understand the molecular basis of how sarcolipin uncoupling of SERCA regulates muscle oxidative metabolism. Using genetically engineered sarcolipin (SLN) mouse models and primary muscle cells, we demonstrate that SLN plays a crucial role in mitochondrial biogenesis and oxidative metabolism in muscle. Loss of SLN severely compromised muscle oxidative capacity without affecting fiber-type composition. Mice overexpressing SLN in fast-twitch glycolytic muscle reprogrammed mitochondrial phenotype, increasing fat utilization and protecting against high-fat diet-induced lipotoxicity. We show that SLN affects cytosolic Ca2+ transients and activates the Ca2+/calmodulin-dependent protein kinase II (CamKII) and PGC1α axis to increase mitochondrial biogenesis and oxidative metabolism. These studies provide a fundamental framework for understanding the role of sarcoplasmic reticulum (SR)-Ca2+ cycling as an important factor in mitochondrial health and muscle metabolism. We propose that SLN can be targeted to enhance energy expenditure in muscle and prevent metabolic disease.

Skeletal Muscle Thermogenesis and Its Role in Whole Body Energy Metabolism.

Obesity and diabetes has become a major epidemic across the globe. Controlling obesity has been a challenge since this would require either increased physical activity or reduced caloric intake; both are difficult to enforce. There has been renewed interest in exploiting pathways such as uncoupling protein 1 (UCP1)-mediated uncoupling in brown adipose tissue (BAT) and white adipose tissue to increase energy expenditure to control weight gain. However, relying on UCP1-based thermogenesis alone may not be sufficient to control obesity in humans. On the other hand, skeletal muscle is the largest organ and a major contributor to basal metabolic rate and increasing energy expenditure in muscle through nonshivering thermogenic mechanisms, which can substantially affect whole body metabolism and weight gain. In this review we will describe the role of Sarcolipin-mediated uncoupling of Sarcoplasmic Reticulum Calcium ATPase (SERCA) as a potential mechanism for increased energy expenditure both during cold and diet-induced thermogenesis.

Both brown adipose tissue and skeletal muscle thermogenesis processes are activated during mild to severe cold adaptation in mice.

Thermogenesis is an important homeostatic mechanism essential for survival and normal physiological functions in mammals. Both brown adipose tissue (BAT) (i.e. uncoupling protein 1 (UCP1)-based) and skeletal muscle (i.e. sarcolipin (SLN)-based) thermogenesis processes play important roles in temperature homeostasis, but their relative contributions differ from small to large mammals. In this study, we investigated the functional interplay between skeletal muscle- and BAT-based thermogenesis under mild versus severe cold adaptation by employing UCP1-/- and SLN-/- mice. Interestingly, adaptation of SLN-/- mice to mild cold conditions (16 °C) significantly increased UCP1 expression, suggesting increased reliance on BAT-based thermogenesis. This was also evident from structural alterations in BAT morphology, including mitochondrial architecture, increased expression of electron transport chain proteins, and depletion of fat droplets. Similarly, UCP1-/- mice adapted to mild cold up-regulated muscle-based thermogenesis, indicated by increases in muscle succinate dehydrogenase activity, SLN expression, mitochondrial content, and neovascularization, compared with WT mice. These results further confirm that SLN-based thermogenesis is a key player in muscle non-shivering thermogenesis (NST) and can compensate for loss of BAT activity. We also present evidence that the increased reliance on BAT-based NST depends on increased autonomic input, as indicated by abundant levels of tyrosine hydroxylase and neuropeptide Y. Our findings demonstrate that both BAT and muscle-based NST are equally recruited during mild and severe cold adaptation and that loss of heat production from one thermogenic pathway leads to increased recruitment of the other, indicating a functional interplay between these two thermogenic processes.

Role of SERCA Pump in Muscle Thermogenesis and Metabolism.

