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Brown fat expresses two PGC-1α isoforms (PGC-1α and NT-PGC-1α) and both play a central role in the regulation of cellular energy metabolism and adaptive thermogenesis by interacting with a wide range of transcription factors including PPARγ, PPARα, ERRα, and NRF1. PGC-1α consists of 797 amino acids, whereas alternative splicing of the PGC-1α gene produces a shorter protein called NT-PGC-1α (aa 1-270). We report in this paper that transcriptional activity of PGC-1α and NT-PGC-1α is differently affected by the transcriptional regulator, Twist-1. Twist-1 suppresses PGC-1α but not NT-PGC-1α. The inhibition of PGC-1α activity by Twist-1 is mediated by direct interaction through the C-terminal region of PGC-1α (aa 353-797). Thus, the absence of the corresponding C-terminal domain in NT-PGC-1α allows NT-PGC-1α to be free from Twist-1-mediated inhibition. Overexpression of Twist-1 in brown adipocytes suppresses transcription of a subset of PGC-1α-target genes involved in mitochondrial fatty acid oxidation and uncoupling (CPT1β, UCP1, and ERRα). In contrast, NT-PGC-1α-mediated induction of these genes is unaffected by Twist-1. These findings show that differences in inhibitory protein-protein interactions of PGC-1α and NT-PGC-1α with Twist-1 lead to differential regulation of their function by Twist-1. [ABSTRACT FROM AUTHOR]

OBJECTIVE -- The sympathetic nervous system and thiazolidinediones control lipid metabolism and have been implicated in body weight regulation. This study was conducted to determine whether the simultaneous activation of these two signaling systems might synergize to exert beneficial effects on the expression of key genes involved in lipid metabolism and mitochondrial biogenesis in subcutaneous fat in nondiabetic subjects. RESEARCH DESIGN AND METHODS -- A total of 57 women and men were randomized into four groups: 1) placebo/placebo (PP), 2) ephedrine HCl (25 mg, 3 times daily) plus caffeine (200 mg, 3 times daily)/placebo (ECP), 3) placebo/pioglitazone (45 mg) (PPio), and 4) ephedrine plus caffeine/pioglitazone (ECPio) for 16 weeks. Adipose tissue samples were obtained after 12 weeks of treatment to determine gene expression. RESULTS -- Body fat decreased by 6.0 and 4.6% in the ECP and ECPio groups, respectively, while remaining unchanged in the PPio and PP groups. Triglyceride levels decreased by -7.7, -24, -15.2, and -41 mg/dl after 16 weeks treatment in the PP, PPio, ECP, and ECPio groups, respectively. This indicates that pioglitazone groups with or without EC (ephedrine HCl plus caffeine) decreased triglycerides, and EC groups with or without pioglitazone decreased body weight. The mRNA for sirtuin 1 and CD36 increased only in the ECPio group. Carnitine palmitoyltransferase-1, medium-chain acyl CoA dehydrogenase, and malonyl-CoA decarboxylase increased with PPio and ECPio. Stearoyl-CoA desaturase decreased with ECP. CONCLUSIONS -- Combined activation of peroxisome proliferator-activated receptor-γ and β-adrenergic receptors has beneficial effects on body weight, plasma triglycerides, and lipid metabolism in subcutaneous fat by increasing the expression of genes required for fatty acid catabolism. [ABSTRACT FROM AUTHOR]

Siberian hamsters (Phodopus sungorus) exhibit a naturally occurring, reversible seasonal obesity with body fat peaking in long ‘summerlike’ days (LDs) and reaching a nadir in short ‘winterlike’ days (SDs). These SD-induced decreases in adiposity are mediated largely via sympathetic nervous system (SNS) innervation of white adipose tissue (WAT), as indicated by increased WAT norepinephrine (NE) turnover. We examined whether SDs also increase sensitivity to NE-stimulated lipolysis. This was accomplished by measuring NE- and β3-adrenoceptor (β3-AR) agonist (BRL-37344)-induced lipolysis (glycerol release) as well as NE-induced cAMP accumulation by inguinal, epididymal, and retroperitoneal WAT (IWAT, EWAT, and RWAT) in isolated adipocytes of LD- and SD-housed hamsters. SDs increased potency/efficacy of NE-triggered lipolysis in a temporally and fat pad-specific manner. Thus when WAT pad mass decreased most rapidly (5 wk of SDs), potency (sensitivity/ECso) and efficacy (maximal response asymptote) of NE-stimulated lipolysis were increased for all WAT pads and also at 10 wk for IWAT compared with their LD counter-parts. SD enhancement of lipolysis was similar for NE and BRL-37344 in IWAT adipocytes. These results, coupled with our previous demonstration that SDs upregulate WAT β3-AR mRNA expression, suggest that increased β3-ARs mediated the SD-induced increased NE sensitivity. NE-stimulated adipocyte accumulation of cAMP was greater after 5 wk of SDs for IWAT and EWAT and after 10 wk of SDs for IWAT compared with LDs, with no photoperiod effect for RWAT. Therefore, the SD-induced increase in SNS drive to WAT and increased sensitivity to this drive may work together to increase lipolysis in SDs. [ABSTRACT FROM AUTHOR]

AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDPbound conformation of Gαi > Gαo. AGS3 and related proteins may influence signal strength or duration as well as the adaptation of the signaling system associated with sustained stimulation. To address these issues, we determined the effect of AGS3 on the integration of stimulatory (Gαs-mediated vasoactive intestinal peptide receptor) and inhibitory (Gαi-mediated α2-adrenergic receptor (α2-AR)) signals to adenylyl cyclase in Chinese hamster ovary cells. AGS3-SHORT and AGS3-LONG did not alter the VIP-induced increase in cAMP or the inhibitory effect of α2-AR activation. System adaptation was addressed by determining the influence of AGS3 on the sensitization of adenylyl cyclase that occurs following prolonged activation of a Gαi-coupled receptor. Incubation of cells with the α2-AR agonist UK14304 (1 µM) for 18 h resulted in a ∼l.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated Gαi3. Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of Gαi3 decay. A mutant AGS3-SHORT incapable of binding G-protein was inactive. These data suggest that AGS3 and perhaps other G-protein regulatory motif-containing proteins increase the stability of Gαi in the membrane, which influences the adaptation of the cell to prolonged activation of Gαi-coupled receptors. [ABSTRACT FROM AUTHOR]

Commercial rearing conditions expose teleost fish to numerous acute and chronic stressors that may precipitate dramatic production losses due to infectious diseases. Chemical mediators released in response to acute stress include the catecholamines, epinephrine and norepinephrine. Mammalian lymphocytes and macrophages express beta-adrenergic receptors (AR) that can bind catecholamines, leading to changes in cell function. In this study, radioligand binding assays demonstrated the presence of β-AR in membranes isolated from head kidney and spleen leukocytes of the channel catfish, Ictalurus punctatus. Competition with subtype selective antagonists CGP-20,712 (β1) and ICI-118,551 (β2) suggested that the β2-adrenergic receptor is the primary receptor subtype present on these membranes. These data along with the HPLC-quantification of catecholamines in plasma of I. punctatus lend further support to the contention that crosstalk between the neuroendocrine and immune systems in lower vertebrates is mediated in part by stress-related biogenic amines like epinephrine and norepinephrine. [Copyright &y& Elsevier]

Examines the activation of inhibitory G-protein alpha-subunits by partial agonists of the serotonin human 5-HT1A receptor. Transfection of plasma membrane; Concentration-dependent labeling of G-protein with 32P-AA-GTP produced by full and partial agonists; Capability of agonists of inducing selective coupling of occupied receptor to available G-protein.

