SUMMARY While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here, we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.

Bruss MD, Khambatta CF, Ruby MA, Aggarwal I, Hellerstein MK. Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates. Am J Physiol Endocrinol Metab 298: E108-E116, 2010. First published November 3, 2009 zdoi:10.1152/ajpendo.00524.2009.-Calorie restriction (CR) increases longevity and retards the development of many chronic diseases, but the underlying metabolic signals are poorly understood. Increased fatty acid (FA) oxidation and reduced FA synthesis have been hypothesized to be important metabolic adaptations to CR. However, at metabolic steady state, FA oxidation must match FA intake plus synthesis; moreover, FA intake is low, not high, during CR. Therefore, it is not clear how FA dynamics are altered during CR. Accordingly, we measured food intake patterns, whole body fuel selection, endogenous FA synthesis, and gene expression in mice on CR. Within 2 days of CR being started, a shift to a cyclic, diurnal pattern of whole body FA metabolism occurred, with an initial phase of elevated endogenous FA synthesis [respiratory exchange ratio (RER) Ͼ1.10, lasting 4 -6 h after food provision], followed by a prolonged phase of FA oxidation (RER ϭ 0.70, lasting 18 -20 h). CR mice oxidized four times as much fat per day as ad libitum (AL)-fed controls (367 Ϯ 19 vs. 97 Ϯ 14 mg/day, P Ͻ O.001) despite reduced energy intake from fat. This increase in FA oxidation was balanced by a threefold increase in adipose tissue FA synthesis compared with AL. Expression of FA synthase and acetyl-CoA carboxylase mRNA were increased in adipose and liver in a timedependent manner. We conclude that CR induces a surprising metabolic pattern characterized by periods of elevated FA synthesis alternating with periods of FA oxidation disproportionate to dietary FA intake. This pattern may have implications for oxidative damage and disease risk. fat synthesis; lipogenesis; palmitoleate; heavy water CALORIE RESTRICTION (CR) delays the development of chronic disease and prolongs lifespan in mice (1,17,27,34). These effects correlate with a rapid induction in the expression of certain genes that persist as long as animals remain on CR (10, 36) even after energy balance is restored. These observations suggest the presence of a chronic signal of reduced energy availability that persists after energy balance has been reestablished. However, the underlying metabolic signals and adaptations responsible are not fully understood.Mice on CR regimens have been reported to exhibit increased expression of genes for fatty acid (FA) oxidation and decreased expression of genes for FA synthesis compared with ad libitum (AL)-fed controls (6,7,30,38). Due to differential entry points into the electron transport chain, a metabolic shift from carbohydrate to FA oxidation may reduce the production of reactive oxygen species (ROS) (15). A shift to FA oxidation thereby represents a potential mechanism for reduced oxidative damage, which has been proposed as a potential explanation for the health benefits of CR (14,15,29,35). It has also be...

T he signaling pathways that mediate insulin's many actions remain incompletely understood, but the following sequence has been well characterized: insulin binds to its receptor, leading to receptor autophosphorylation and activation of receptor tyrosine kinase, which in turn results in tyrosine phosphorylation of endogenous substrates including insulin receptor substrate proteins. These docking proteins engage downstream signaling molecules such as phosphatidylinositol (PI) 3-kinase (1-3). PI 3-kinase catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate on the D3 position of inositol, and the resultant PI 3,4,5-trisphosphate binds and activates more distal signaling proteins, including phosphoinositide-dependent kinase-1 and Akt, a serine/threonine kinase. Akt has been implicated as a key signaling protein for several of insulin's actions, including activation of glycogen synthesis, protein synthesis, and GLUT4 translocation to the cell surface, thereby increasing glucose transport (1-5). Identification of the intermediate signaling steps linking Akt to insulin's diverse actions remains incomplete.Recent research using 3T3-L1 adipocytes demonstrated that insulin leads to the phosphorylation of multiple proteins that contain one or more consensus sequences for phosphorylation by Akt (6). Among these Akt substrates was a 160-kDa protein, named Akt substrate of 160 kDa (AS160), which contained six Akt consensus sequences that become phosphorylated in insulin-treated adipocytes. AS160 also includes a GTPase-activating domain for small G-proteins, known as Rabs, which participate in vesicular trafficking (7). A point mutation of two or more of the consensus phosphorylation sites for Akt resulted in a marked decline in insulin-stimulated GLUT4 redistribution to the cell surface. Thus, in 3T3-L1 adipocytes, phosphorylation of AS160 was strongly implicated as an intermediate step linking insulin's activation of Akt to increased glucose transport. More recently, Zeigerer et al. (8) demonstrated that AS160 is important for insulin's activation of GLUT4 vesicle exocytosis without altering insulin-mediated inhibition of GLUT4 internalization.Insulin also activates Akt in skeletal muscle (9,10), and AS160 is expressed by this tissue (6), which is a major target for insulin action. An important question is this: Does insulin, in skeletal muscle, lead to increased phosphorylation of AS160 and/or other proteins containing the Akt phosphomotif? Accordingly, our first aim was to AICAR, 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside; AS160, Akt substrate of 160 kDa; KHB, Krebs-Henseleit buffer; PAS, phospho-(Ser/Thr) Akt substrate; PI, phosphatidylinositol; TBST, Tris-buffered saline plus Tween.

