PKCλ is implicated as a downstream effector of PI3K in insulin action. We show here that mice that lack PKCλ specifically in the liver (L-λKO mice), produced with the use of the Cre-loxP system, exhibit increased insulin sensitivity as well as a decreased triglyceride content and reduced expression of the sterol regulatory element–binding protein-1c (SREBP-1c) gene in the liver. Induction of the hepatic expression of Srebp1c and of its target genes involved in fatty acid/triglyceride synthesis by fasting and refeeding or by hepatic expression of an active form of PI3K was inhibited in L-λKO mice compared with that in control animals. Expression of Srebp1c induced by insulin or by active PI3K in primary cultured rat hepatocytes was inhibited by a dominant-negative form of PKCλ and was mimicked by overexpression of WT PKCλ. Restoration of PKCλ expression in the liver of L-λKO mice with the use of adenovirus-mediated gene transfer corrected the metabolic abnormalities of these animals. Hepatic PKCλ is thus a determinant of hepatic lipid content and whole-body insulin sensitivity.
Michihiro Matsumoto, Wataru Ogawa, Kazunori Akimoto, Hiroshi Inoue, Kazuaki Miyake, Kensuke Furukawa, Yoshitake Hayashi, Haruhisa Iguchi, Yasushi Matsuki, Ryuji Hiramatsu, Hitoshi Shimano, Nobuhiro Yamada, Shigeo Ohno, Masato Kasuga, Tetsuo Noda
Thyroid hormone action is mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. DNA-binding is presumed to be essential for all nuclear actions of thyroid hormone. To test this hypothesis in vivo, the DNA-binding domain of TR-β was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-β DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homozygous mutant (TR-βGS/GS) mice displayed abnormal T3 regulation of the hypothalamic-pituitary-thyroid axis and retina identical to abnormalities previously observed in TR-β KO (TR-β–/–) mice. However, TR-βGS/GS mutant mice maintained normal hearing at certain frequencies and did not display significant outer hair cell loss, in contrast to TR-β–/– mice. DNA-binding, therefore, is essential for many functions of the TR, including retinal development and negative feedback regulation by thyroid hormone of the hypothalamic-pituitary-thyroid axis. Inner ear development, although not completely normal, can occur in the absence of TR DNA-binding, suggesting that an alternative and perhaps novel thyroid hormone-signaling pathway may mediate these effects.
Nobuyuki Shibusawa, Koshi Hashimoto, Amisra A. Nikrodhanond, M. Charles Liberman, Meredithe L. Applebury, Xiao Hui Liao, Janet T. Robbins, Samuel Refetoff, Ronald N. Cohen, Fredric E. Wondisford
The type 2 iodothyronine deiodinase (D2) is an integral membrane ER-resident selenoenzyme that activates the pro-hormone thyroxine (T4) and supplies most of the 3,5,3′-triiodothyronine (T3) that is essential for brain development. D2 is inactivated by selective conjugation to ubiquitin, a process accelerated by T4 catalysis and essential for the maintenance of T3 homeostasis. A yeast two-hybrid screen of a human-brain library with D2 as bait identified von Hippel–Lindau protein–interacting deubiquitinating enzyme-1 (VDU1). D2 interaction with VDU1 and VDU2, a closely related deubiquitinase, was confirmed in mammalian cells. Both VDU proteins colocalize with D2 in the ER, and their coexpression prolongs D2 half-life and activity by D2 deubiquitination. VDU1, but not VDU2, is markedly increased in brown adipocytes by norepinephrine or cold exposure, further amplifying the increase in D2 activity that results from catecholamine-stimulated de novo synthesis. Thus, deubiquitination regulates the supply of active thyroid hormone to brown adipocytes and other D2-expressing cells.