Investigates the effect of bile acids on cyclic adenosine monophosphate (cAMP) synthesis in isolated hamster hepatocytes. Effect of ursodeoxycholic acid (UDCA) and its conjugates; Interaction with glucagon; Mechanism of action.

The original description of dietary methionine restriction (MR) used semipurified diets to limit methionine intake to 20% of normal levels, and this reduction in dietary methionine increased longevity by ∼30% in rats. The MR diet also produces paradoxical increases in energy intake and expenditure and limits fat deposition while reducing tissue and circulating lipids and enhancing overall insulin sensitivity. In the years following the original 1993 report, a comprehensive effort has been made to understand the nutrient sensing and signaling systems linking reduced dietary methionine to the behavioral, physiological, biochemical, and transcriptional components of the response. Recent work has shown that transcriptional activation of hepatic fibroblast growth factor 21 (FGF21) is a key event linking the MR diet to many but not all components of its metabolic phenotype. These findings raise the interesting possibility of developing therapeutic, MR-based diets that produce the beneficial effects of FGF21 by nutritionally modulating its transcription and release. [ABSTRACT FROM AUTHOR]

Objective: Sitagliptin and other dipeptidyl peptidase (DPP)‐4 inhibitors/gliptins are antidiabetic drugs known to improve lipid profile, and confer anti‐inflammatory and anti‐fibrotic effects, which are independent of their hypoglycemic effects. However, in our previous short-term (35 days) studies, we showed that sitagliptin accentuates the hepato-inflammatory effects of high dietary cholesterol (Cho) in male Sprague–Dawley rats. Since most type 2 diabetics also present with lipid abnormalities and use DPP-4 inhibitors for glucose management, the present study was conducted to assess the impact of sitagliptin during long-term (98 days) feeding of a high Cho diet. An additional component of the present investigation was the inclusion of other gliptins to determine if hepatic steatosis, necro‐inflammation, and fibrosis were specific to sitagliptin or are class effects. Methods: Adult male Sprague–Dawley rats were fed control or high Cho (2.0%) diets, and gavaged daily (from day 30 through 98) with vehicle or DPP-4 inhibitors (sitagliptin or alogliptin or saxagliptin). On day 99 after a 4 h fast, rats were euthanized. Blood and liver samples were collected to measure lipids and cytokines, and for histopathological evaluation, determination of hepatic lesions (steatosis, necrosis, inflammation, and fibrosis) using specific staining and immunohistochemical methods. Results: Compared to controls, the high Cho diet produced a robust increase in NASH like phenotype that included increased expression of hepatic (Tnfa, Il1b, and Mcp1) and circulatory (TNFα and IL-1β) markers of inflammation, steatosis, necrosis, fibrosis, and mononuclear cell infiltration. These mononuclear cells were identified as macrophages and T cells, and their recruitment in the liver was facilitated by marked increases in endothelium‐expressed cell adhesion molecules. Importantly, treatment with DPP‐4 inhibitors (3 tested) neither alleviated the pathologic responses induced by high Cho diet nor improved lipid profile. Conclusions: The potential lipid lowering effects of DPP-4 inhibitors were diminished by high Cho (a significant risk factor for inducing liver damage). The robust inflammatory responses induced by high Cho feeding in long-term experiment were not exacerbated by DPP-4 inhibitors and a consistent hepatic inflammatory environment persisted, implying a prospective physiological adaptation. [ABSTRACT FROM AUTHOR]

Aims/Hypothesis: High fat diet (HFD)-induced insulin resistance (IR) is partially characterized by reduced skeletal muscle mitochondrial function and peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1α) expression. Our previous study showed that a high dose of the bioflavonoid quercetin exacerbated HFD-induced IR; yet, others have demonstrated that quercetin improves insulin sensitivity. The aim of this study was to investigate whether differing doses of quercetin act in a time-dependent manner to attenuate HFD-induced IR in association with improved skeletal muscle mitochondrial function and PGC1α expression. Methods: C57BL/6J mice were fed HFD for 3 or 8 wks, with or without a low (50 ug/day; HF+50Q) or high (600 ug/day, HF+600Q) dose of quercetin. Whole body and metabolic phenotypes and insulin sensitivity were assessed. Skeletal muscle metabolomic analysis of acylcarnitines and PGC1α mRNA expression via qRT-PCR were measured. Results: Quercetin at 50 ug/day for 8 wk attenuated HFD-induced increases in fat mass, body weight and IR and increased PGC1α expression, whereas 600 ug/day of quercetin exacerbated fat mass accumulation without altering body weight, IR or PGC1α. PGC1α expression correlated with acylcarnitine levels similarly in HF and HF+600Q; these correlations were not present in HF+50Q. At both time points, energy expenditure increased in HF+50Q and decreased in HF+600Q, independent of PGC1α and IR. Conclusions/Interpretation: Chronic dietary quercetin supplementation at low but not higher dose ameliorates the development of diet-induced IR while increasing PGC1α expression in muscle, suggesting that skeletal muscle may be an important target for the insulin-sensitizing effects of a low dose of quercetin. [ABSTRACT FROM AUTHOR]

The article discusses the transcriptional and metabolic responses to essential amino acid (EAA) deprivation or restriction. Information on the geometric framework model of nutrition is provided, as well as the protein leverage hypothesis. It notes the important role of EAAs in mediating the effects of dietary protein on metabolism and energy balance. Metabolic responses to methionine resistance are hyperphagia, reduced fat deposition and increased plasma adiponectin.