β1- and β2-adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that β1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β-arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1- and β2-adrenoceptor signaling.

SUMMARY It is proposed that caloric restriction (CR) increases mitochondrial biogenesis. However, it is not clear why CR increases an energetically costly biosynthetic process. We hypothesized that 40% CR would decrease mitochondrial protein synthesis and would be regulated by translational rather than transcriptional mechanisms. We assessed cumulative mitochondrial protein synthesis over 6 weeks and its transcriptional and translational regulation in the liver, heart, and skeletal muscle of young (6 mo), middle (12 mo), and old (24 mo) male B6D2F1 mice that were lifelong CR or ad lib (AL) controls. Mitochondrial protein synthesis was not different between AL and CR (Fractional synthesis over 6-weeks (range): liver 91 – 100%, heart 74–85% skeletal muscle 53–72%) despite a decreased cellular proliferation in liver and heart with CR. With CR there was an increase in AMP activated protein kinase (AMPK) phosphorylation:total (P:T) in heart and liver, and an increase in peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) mRNA in all tissues, but not protein. Ribosomal protein S6 (RpS6) was decreased with CR. In conclusion, CR maintained mitochondrial protein synthesis while decreasing cellular proliferation during a time of energetic stress, which is consistent with the concept that CR increases somatic maintenance. Alternative mechanisms to global translation initiation may be responsible for selective translation of mitochondrial proteins.

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

Increased protein synthesis is proposed as a mechanism of life-span extension during caloric restriction (CR). We hypothesized that CR does not increase protein synthesis in all tissues and protein fractions and that any increased protein synthesis with CR would be due to an increased anabolic effect of feeding. We used short- (4 hours) and long-term (6 weeks) methods to measure in vivo protein synthesis in lifelong ad libitum (AL) and CR mice. We did not detect an acute effect of feeding on protein synthesis while liver mitochondrial protein synthesis was lower in CR mice versus AL mice. Mammalian target of rapamycin (mTOR) signaling was repressed in liver and heart from CR mice indicative of energetic stress and suppression of growth. Our main findings were that CR did not increase rates of mixed protein synthesis over the long term or in response to acute feeding, and protein synthesis was maintained despite decreased mTOR signaling.

Complex diseases result from molecular changes induced by multiple genetic factors and the environment. To derive a systems view of how genetic loci interact in the context of tissue-specific molecular networks, we constructed an F2 intercross comprised of >500 mice from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mouse strains made genetically obese by the Leptinob/ob mutation (Lepob). High-density genotypes, diabetes-related clinical traits, and whole-transcriptome expression profiling in five tissues (white adipose, liver, pancreatic islets, hypothalamus, and gastrocnemius muscle) were determined for all mice. We performed an integrative analysis to investigate the inter-relationship among genetic factors, expression traits, and plasma insulin, a hallmark diabetes trait. Among five tissues under study, there are extensive protein–protein interactions between genes responding to different loci in adipose and pancreatic islets that potentially jointly participated in the regulation of plasma insulin. We developed a novel ranking scheme based on cross-loci protein-protein network topology and gene expression to assess each gene's potential to regulate plasma insulin. Unique candidate genes were identified in adipose tissue and islets. In islets, the Alzheimer's gene App was identified as a top candidate regulator. Islets from 17-week-old, but not 10-week-old, App knockout mice showed increased insulin secretion in response to glucose or a membrane-permeant cAMP analog, in agreement with the predictions of the network model. Our result provides a novel hypothesis on the mechanism for the connection between two aging-related diseases: Alzheimer's disease and type 2 diabetes.