In muscle cells, the sarcoplasmic reticulum (SR) not only acts as a Ca2+ store, but also regulates the contractile characteristics of the muscle. Ca2+ release from the SR is the primary mechanism for activating muscle contraction and reuptake of Ca2+ by the sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump causes muscle relaxation. The SERCA pump isoforms are encoded by three genes, SERCA 1, 2, and 3, which are differentially expressed in muscle and determine SR Ca2+ dynamics by affecting the rate and amount of Ca2+ uptake, thereby affecting SR store and release of Ca2+ in muscle. In muscle, small molecular weight proteins, including Phospholamban (PLB) and Sarcolipin (SLN), also regulate the SERCA pump. Regulation of the SERCA pump by PLB or SLN affects cytosolic Ca2+ dynamics and changes in cytosolic Ca2+ not only affect contractile function, but also mitochondrial ATP production. Recent studies have shown that alterations in cytosolic Ca2+ affects Ca2+ entry into mitochondria and ATP production; thus, Ca2+ serves as an integrating signal between muscle contraction-dependent energy demand and mitochondrial energy production. In addition, changes in cytosolic Ca2+ can affect Ca2+ signaling pathways modulating gene expression and muscle growth. An emerging area of research shows that SR Ca2+ cycling is also a player in muscle-based nonshivering thermogenesis. Recent data shows that SERCA uncoupling by SLN leads to increased ATP hydrolysis and heat production. Our studies, using genetically altered mouse models of SLN, show that SLN/SERCA interaction plays an important role in muscle thermogenesis and metabolism, which will be discussed here, in great length. © 2017 American Physiological Society. Compr Physiol 7:879-890, 2017.

Fat-specific Dicer deficiency accelerates aging and mitigates several effects of dietary restriction in mice.

Aging increases the risk of type 2 diabetes, and this can be prevented by dietary restriction (DR). We have previously shown that DR inhibits the downregulation of miRNAs and their processing enzymes - mainly Dicer - that occurs with aging in mouse white adipose tissue (WAT). Here we used fat-specific Dicer knockout mice (AdicerKO) to understand the contributions of adipose tissue Dicer to the metabolic effects of aging and DR. Metabolomic data uncovered a clear distinction between the serum metabolite profiles of Lox control and AdicerKO mice, with a notable elevation of branched-chain amino acids (BCAA) in AdicerKO. These profiles were associated with reduced oxidative metabolism and increased lactate in WAT of AdicerKO mice and were accompanied by structural and functional changes in mitochondria, particularly under DR. AdicerKO mice displayed increased mTORC1 activation in WAT and skeletal muscle, where Dicer expression is not affected. This was accompanied by accelerated age-associated insulin resistance and premature mortality. Moreover, DR-induced insulin sensitivity was abrogated in AdicerKO mice. This was reverted by rapamycin injection, demonstrating that insulin resistance in AdicerKO mice is caused by mTORC1 hyperactivation. Our study evidences a DR-modulated role for WAT Dicer in controlling metabolism and insulin resistance.

Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance.

Metabolic syndrome is a cluster of metabolic risk factors such as obesity, diabetes and cardiovascular diseases. Mitochondria is the main site of ATP production and its dysfunction leads to decreased oxidative phosphorylation, resulting in lipid accumulation and insulin resistance. Our group has demonstrated that kinins can modulate glucose and lipid metabolism as well as skeletal muscle mass. By using B2 receptor knockout mice (B2R-/-) we investigated whether kinin action affects weight gain and physical performance of the animals. Our results show that B2R-/- mice are resistant to high fat diet-induced obesity, have higher glucose tolerance as well as increased mitochondrial mass. These features are accompanied by higher energy expenditure and a lower feed efficiency associated with an increase in the proportion of type I fibers and intermediary fibers characterized by higher mitochondrial content and increased expression of genes related to oxidative metabolism. Additionally, the increased percentage of oxidative skeletal muscle fibers and mitochondrial apparatus in B2R-/- mice is coupled with a higher aerobic exercise performance. Taken together, our data give support to the involvement of kinins in skeletal muscle fiber type distribution and muscle metabolism, which ultimately protects against fat-induced obesity and improves aerobic exercise performance.

Early pharmacological inhibition of angiotensin-I converting enzyme activity induces obesity in adulthood.

We have investigated early programming of body mass in order to understand the multifactorial etiology of obesity. Considering that the renin-angiotensin system (RAS) is expressed and functional in the white adipose tissue (WAT) and modulates its development, we reasoned whether early transitory inhibition of angiotensin-I converting enzyme activity after birth could modify late body mass development. Therefore, newborn Wistar rats were treated with enalapril (10 mg/kg of body mass) or saline, starting at the first day of life until the age of 16 days. Between days ninetieth and hundred and eightieth, a group of these animals received high fat diet (HFD). Molecular, biochemical, histological, and physiological data were collected. Enalapril treated animals presented hyperphagia, overweight, and increased serum level of triglycerides, total cholesterol and leptin, in adult life. Body composition analyses revealed higher fat mass with increased adipocyte size in these animals. Molecular analyses revealed that enalapril treatment increases neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) gene expression in hypothalamus, fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) gene expression in retroperitoneal WAT, and decreases peroxixome proliferators-activated receptor (PPAR)γ, PPARα, uncoupling protein (UCP)2, and UCP3 gene expression in WAT. The results of the current study indicate that enalapril administration during early postnatal development increases body mass, adiposity and serum lipids in adulthood associated with enhanced food intake and decreased metabolic activity in WAT, predisposing to obesity in adulthood.