G protein-coupled receptors mediate cell responses to extra- cellular stimuli and likely function in the context of a larger signal transduction complex. Utilizing the third intracellular loop of a G protein-coupled receptor in glutathione S-transferase pulldown assays from rat brain lysates coupled with high sensitivity detection methods and subsequent functional studies, we report the identification of SET as a regulator of muscarinic receptor signaling. SET is a putative oncogene reported to inhibit protein phosphatase 2A and regulate gene transcription. SET binds the carboxyl region of the M3-muscarinic receptor i3 loop, and endogenous SET co-immunoprecipitates with intact M3 muscarinic receptor expressed in cells. Small interfering RNA knockdown of endogenous SET in Chinese hamster ovary cells stably expressing the M3 muscarinic receptor augmented receptor-mediated mobilization of intracellular calcium by ~35% with no change in agonist EC50, indicating that interaction of SET with the M3 muscarinic receptor reduces its signaling capacity. SET knockdown had no effect on the mobilization of intracellular calcium by the P2-purinergic receptor, ionomycin, or a direct activator of phospholipase C, indicating a specific regulation of M3 muscarinic receptor signaling. These data provide expanded functionality for SET and a previously unrecognized mechanism for regulation of GPCR signaling capacity. [ABSTRACT FROM AUTHOR]

Abstract: We addressed the role of the G-protein regulatory (GPR) motif-containing Leu–Gly–Asn-enriched protein (LGN) and G-proteins (Giα3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Giα3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Giα3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Giα. The influence of LGN and Giα3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry. [Copyright &y& Elsevier]

We demonstrated recently that increasing the levels of cAMP allows opioids to modulate GABAergic synaptic transmission between the nucleus of the tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV). Using a combination of electrophysiological, immunohistochemical and biochemical approaches, we provide evidence that vagal afferent fibres dampen cAMP levels within the vagal brainstem circuits via tonic activation of group II metabotropic glutamate receptors (mGluRs). Whole-cell patch-clamp recordings were made from identified neurons of the rat DMV. Following chronic vagal deafferentation, the opioid agonist methionine-enkephalin (ME) inhibited the amplitude of evoked IPSC (eIPSC) in 32 of 33 neurons, without exogenous enhancement of cAMP levels. The ME-induced inhibition was prevented by the group II mGluR-selective agonist APDC. Following perfusion with the group II mGluR-selective antagonist EGLU, ME inhibited eIPSC amplitude in brainstem slices of control rats. Immunohistochemical experiments revealed that, following vagal deafferentation, μ-opioid receptors were colocalized on GABAergic profiles apposing DMV neurons; the number of colocalized profiles was significantly decreased by pretreatment with APDC. Radioimmunoassay and Western blot analysis showed that cAMP and phosphorylated cyclic AMP response element binding protein (pCREB) levels in the dorsal vagal complex were increased following vagal deafferentation. Our data show that by tonically dampening the levels of cAMP within the GABAergic synaptic contacts, activated group II mGluRs prevent the modulation of this synapse by endogenous opioids. These data suggest that the plasticity, hence the response, of central circuits controlling the vagal motor outflow to visceral organs is modulated and finely tuned by vagal afferent fibres. [ABSTRACT FROM AUTHOR]

Low-protein (LP) diets extend lifespan through a comprehensive improvement in metabolic health across multiple tissues and organs. Many of these metabolic responses to protein restriction are secondary to transcriptional activation and release of FGF21 from the liver. However, the effects of an LP diet on the kidney in the context of aging has not been examined. Therefore, the goal of the current study was to investigate the impact of chronic consumption of an LP diet on the kidney in aging mice lacking FGF21. Wild-type (WT; C57BL/6J) and FGF21 knockout (KO) mice were fed a normal protein diet (20% casein) or an LP (5% casein) diet ad libitum from 3 to 22 mo of age. The LP diet led to a decrease in kidney weight and urinary albumin-to-creatinine ratio in both WT and FGF21 KO mice. Although the LP diet produced only mild fibrosis and infiltration of leukocytes in WT kidneys, the effects were significantly exacerbated by the absence of FGF21. Accordingly, transcriptomic analysis showed that inflammation-related pathways were significantly enriched and upregulated in response to LP diet in FGF21 KO mice but not WT mice. Collectively, these data demonstrate that the LP diet negatively affected the kidney during aging, but in the absence of FGF21, the LP diet-induced renal damage and inflammation were significantly worse, indicating a protective role of FGF21 in the kidney. [ABSTRACT FROM AUTHOR]

Background: Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR).Objective: We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR.Methods: Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR.Results: We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR.Conclusions: Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice. [ABSTRACT FROM AUTHOR]

The initial sensing of dietary methionine restriction (MR) occurs in the liver where it activates an integrated stress response (ISR) that quickly reduces methionine utilization. The ISR program is regulated in part by ATF4, but ATF4's prototypical upstream regulator, eIF2α, is not acutely activated by MR. Bioinformatic analysis of RNAseq and metabolomics data from liver samples harvested 3 h and 6 h after initiating MR shows that general translation is inhibited at the level of ternary complex formation by an acute 50% reduction of hepatic methionine that limits formation of initiator methionine tRNA. The resulting ISR is induced by selective expression of ATF4 target genes that mediate adaptation to reduced methionine intake and return hepatic methionine to control levels within 4 days of starting the diet. Complementary in vitro experiments in HepG2 cells after knockdown of ATF4, or inhibition of mTOR or Erk1/2 support the conclusion that the early induction of genes by MR is partially dependent on ATF4 and regulated by both mTOR and Erk1/2. Taken together, these data show that initiation of dietary MR induces an mTOR- and Erk1/2-dependent stress response that is linked to ATF4 by the sharp, initial drop in hepatic methionine and resulting repression of translation pre-initiation. [ABSTRACT FROM AUTHOR]

Deals with a study that suggests that lipopolysaccharide (LPS) tolerance induces selective alterations in eicosanoid regulation of cAMP formation. LPS tolerance phenomenon; Function of cAMP in several macrophage functions; Results of the study.

Presents information pertaining to the stimulation of the cyclic adenosine 3'-5'-phosphate (cAMP) production caused by the increased prostacyclin and PGE2 with reference to the macrophages from endotoxin-tolerant rats. What the tolerant macrophages exhibit; Information on the incubation of macrophages; Performance of the assay.

Background: Both cholesterol (Cho) and methionine (Met, a precursor for homocysteine) are risk factors for fatty liver disease. Since Western diets are rich in Cho and Met, we investigated the hepatic effects of feeding a diet enriched in Met and Cho. Further, based on the reported anti-oxidative and lipid lowering properties of sitagliptin (an antidiabetic drug), we tested whether it could counteract the negative effects of high Cho and Met. We therefore hypothesized that sitagliptin would ameliorate the development of liver pathology that is produced by feeding diets rich in either Cho, Met, or both. Methods: Male Sprague Dawley rats were fed ad libitum a) control diet, or b) high Met or c) high Cho, or d) high Met + high Cho diets for 35 days. From day 10 to 35, 50% of rats in each dietary group were gavaged with either vehicle or an aqueous suspension of sitagliptin (100 mg/kg/day). Liver samples were harvested for histological, molecular, and biochemical analyses. Results: The high Cho diet produced significant hepatic steatosis which was unaffected by sitagliptin. Contrary to expectation, sitagliptin exacerbated expression of hepatic markers of oxidative stress and fibrosis in rats fed high Cho. Corresponding increases in 4-hydroxynonenal adducts and collagen deposition were demonstrated by immunohistochemistry and sirius red staining. These hepatic changes were absent in rats on the high Met diet and they were comparable to controls. The inclusion of Met in the high Cho diet resulted in significant reduction of the hepatic steatosis, oxidative stress, and fibrosis produced by high Cho alone. Conclusion: Sitagliptin exacerbated the effects of high Cho on both oxidative stress and fibrosis, resulting in NASH like symptoms that were significantly reversed by the inclusion of Met. [ABSTRACT FROM AUTHOR]