Cyntia Curcio-Morelli, Ann Marie Zavacki, Marcelo Christofollete, Balazs Gereben, Beatriz C.G. de Freitas, John W. Harney, Zaibo Li, Guan Wu, Antonio C. Bianco
NO prevents atherogenesis and inflammation in vessel walls by inhibition of cell proliferation and cytokine-induced endothelial expression of adhesion molecules and proinflammatory cytokines. Reduced NO production due to inhibition of either eNOS or iNOS may therefore reinforce atherosclerosis. Patients with end-stage renal failure show markedly increased mortality due to atherosclerosis. In the present study we tested the hypothesis that uremic toxins are responsible for reduced iNOS expression. LPS-induced iNOS expression in mononuclear leukocytes was studied using real-time PCR. The iNOS expression was blocked by addition of plasma from patients with end-stage renal failure, whereas plasma from healthy controls had no effect. Hemofiltrate obtained from patients with end-stage renal failure was fractionated by chromatographic methods. The chromatographic procedures revealed a homogenous fraction that inhibits iNOS expression. Using gas chromatography/mass spectrometry, this inhibitor was identified as phenylacetic acid. Authentic phenylacetic acid inhibited iNOS expression in a dose-dependent manner. In healthy control subjects, plasma concentrations were below the detection level, whereas patients with end-stage renal failure had a phenylacetic acid concentration of 3.49 ± 0.33 mmol/l (n = 41). It is concluded that accumulation of phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression. That mechanism may contribute to increased atherosclerosis and cardiovascular morbidity in patients with end-stage renal failure.
J. Jankowski, M. van der Giet, V. Jankowski, S. Schmidt, M. Hemeier, B. Mahn, G. Giebing, M. Tölle, H. Luftmann, H. Schlüter, W. Zidek, M. Tepel
Obesity is closely associated with the metabolic syndrome, a combination of disorders including insulin resistance, diabetes, dyslipidemia, and hypertension. A role for local glucocorticoid reamplification in obesity and the metabolic syndrome has been suggested. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regenerates active cortisol from inactive 11-keto forms, and aP2-HSD1 mice with relative transgenic overexpression of this enzyme in fat cells develop visceral obesity with insulin resistance and dyslipidemia. Here we report that aP2-HSD1 mice also have high arterial blood pressure (BP). The mice have increased sensitivity to dietary salt and increased plasma levels of angiotensinogen, angiotensin II, and aldosterone. This hypertension is abolished by selective angiotensin II receptor AT-1 antagonist at a low dose that does not affect BP in non-Tg littermates. These findings suggest that activation of the circulating renin-angiotensin system (RAS) develops in aP2-HSD1 mice. The long-term hypertension is further reflected by an appreciable hypertrophy and hyperplasia of the distal tubule epithelium of the nephron, resembling salt-sensitive or angiotensin II–mediated hypertension. Taken together, our findings suggest that overexpression of 11β-HSD1 in fat is sufficient to cause salt-sensitive hypertension mediated by an activated RAS. The potential role of adipose 11β-HSD1 in mediating critical features of the metabolic syndrome extends beyond obesity and metabolic complications to include the most central cardiovascular feature of this disorder.
Hiroaki Masuzaki, Hiroshi Yamamoto, Christopher J. Kenyon, Joel K. Elmquist, Nicholas M. Morton, Janice M. Paterson, Hiroshi Shinyama, Matthew G.F. Sharp, Stewart Fleming, John J. Mullins, Jonathan R. Seckl, Jeffrey S. Flier
It has been found that 4-estren-3α,17β-diol, a synthetic ligand for the estrogen receptor (ER) or androgen receptor (AR), which does not affect classical transcription, reverses bone loss in ovariectomized females or orchidectomized males without affecting the uterus or seminal vesicles, demonstrating that the classical genotropic actions of sex steroid receptors are dispensable for their bone-protective effects, but indispensable for their effects on reproductive organs. We have now investigated the mechanism of action of this compound. We report that, identically to 17β-estradiol or dihydrotestosterone, but differently from raloxifene, estren alters the activity of Elk-1, CCAAT enhancer binding protein–β (C/EBPβ), and cyclic adenosine monophosphate–response element binding protein (CREB), or c-Jun/c-Fos by an extranuclear action of the ER or AR, resulting in activation of the Src/Shc/ERK pathway or downregulation of JNK, respectively. All of these effects are non–sex specific, require only the ligand-binding domain of the receptor, and are indispensable for the antiapoptotic action of these ligands on osteoblastic and HeLa cells. Moreover, administration of 17β-estradiol or 4-estren-3α,17β-diol to ovariectomized mice induces phosphorylation of ERKs, Elk-1, and C/EBPβ, downregulates c-Jun, and upregulates the expression of egr-1, an ERK/SRE target gene. Kinase-initiated regulation of commonly used transcription factors offers a molecular explanation for the profound skeletal effects of sex steroid receptor ligands, including synthetic ones that are devoid of classical transcriptional activity.