Role of vascular Kinin B1 and B2 receptors in endothelial nitric oxide metabolism.

Kinin B(1) and B(2) receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B(1) (B(1)(-/-)) and B(2) receptor (B(2)(-/-)) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B(1)(-/-) and B(2)(-/-) in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B(1)(-/-) and B(2)(-/-). In contrast, in the presence of l-arginine, Ca(2+) and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B(1)(-/-) and B(2)(-/-). The present study demonstrated that targeted deletion of B(1) or B(2) receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B(1)(-/-) and B(2)(-/-) vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.

Myelopoiesis modulation by ACE hyperfunction in kinin B(1) receptor knockout mice: relationship with AcSDKP levels.

Angiotensin I-converting enzyme (ACE), a common element of renin-angiotensin system (RAS) and kallikrein-kinin system (KKS), is involved in myelopoiesis modulation, mainly by cleaving the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). Based on this finding and in our results showing B1 and B2 kinin receptors expression in murine bone marrow (BM) cells, we evaluated the ACE influence on myelopoiesis of kinin B1 receptor knockout mice (B1KO) using long-term bone marrow cultures (LTBMCs). Captopril and AcSDKP were used as controls. Enhanced ACE activity, expressed by non-hematopoietic cells (Ter-199(-) and CD45(-)), was observed in B1KO LTBMCs when compared to wild-type (WT) cells. ACE hyperfunction in B1KO cells was maintained when LTBMCs from B1KO mice were treated with captopril (1.0microM) or AcSDKP (1.0nM). Although no alterations were observed in ACE mRNA and protein levels under these culture conditions, 3.0nM of AcSDKP increased ACE mRNA levels in WT LTBMCs. No alteration in the number of GM-CFC was seen in B1KO mice compared to WT animals, even when the former were treated with AcSDKP (10microg/kg) or captopril (100mg/kg) for 4 consecutive days. Hematological data also revealed no differences between WT and B1KO mice under basal conditions. When the animals received 4 doses of lipopolysaccharide (LPS), a decreased number of blood cells was detected in B1KO mice in relation to WT. We also found a decreased percentage of Gr1(+)/Mac-1(+), Ter119(+), B220(+), CD3(+), and Lin(-)Sca1(+)c-Kit(+) (LSK) cells in the BM of B1KO mice compared to WT animals. Low AcSDKP levels were observed in BM cultures from B1KO in comparison to WT cultures. We conclude that ACE hyperfunction in B1KO mice resulted in faster hydrolysis of AcSDKP peptide, which in turn decreased in BM tissues allowing HSC to enter the S stage of the cell cycle.

Kinin B1 receptor deficiency leads to leptin hypersensitivity and resistance to obesity.

OBJECTIVE: Kinins mediate pathophysiological processes related to hypertension, pain, and inflammation through the activation of two G-protein-coupled receptors, named B(1) and B(2). Although these peptides have been related to glucose homeostasis, their effects on energy balance are still unknown. RESEARCH DESIGN AND METHODS: Using genetic and pharmacological strategies to abrogate the kinin B(1) receptor in different animal models of obesity, here we present evidence of a novel role for kinins in the regulation of satiety and adiposity. RESULTS: Kinin B(1) receptor deficiency in mice (B(1)(-/-)) resulted in less fat content, hypoleptinemia, increased leptin sensitivity, and robust protection against high-fat diet-induced weight gain. Under high-fat diet, B(1)(-/-) also exhibited reduced food intake, improved lipid oxidation, and increased energy expenditure. Surprisingly, B(1) receptor deficiency was not able to decrease food intake and adiposity in obese mice lacking leptin (ob/ob-B(1)(-/-)). However, ob/ob-B(1)(-/-) mice were more responsive to the effects of exogenous leptin on body weight and food intake, suggesting that B(1) receptors may be dependent on leptin to display their metabolic roles. Finally, inhibition of weight gain and food intake by B(1) receptor ablation was pharmacologically confirmed by long-term administration of the kinin B(1) receptor antagonist SSR240612 to mice under high-fat diet. CONCLUSIONS: Our data suggest that kinin B(1) receptors participate in the regulation of the energy balance via a mechanism that could involve the modulation of leptin sensitivity.