Objective: Hypercholesterolemia is associated with the development of a pro-inflammatory state and is a documented risk factor for progression to insulin resistance, nonalcoholic fatty liver and cardiovascular diseases. Sitagliptin is an incretin enhancer that improves glucose tolerance by inhibiting dipeptidyl peptidase-4, but it also has reported anti-inflammatory effects. The current study was thus undertaken to examine the interactions of dietary Cholesterol (Cho) and sitagliptin on markers of inflammation. Methods: Male Sprague–Dawley rats were provided diets high in Cho and gavaged with vehicle or an aqueous suspension of sitagliptin (100 mg/kg/day) from day 10 through day 35. Molecular methods were used to analyze the lipid profile and inflammatory markers in liver and serum samples. H&E-stained liver sections were used for histopathological evaluation. Hepatic influx of mononuclear cells and necrosis were assessed by immunohistochemistry. Results: Sitagliptin reduced triglyceride and Cho levels in serum of rats on the control diet but these effects were abrogated in rats on the high-Cho diet. Sitagliptin produced a significant increase in the expression of hepatic inflammatory markers (Tnfa, Il1b, and Mcp1) and a corresponding increase in serum TNFα and IL-1β in rats on the high-Cho diet, but it had no effect on rats on the control diet. Additionally, sitagliptin had no effect on liver morphology in rats on the control diet, but it produced hepatic histopathological changes indicative of necrosis and mononuclear cell infiltration in rats on the high-Cho diet. These mononuclear cells were identified as macrophages and T cells. Conclusion: When provided in the context of a high-Cho diet, these findings reveal previously unrecognized hepato-inflammatory effects of sitagliptin that are accompanied by evidence of hepatic necrosis and mononuclear cell infiltration. [ABSTRACT FROM AUTHOR]

Highlights • The liver is the key anatomical site for sensing of dietary methionine restriction. • The liver responds to dietary methionine restriction by releasing FGF21. • FGF21 mediates many of the metabolic effects of dietary methionine restriction. Abstract Dietary methionine restriction (MR) is implemented using a semi-purified diet that reduces methionine by ∼80% and eliminates dietary cysteine. Within hours of its introduction, dietary MR initiates coordinated series of transcriptional programs and physiological responses that include increased energy intake and expenditure, decreased adiposity, enhanced insulin sensitivity, and reduction in circulating and tissue lipids. Significant progress has been made in cataloguing the physiological responses to MR in males but not females, but identities of the sensing and communication networks that orchestrate these responses remain poorly understood. Recent work has implicated hepatic FGF21 as an important mediator of MR, but it is clear that other mechanisms are also involved. The goal of this review is to explore the temporal and spatial organization of the responses to dietary MR as a model for understanding how nutrient sensing systems function to integrate complex transcriptional, physiological, and behavioral responses to changes in dietary composition. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) produces concurrent increases in energy intake and expenditure, but the proportionately larger increase in energy expenditure (EE) effectively limits weight gain and adipose tissue accretion over time. Increased hepatic fibroblast growth factor-21 (FGF21) is essential to MR-dependent increases in EE, but it is unknown whether the downregulation of hepatic stearoyl-coenzyme A desaturase-1 (SCD1) by MR could also be a contributing factor. Global deletion of SCD1 mimics cold exposure in mice housed at 23 °C by compromising the insular properties of the skin. The resulting cold stress increases EE, limits fat deposition, reduces hepatic lipids, and increases insulin sensitivity by activating thermoregulatory thermogenesis. To examine the efficacy of MR in the absence of SCD1 and without cold stress, the biological efficacy of MR in Scd1−/− mice housed near thermoneutrality (28 °C) was evaluated. Compared with wild-type mice on the control diet, Scd1−/− mice were leaner, had higher EE, lower hepatic and serum triglycerides, and lower serum leptin and insulin. Although dietary MR increased adipose tissue UCP1 expression, hepatic Fgf21 messenger RNA, 24 h EE, and reduced serum triglycerides in Scd1−/− mice, it failed to reduce adiposity or produce any further reduction in hepatic triglycerides, serum insulin, or serum leptin. These findings indicate that even when thermal stress is minimized, global deletion of SCD1 mimics and effectively masks many of the metabolic responses to dietary MR. However, the retention of several key effects of dietary MR in this model indicates that SCD1 is not a mediator of the biological effects of the diet. [ABSTRACT FROM AUTHOR]

An association between arteriosclerosis and homocysteine (Hcy) was first demonstrated in 1969. Hcy is a sulfur containing amino acid derived from the essential amino acid methionine (Met). Hyperhomocysteinemia (HHcy) was subsequently shown in several age-related pathologies such as osteoporosis, Alzheimer's disease, Parkinson's disease, stroke, and cardiovascular disease (CVD). Also, Hcy is associated with (but not limited to) cancer, aortic aneurysm, hypothyroidism and end renal stage disease to mention some. The circulating levels of Hcy can be increased by defects in enzymes of the metabolism of Met, deficiencies of vitamins B6, B12 and folate or by feeding Met enriched diets. Additionally, some of the pharmaceuticals currently in clinical practice such as lipid lowering, and anti-Parkinsonian drugs are known to elevate Hcy levels. Studies on supplementation with folate, vitamins B6 and B12 have shown reduction in Hcy levels but concomitant reduction in certain associated pathologies have not been definitive. The enormous importance of Hcy in health and disease is illustrated by its prevalence in the medical literature (e.g. > 22,000 publications). Although there are compelling data in favor of Hcy as a modifiable risk factor, the debate regarding the significance of Hcy mediated health effects is still ongoing. Despite associations between increased levels of Hcy with several pathologies being well documented, whether it is a causative factor, or an effect remains inconclusive. The present review though not exhaustive, is focused on several important aspects of Hcy metabolism and their relevance to health. [ABSTRACT FROM AUTHOR]