Stavroula Kousteni, Li Han, Jin-Ran Chen, Maria Almeida, Lilian I. Plotkin, Teresita Bellido, Stavros C. Manolagas
To elucidate the role of leptin in regulating neuroendocrine and metabolic function during an acute fast, six to eight healthy, lean men were studied under four separate conditions: a baseline fed state and three 72-hour fasting studies with administration of either placebo, low-dose recombinant-methionyl human leptin (r-metHuLeptin), or replacement-dose r-metHuLeptin designed to maintain serum leptin at levels similar to those in the fed state. Replacement-dose r-metHuLeptin administered during fasting prevents the starvation-induced changes in the hypothalamic-pituitary-gonadal axis and, in part, the hypothalamic-pituitary-thyroid axis and IGF-1 binding capacity in serum. Thus, in normal men, the fall in leptin with fasting may be both necessary and sufficient for the physiologic adaptations of these axes, which require leptin levels above a certain threshold for activation. In contrast to findings in mice, fasting-induced changes in the hypothalamic-pituitary-adrenal, renin-aldosterone, and growth hormone–IGF-1 axes as well as fuel utilization may be independent of leptin in humans. The role of leptin in normalizing several starvation-induced neuroendocrine changes may have important implications for the pathophysiology and treatment of eating disorders and obesity.
Jean L. Chan, Kathleen Heist, Alex M. DePaoli, Johannes D. Veldhuis, Christos S. Mantzoros
Rapid oscillations of visceral lipolysis have been reported. To examine the putative role of the CNS in oscillatory lipolysis, we tested the effects of β3-blockade on pulsatile release of FFAs. Arterial blood samples were drawn at 1-minute intervals for 120 minutes from fasted, conscious dogs (n = 7) during the infusion of saline or bupranolol (1.5 μg/kg/min), a high-affinity β3-blocker. FFA and glycerol time series were analyzed and deconvolution analysis was applied to estimate the rate of FFA release. During saline infusion FFAs and glycerol oscillated in phase at about eight pulses/hour. Deconvolution analysis showed bursts of lipolysis (nine pulses/hour) with time-dependent variation in burst frequency. Bupranolol completely removed rapid FFA and glycerol oscillations. Despite removal of lipolytic bursts, plasma FFAs (0.31 mM) and glycerol (0.06 mM) were not totally suppressed and deconvolution analysis revealed persistent non-oscillatory lipolysis (0.064 mM/min). These results show that lipolysis in the fasting state consists of an oscillatory component, which appears to be entirely dependent upon sympathetic innervation of the adipose tissue, and a non-oscillatory, constitutive component, which persists despite β3-blockade. The extinction of lipid fuel bursts by β3-blockade implies a role for the CNS in the maintenance of cyclic provision of lipid fuels.
Katrin Hücking, Marianthe Hamilton-Wessler, Martin Ellmerer, Richard N. Bergman
Shouhong Xuan, Tadahiro Kitamura, Jun Nakae, Katerina Politi, Yoshiaki Kido, Peter E. Fisher, Manrico Morroni, Saverio Cinti, Morris F. White, Pedro L. Herrera, Domenico Accili, Argiris Efstratiadis
Shoshana Yakar, Clifford J. Rosen, Wesley G. Beamer, Cheryl L. Ackert-Bicknell, Yiping Wu, Jun-Li Liu, Guck T. Ooi, Jennifer Setser, Jan Frystyk, Yves R. Boisclair, Derek LeRoith