ACE activity is modulated by kinin B2 receptor.

Angiotensin-converting enzyme (ACE) is an ectoprotein able to modulate the activity of a plethora of compounds, among them angiotensin I and bradykinin. Despite several decades of research, new aspects of the mechanism of action of ACE have been elucidated, expanding our understanding of its role not only in cardiovascular regulation but also in different areas. Recent findings have ascribed an important role for ACE/kinin B(2) receptor heterodimerization in the pharmacological properties of the receptor. In this work, we tested the hypothesis that this interaction also affects ACE enzymatic activity. ACE catalytic activity was analyzed in Chinese hamster ovary cell monolayers coexpressing the somatic form of the enzyme and the receptor coding region using as substrate the fluorescence resonance energy transfer peptide Abz-FRK(Dnp)P-OH. Results show that the coexpression of the kinin B(2) receptor leads to an augmentation in ACE activity. In addition, this effect could be blocked by the B(2) receptor antagonist icatibant. The hypothesis was also tested in endothelial cells, a more physiological system, where both proteins are naturally expressed. Endothelial cells from genetically ablated kinin B(2) receptor mice showed a decreased ACE activity when compared with wild-type mice cells. In summary, this is the first report showing that the ACE/kinin B(2) receptor interaction modulates ACE activity. Taking into account the interplay among ACE, ACE inhibitors, and kinin receptors, we believe that these results will shed new light into the arena of the controversial search for the mechanism controlling these interactions.

Swimming training exacerbates pathological cardiac hypertrophy in kinin B2 receptor-deficient mice.

Kallikrein-kinin system exerts cardioprotective effects against pathological hypertrophy. These effects are modulated mainly via B2 receptor activation. Chronic physical exercise can induce physiological cardiac hypertrophy characterized by normal organization of cardiac structure. Therefore, the aim of this work was to verify the influence of kinin B2 receptor deletion on physiological hypertrophy to exercise stimulus. Animals were submitted to swimming practice for 5 min or for 60 min, 5 days a week, during 1 month and several cardiac parameters were evaluated. Results showed no significantly difference in heart weight between both groups, however an increased left ventricle weight and myocyte diameter were observed after the 60 min swimming protocol, which was more pronounced in B2(-/-) mice. In addition, sedentary B2(-/-) animals presented higher left ventricle mass when compared to wild-type (WT) mice. An increase in capillary density was observed in exercised animals, however the effect was less pronounced in B2(-/-) mice. Collagen, a marker of pathological hypertrophy, was increased in B2(-/-) mice submitted to swimming protocol, as well as left ventricular thickness, suggesting that these animals do not respond with physiological hypertrophy for this kind of exercise. In conclusion, our data suggest an important role for the kinin B2 receptor in physiological cardiac hypertrophy.

Increase in kinins on post-exercise hypotension in normotensive and hypertensive volunteers.

Post-exercise hypotension is an important event for blood pressure regulation, especially in hypertensive individuals. Although post-exercise hypotension is a well-known phenomenon, the mechanism responsible is still unclear. The kallikrein-kinin system is involved in blood pressure control, but its role in post-exercise hypotension has not yet been investigated. Thus, the purpose of this study was to investigate the involvement of the vasodilators bradykinin and des-Arg(9)-BK and kallikrein activity in post-exercise hypotension promoted by 35 min of cycle ergometer (CE) or circuit weight-training (CWT) bouts in normotensive and hypertensive individuals. A significant decrease in mean arterial pressure at 45 and 60 min after CE and 45 min after CWT was observed in normotensive individuals. Hypertensive values of mean arterial pressure were significantly reduced at 45 and 60 min after CE and at 60 min after CWT. Before exercise, plasma bradykinin concentrations and kallikrein activity were higher in hypertensive compared to normotensive volunteers. Kinin levels increased in the groups evaluated at the end of the training period and 60 min post-exercise. These data suggest that the kallikrein-kinin system may be involved in post-exercise hypotension in normotensive and hypertensive individuals subjected to CE and CWT bouts.