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) produces a coordinated series of transcriptional responses in peripheral tissues that limit fat accretion, remodel lipid metabolism in liver and adipose tissue, and improve overall insulin sensitivity. Hepatic sensing of reduced methionine leads to induction and release of fibroblast growth factor 21 (FGF21), which acts centrally to increase sympathetic tone and activate thermogenesis in adipose tissue. FGF21 also has direct effects in adipose to enhance glucose uptake and oxidation. However, an understanding of how the liver senses and translates reduced dietary methionine into these transcriptional programs remains elusive. A comprehensive systems biology approach integrating transcriptomic and metabolomic readouts in MR-treated mice confirmed that three interconnected mechanisms (fatty acid transport and oxidation, tricarboxylic acid cycle, and oxidative phosphorylation) were activated in MR-treated inguinal adipose tissue. In contrast, the effects of MR in liver involved up-regulation of anti-oxidant responses driven by the nuclear factor, erythroid 2 like 2 transcription factor, NFE2L2. Metabolomic analysis provided evidence for redox imbalance, stemming from large reductions in the master anti-oxidant molecule glutathione coupled with disproportionate increases in ophthalmate and its precursors, glutamate and 2-aminobutyrate. Thus, cysteine and its downstream product, glutathione, emerge as key early hepatic signaling molecules linking dietary MR to its metabolic phenotype. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) produces a rapid and persistent remodeling of white adipose tissue (WAT), an increase in energy expenditure (EE), and enhancement of insulin sensitivity. Recent work established that hepatic expression of FGF21 is robustly increased by MR. Fgf21-/- mice were used to test whether FGF21 is an essential mediator of the physiological effects of dietary MR. The MR-induced increase in energy intake and EE and activation of thermogenesis in WAT and brown adipose tissue were lost in Fgf21-/- mice. However, dietary MR produced a comparable reduction in body weight and adiposity in both genotypes because of a negative effect of MR on energy intake in Fgf21-/- mice. Despite the similar loss in weight, dietary MR produced a more significant increase in in vivo insulin sensitivity in wild-type than in Fgf21-/- mice, particularly in heart and inguinal WAT. In contrast, the ability of MR to regulate lipogenic and integrated stress response genes in liver was not compromised in Fgf21-/- mice. Collectively, these findings illustrate that FGF21 is a critical mediator of the effects of dietary MR on EE, remodeling of WAT, and increased insulin sensitivity but not of its effects on hepatic gene expression. [ABSTRACT FROM AUTHOR]

The β3-adrenergic receptor (AR) signaling pathway is a major component of adaptive thermogenesis in brown and white adipose tissue during cold acclimation. The β3-AR signaling highly induces the expression of transcriptional coactivator PGC-1α and its splice variant N-terminal (NT)-PGC-1α, which in turn activate the transcription program of adaptive thermogenesis by co-activating a number of transcription factors. We previously reported that NT-PGC-1α is able to increase mitochondrial number and activity in cultured brown adipocytes by promoting the expression of mitochondrial and thermogenic genes. In the present study, we performed genome-wide profiling of NT-PGC-1α-responsive genes in brown adipocytes to identify genes potentially regulated by NT-PGC-1α. Canonical pathway analysis revealed that a number of genes upregulated by NT-PGC-1α are highly enriched in mitochondrial pathways including fatty acid transport and β-oxidation, TCA cycle and electron transport system, thus reinforcing the crucial role of NT-PGC-1α in the enhancement of mitochondrial function. Moreover, canonical pathway analysis of NT-PGC-1α-responsive genes identified several metabolic pathways including glycolysis and fatty acid synthesis. In order to validate the identified genes in vivo, we utilized the FL-PGC-1α-/- mouse that is deficient in full-length PGC-1α (FL-PGC-1α) but expresses a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α254). The β3-AR-induced increase of NT-PGC-1α254 in FL-PGC-1α-/- brown and white adipose tissue was closely associated with elevated expression of genes involved in thermogenesis, mitochondrial oxidative metabolism, glycolysis and fatty acid synthesis. Increased adipose tissue thermogenesis by β3-AR activation resulted in attenuation of adipose tissue expansion in FL-PGC-1α-/- adipose tissue under the high-fat diet condition. Together, the data strengthen our previous findings that NT-PGC-1α regulates mitochondrial genes involved in thermogenesis and oxidative metabolism in brown and white adipocytes and further suggest that NT-PGC-1α regulates a broad spectrum of genes to meet cellular needs for adaptive thermogenesis. [ABSTRACT FROM AUTHOR]

Restricting availability of essential amino acids (EAAs) limits aminoacylation of tRNAs by their cognate EAAs and activates the nutrient-sensing kinase, general control nonderepressible 2 (GCN2). Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2), altering gene-specific translation and initiating a transcriptional program collectively described as the integrated stress response (ISR). Central GCN2 activation by EAA deprivation is also linked to an acute aversive feeding response. Dietary methionine restriction (MR) produces a well-documented series of physiological responses (increased energy intake and expenditure, decreased adiposity, and increased insulin sensitivity), but the role of GCN2 in mediating them is unknown. Using Gcn2(-/-) mice, we found that the absence of GCN2 had no effect on the ability of MR to reduce body weight or adiposity, increase energy intake and expenditure, increase hepatic transcription and release of fibroblast growth factor 21, or improve insulin sensitivity. Interestingly, hepatic eIF2 phosphorylation by MR was uncompromised in Gcn2(-/-) mice. Instead, protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) was activated in both intact and Gcn2(-/-) mice. PERK activation corresponded with induction of the ISR and the nuclear respiratory factor 2 antioxidant program but not ER stress. These data uncover a novel glutathione-sensing mechanism that functions independently of GCN2 to link dietary MR to its metabolic phenotype. [ABSTRACT FROM AUTHOR]

Dietary leucine was incrementally restricted to test whether limiting this essential amino acid (EAA) would fully reproduce the beneficial responses produced by dietary methionine restriction. Restricting leucine by 85% increased energy intake and expenditure within 5 to 7 days of its introduction and reduced overall accumulation of adipose tissue. Leucine restriction (LR) also improved glucose tolerance, increased hepatic release of fibroblast growth factor 21 into the blood stream, and enhanced insulin-dependent activation of Akt in liver. However, LR had no effect on hepatic lipid levels and failed to lower lipogenic gene expression in the liver. LR did affect remodeling of white and brown adipose tissues, increasing expression of both thermogenic and lipogenic genes. These findings illustrate that dietary LR reproduces many but not all of the physiological responses of methionine restriction. The primary differences occur in the liver, where methionine and LR cause opposite effects on tissue lipid levels and expression of lipogenic genes. Altogether, these findings suggest that the sensing systems which detect and respond to dietary restriction of EAAs act through mechanisms that both leucine and methionine are able to engage, and in the case of hepatic lipid metabolism, may be unique to specific EAAs such as methionine. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) produces a coordinated series of biochemical and physiological responses that improve biomarkers of metabolic health, increase energy expenditure, limit fat accretion and improve overall insulin sensitivity. Inguinal white adipose tissue (IWAT) is a primary target and site of action where the diet initiates transcriptional programs linked to enhancing both synthesis and oxidation of lipid. Using a combination of ex vivo approaches to assess dietary effects on cell morphology and function, we report that dietary MR produced a fourfold increase in multilocular, UCP1-expressing cells within this depot in conjunction with significant increases in mitochondrial content, size and cristae density. Dietary MR increased expression of multiple enzymes within the citric acid cycle, as well as respiratory complexes I, II and III. The physiological significance of these responses, evaluated in isolated mitochondria by high-resolution respirometry, was a significant increase in respiratory capacity measured using multiple substrates. The morphological, transcriptional and biochemical remodeling of IWAT mitochondria enhances the synthetic and oxidative capacity of this tissue and collectively underlies its expanded role as a significant contributor to the overall increase in metabolic flexibility and uncoupled respiration produced by the diet. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) by 80% increases energy expenditure (EE), reduces adiposity, and improves insulin sensitivity. We propose that the MR-induced increase in EE limits fat deposition by increasing sympathetic nervous system-dependent remodeling of white adipose tissue and increasing uncoupling protein 1 (UCP1) expression in both white and brown adipose tissue. In independent assessments of the role of UCP1 as a mediator of MR's effects on EE and insulin sensitivity, EE did not differ between wild-type (WT) and Ucp1-/- mice on the control diet, but MR increased EE by 31% and reduced adiposity by 25% in WT mice. In contrast, MR failed to increase EE or reduce adiposity in Ucp1-/-mice. However, MR was able to increase overall insulin sensitivity by 2.2-fold in both genotypes. Housing temperatures used to minimize (28°C) or increase (23°C) sympathetic nervous system activity revealed temperature-independent effects of the diet on EE. Metabolomics analysis showed that genotypic and dietary effects on white adipose tissue remodeling resulted in profound increases in fatty acid metabolism within this tissue. These findings establish that UCP1 is required for the MR-induced increase in EE but not insulin sensitivity and suggest that diet-induced improvements in insulin sensitivity are not strictly derived from dietary effects on energy balance. [ABSTRACT FROM AUTHOR]

To understand the physiological significance of the reduction in fasting insulin produced by dietary methionine restriction (MR), hyperinsulinemic-euglycemic clamps were used to examine the effect of the diet on overall and tissue-specific insulin sensitivity in mice. The steady-state glucose infusion rate was threefold higher in the MR group and consistent with the 2.5- to threefold increase in 2-deoxyglucose uptake in skeletal muscle, heart, and white adipose tissue. Dietary MR enhanced suppression of hepatic glucose production by insulin, enhanced insulin-dependent Akt phosphorylation in the liver, and increased hepatic expression and circulating fibroblast growth factor 21 (FGF-21) by fourfold. Limitation of media methionine recapitulated amplification of Akt phosphorylation by insulin in HepG2 cells but not in 3T3-L1 adipocytes or C2C12 myotubes. Amplification of insulin signaling in HepG2 cells by MR was associated with reduced glutathione, where it functions as a co-factor for phosphatase and tensin homolog. In contrast, FGF-21, but not restricting media methionine, enhanced insulin-dependent Akt phosphorylation in 3T3-L1 adipocytes. These findings provide a potential mechanism for the diet-induced increase in insulin sensitivity among tissues that involves a direct effect of methionine in liver and an indirect effect in adipose tissue through MR-dependent increases in hepatic transcription and release of FGF-21. [ABSTRACT FROM AUTHOR]

Methionine restriction ( MR) decreases body weight and adiposity and improves glucose homeostasis in rodents. Similar to caloric restriction, MR extends lifespan, but is accompanied by increased food intake and energy expenditure. Most studies have examined MR in young animals; therefore, the aim of this study was to investigate the ability of MR to reverse age-induced obesity and insulin resistance in adult animals. Male C57BL/6J mice aged 2 and 12 months old were fed MR (0.172% methionine) or control diet (0.86% methionine) for 8 weeks or 48 h. Food intake and whole-body physiology were assessed and serum/tissues analyzed biochemically. Methionine restriction in 12-month-old mice completely reversed age-induced alterations in body weight, adiposity, physical activity, and glucose tolerance to the levels measured in healthy 2-month-old control-fed mice. This was despite a significant increase in food intake in 12-month-old MR-fed mice. Methionine restriction decreased hepatic lipogenic gene expression and caused a remodeling of lipid metabolism in white adipose tissue, alongside increased insulin-induced phosphorylation of the insulin receptor ( IR) and Akt in peripheral tissues. Mice restricted of methionine exhibited increased circulating and hepatic gene expression levels of FGF21, phosphorylation of eIF2a, and expression of ATF4, with a concomitant decrease in IRE1α phosphorylation. Short-term 48-h MR treatment increased hepatic FGF21 expression/secretion and insulin signaling and improved whole-body glucose homeostasis without affecting body weight. Our findings suggest that MR feeding can reverse the negative effects of aging on body mass, adiposity, and insulin resistance through an FGF21 mechanism. These findings implicate MR dietary intervention as a viable therapy for age-induced metabolic syndrome in adult humans. [ABSTRACT FROM AUTHOR]

Enhanced fibroblast growth factor 21 (FGF21) production and circulation has been linked to the metabolic adaptation to starvation. Here, we demonstrated that hepatic FGF21 expression is induced by dietary protein restriction, but not energy restriction. Circulating FGF21 was increased 10-fold in mice and rats fed a low-protein (LP) diet. In these animals, liver Fgf21 expression was increased within 24 hours of reduced protein intake. In humans, circulating FGF21 levels increased dramatically following 28 days on a LP diet. LP-induced increases in FGF21 were associated with increased phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the liver, and both baseline and LP-induced serum FGF21 levels were reduced in mice lacking the eIF2α kinase general control nonderepressible 2 (GCN2). Finally, while protein restriction altered food intake, energy expenditure, and body weight gain in WT mice, FGF21-deficient animals did not exhibit these changes in response to a LP diet. These and other data demonstrate that reduced protein intake underlies the increase in circulating FGF21 in response to starvation and a ketogenic diet and that FGF21 is required for behavioral and metabolic responses to protein restriction. FGF21 therefore represents an endocrine signal of protein restriction, which acts to coordinate metabolism and growth during periods of reduced protein intake. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) and calorie restriction (CR) each improve metabolic health and extend life span. We need comprehensive transcriptome profiling and systems biology analysis to interrogate the unique and overlapping molecular responses in rats provided these dietary regimens for 20 mo after weaning. Microarray analysis was conducted on inguinal white adipose (IWAT), brown adipose tissue (BAT), liver, and skeletal muscle. Compared to controls, CR-induced transcriptomic responses (absolute fold change ≥ 1.5 and P ≤0.05) were comparable in IWAT, BAT, and liver (~800 genes). MR-induced effects were largely restricted to IWAT and liver (~2400 genes). Pathway enrichment and gene-coexpression analyses showed that induction of fatty acid synthesis in IWAT was common to CR and MR, whereas immunity and proinflammatory signaling pathways were specifically down-regulated in MR-treated IWAT and liver (FDR ≤0.07-0.3). BAT demonstrated consistent down- regulation of PPAR-signalling under CR and MR, whereas muscle was largely unaffected. Interactome analysis identified CR-specific down-regulation of cytoskeletal matrix components in IWAT and MR-specific up-regulation of rihosomal genes in liver (FDR≤0.001). Transcriptomic down-regulation of inflammation genes by MR in IWAT was consistent with upstream inhibition of STAT3. Together, these results provide an integrated picture of the breadth of transcriptional responses to MR and CR among key metabolic tissues. [ABSTRACT FROM AUTHOR]

Calorie restriction (CR) without malnutrition increases lifespan and produces significant improvements in biomarkers of metabolic health. The improvements are attributable in part to effects of CR on energy balance, which limit fat accumulation by restricting energy intake. Normal age-associated increases in adiposity and insulin resistance are associated with development of a systemic proinflammatory state, while chronic CR limits fat deposition and expression of inflammatory markers. Dietary methionine restriction (MR) has emerged as an effective CR mimetic because it produces a comparable extension in lifespan. MR also reduces adiposity through a compensatory increase in energy expenditure that effectively limits fat accumulation, but essentially nothing is known about the effects of MR on systemic inflammation. Here, we review the relationships between these two interventions and discuss their transcriptional impact. In addition, using tissues from rats after long-term consumption of CR or MR diets, transcriptional profiling was used to examine retrospectively the systems biology of 59 networks of molecules annotated to inflammation. Transcriptional effects of both diets occurred primarily in white adipose tissue and liver, and the responses to MR were far more robust than those to CR. The primary transcriptional targets of MR in both liver and white adipose tissue were phagocytes and macrophages, where expression of genes associated with immune cell infiltration and quantity was reduced. These findings support the conclusion that anti-inflammatory responses produced by CR and MR are not strictly dependent upon reduced adiposity but are significantly influenced by the metabolic mechanisms through which energy balance is altered. © 2013 BioFactors, 40(1):13-26, 2014 [ABSTRACT FROM AUTHOR]

PGC-1α is an inducible transcriptional coactivator that regulates mitochondrial biogenesis and cellular energy metabolism in skeletal muscle. Recent studies have identified two additional PGC-1α transcripts that are derived from an alternative exon 1 (exon 1b) and induced by exercise. Given that the PGC-1α gene also produces NT-PGC-1α transcript by alternative 3' splicing between exon 6 and exon 7, we have investigated isoform-specific expression ofNT-PGC-1αmRNAin mouse skeletalmuscle during physical exercise with different intensities. We report here that NT-PGC-1α-a mRNA expression derived from a canonical exon 1 (exon 1a) is increased by high-intensity exercise and AMPK activator AICAR in mouse skeletal muscle but not altered by low- and medium-intensity exercise and β2-adrenergic receptor agonist clenbuterol. In contrast, the alternative exon 1b-driven NT-PGC- 1α-b (PGC-1α4) and NT-PGC-1α-c are highly induced by low-, medium-, and high-intensity exercise, AICAR, and clenbuterol. Ectopic expression of NT-PGC-1α-a in C2C12 myotube cells upregulates myosin heavy chain (MHC I,MHC II a) and Glut4, which represent oxidative fibers, and promotes the expression of mitochondrial genes (Cyc1, COX5B, and ATP5B). In line with gene expression data, citrate synthase activity was significantly increased by NT-PGC-1α-a in C2C12 myotube cells. Our results indicate the regulatory role for NT-PGC-1α-a in mitochondrial biogenesis and adaptation of skeletal muscle to endurance exercise. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) produces an integrated series of biochemical and physiological responses that improve biomarkers of metabolic health, limit fat accretion, and enhance insulin sensitivity. Using transcriptional pro ling to guide tissue-specific evaluations of molecular responses to MR, we report that liver and adipose tissue are the primary targets of a transcriptional program that remodeled lipid metabolism in each tissue. The MR diet produced a coordinated downregulation of lipogenic genes in the liver, resulting in a corresponding reduction in the capacity of the liver to synthesize and export lipid. In contrast, the transcriptional response in white adipose tissue (WAT) involved a depot-specific induction of lipogenic and oxidative genes and a commensurate increase in capacity to synthesize and oxidize fatty acids. These responses were accompanied by a significant change in adipocyte morphology, with the MR diet reducing cell size and increasing mitochondrial density across all depots. The coordinated transcriptional remodeling of lipid metabolism between liver and WAT by dietary MR produced an overall reduction in circulating and tissue lipids and provides a potential mechanism for the increase in metabolic flexibility and enhanced insulin sensitivity produced by the diet. [ABSTRACT FROM AUTHOR]

PGC-1α is an inducible transcriptional coactivator that regulates cellular energy metabolism and adaptation to environmental and nutritional stimuli. In tissues expressing PGC-1α, alternative splicing produces a truncated protein (NT-PGC-1α) corresponding to the first 267 amino acids of PGC-1α. Brown adipose tissue also expresses two novel exon 1b-derived isoforms of PGC-1α and NT-PGC-1α, which are 4 and 13 amino acids shorter in the N termini than canonical PGC-1α and NT-PGC-1α, respectively. To evaluate the ability of NT-PGC-1α to substitute for PGC-1α and assess the isoform-specific role of NT-PGC-1α, adaptive thermogenic responses of adipose tissue were evaluated in mice lacking full-length PGC-1α (FL-PGC-1-/-) but expressing slightly shorter but functionally equivalent forms of NT-PGC-1α (NT-PGC-1α254). At room temperature, NT-PGC-1α and NT-PGC-1α254 were produced from conventional exon 1a-derived transcripts in brown adipose tissue of wild type and FL-PGC-1α-/- mice, respectively. However, cold exposure shifted transcription to exon 1b, increasing exon 1b-derived mRNA levels. The resulting transcriptional responses produced comparable increases in energy expenditure and maintenance of core body temperature in WT and FL-PGC-1α-/- mice. Moreover, treatment of the two genotypes with a selective β3-adrenergic receptor agonist produced similar increases in energy expenditure, mitochondrial DNA, and reductions in adiposity. Collectively, these findings illustrate that the transcriptional and physiological responses to sympathetic input are unabridged in FL-PGC-1α-/- mice, and that NT-PGC-1α is sufficient to link β3-androgenic receptor activation to adaptive thermogenesis in adipose tissue. Furthermore, the transcriptional shift from exon 1a to 1b supports isoform-specific roles for NT-PGC-1α in basal and adaptive thermogenesis. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) is a mimetic of chronic dietary restriction (DR) in the sense that MR increases rodent longevity, but without food restriction. We report here that MR also persistently increases total energy expenditure (EE) and limits fat deposition despite increasing weight-specific food consumption. In Fischer 344 (F344) rats consuming control or MR diets for 3, 9, and 20 mo, mean EE was 1.5-fold higher in MR vs. control rats, primarily due to higher EE during the night at all ages. The day-to-night transition produced a twofold higher heat increment of feeding (3.0°C vs. 1.5°C) in MR vs. controls and an exaggerated increase in respiratory quotient (RQ) to values greater than I, indicative of the interconversion of glucose to lipid by de novo lipogenesis. The simultaneous inhibition of glucose utilization and shift to fat oxidation during the day was also more complete in MR (RQ -0.75) vs. controls (RQ -0.85). Dietary MR produced a rapid and persistent increase in uncoupling protein 1 expression in brown (BAT) and white adipose tissue (WAT) in conjunction with decreased leptin and increased adiponectin levels in serum, suggesting that remodeling of the metabolic and endocrine function of adipose tissue may have an important role in the overall increase in EE. We conclude that the hyperphagic response to dietary MR is matched to a coordinated increase in uncoupled respiration, suggesting the engagement of a nutrient-sensing mechanism, which compensates for limited methionine through integrated effects on energy homeostasis. [ABSTRACT FROM AUTHOR]

Dietary methionine restriction (MR) limits fat deposition and decreases plasma leptin, while increasing food consumption, total energy expenditure (EE), plasma adiponectin, and expression of uncoupling protein 1 (UCP1) in brown and white adipose tissue (BAT and WAT). β-adrenergic receptors (3-AR) serve as conduits for sympathetic input to adipose tissue, but their role in mediating the effects of MR on energy homeostasis is unclear. Energy intake, weight, and adiposity were modestly higher in β3-AR-/- mice on the Control diet compared with wild-type (WT) mice, but the hyperphagic response to the MR diet and the reduction in fat deposition did not differ between the genotypes. The absence of β3-ARs also did not diminish the ability of MR to increase total EE and plasma adiponectin or decrease leptin mRNA, but it did block the MR-dependent increase in UCP1 mRNA in BAT but not WAT. In a further study, propranolol was used to antagonize remaining β-adrenergic input (β1- and β2-ARs) in β3-AR-/- mice, and this treatment blocked >50% of the MR-induced increase in total EE and UCP1 induction in both BAT and WAT. We conclude that signaling through β-adrenergic receptors is a component of the mechanism used by dietary MR to increase EE, and that β1- and β2-ARs are able to substitute for β3-ARs in mediating the effect of dietary MR on EE. These findings are consistent with the involvement of both UCP1-dependent and -independent mechanisms in the physiological responses affecting energy balance that are produced by dietary MR. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) plays a central role in the regulation of cellular energy metabolism and metabolic adaptation to environmental and nutritional stimuli. We recently described a novel, biologically active splice variant of PGC-1α (NT-PGC-1α, amino acids 1-270) that retains the ability to interact with and transactivate nuclear hormone receptors through its N-terminal transactivation domain. Whereas PGC-1α is an unstable nuclear protein sensitive to ubiquitin-mediated targeting to the proteasome, NT-PGC-1α is relatively stable and predominantly cytoplasmic, suggesting that its ability to interact with and activate nuclear receptors and transcription factors is dependent upon regulated access to the nucleus. We provide evidence that NT-PGC-1α interacts with the nuclear exportin, CRM1, through a specific leucine-rich domain (nuclear export sequence) that regulates its export to the cytoplasm. The nuclear export of NT-PGC-1α is inhibited by protein kinase A-dependent phosphorylation of Ser-194, Ser-241, and Thr-256 on NT-PGC-1α, which effectively increases its nuclear concentration. Using site-directed mutagenesis to prevent or mimic phosphorylation at these sites, we show that the transcriptional activity of NT-PGC-1α is regulated in part through regulation of its subcellular localization. These findings suggest that the function of NT-PGC-1α as a transcriptional co-activator is regulated by protein kinase A-dependent inhibition of CRM1-mediated export from the nucleus. [ABSTRACT FROM AUTHOR]

The transcriptional co-activator PGC-1α regulates functional plasticity in adipose tissue by linking sympathetic input to the transcriptional program of adaptive thermogenesis. We report here a novel truncated form of PGC-1α(NT-PGC-1α) produced by alternative 3' splicing that introduces an in-frame stop codon into PGC-1α mRNA. The expressed protein includes the first 267 amino acids of PGC-1α and 3 additional amino acids from the splicing insert. NT-PGC-1α contains the transactivation and nuclear receptor interaction domains but is missing key domains involved in nuclear localization, interaction with other transcription factors, and protein degradation. Expression and subcellular localization of NT-PGC-1α are dynamically regulated in the context of physiological signals that regulate fulllength PGC-1α, but the truncated domain structure conveys unique properties with respect to protein-protein interactions, protein stability, and recruitment to target gene promoters. Therefore, NT-PGC-1α is a co-expressed, previously unrecognized form of PGC-1α with functions that are both unique from and complementary to PGC-1α. [ABSTRACT FROM AUTHOR]

Abstract: Insulin and leptin play complementary roles in regulating the consumption, uptake, oxidation and storage of nutrients. Chronic consumption of diets that contain a high proportion of calories from saturated fat induces a progressive deterioration in function of both hormones. Certain rat lines and strains of mice are particularly sensitive to the obesogenic and diabetogenic effects of high fat diets, and have been used extensively to study the developmental progression of insulin and leptin resistance in relation to the increasing adiposity that is characteristic of their response to these diets. Some aspects of the diminished efficacy of each hormone are secondary to increased adiposity but a consensus is emerging to support the view that direct effects of dietary components or their metabolites, independent of the resulting obesity, play important roles in development of insulin and leptin resistance. In this minireview, we will examine the implications of crosstalk between leptin and insulin signaling during the development of diet-induced obesity, emphasizing potential interactions between pathways that occur among target sites, and exploring how these interactions may influence the progression of obesity and diabetes. [Copyright &y& Elsevier]

Objective: The sympathetic nervous system and thiazolidinediones control lipid metabolism and have been implicated in body weight regulation. This study was conducted to determine whether the simultaneous activation of these two signaling systems might synergize to exert beneficial effects on the expression of key genes involved in lipid metabolism and mitochondrial biogenesis in subcutaneous fat in nondiabetic subjects.Research Design and Methods: A total of 57 women and men were randomized into four groups: 1) placebo/placebo (PP), 2) ephedrine HCl (25 mg, 3 times daily) plus caffeine (200 mg, 3 times daily)/placebo (ECP), 3) placebo/pioglitazone (45 mg) (PPio), and 4) ephedrine plus caffeine/pioglitazone (ECPio) for 16 weeks. Adipose tissue samples were obtained after 12 weeks of treatment to determine gene expression.Results: Body fat decreased by 6.0 and 4.6% in the ECP and ECPio groups, respectively, while remaining unchanged in the PPio and PP groups. Triglyceride levels decreased by -7.7, -24, -15.2, and -41 mg/dl after 16 weeks treatment in the PP, PPio, ECP, and ECPio groups, respectively. This indicates that pioglitazone groups with or without EC (ephedrine HCl plus caffeine) decreased triglycerides, and EC groups with or without pioglitazone decreased body weight. The mRNA for sirtuin 1 and CD36 increased only in the ECPio group. Carnitine palmitoyltransferase-1, medium-chain acyl CoA dehydrogenase, and malonyl-CoA decarboxylase increased with PPio and ECPio. Stearoyl-CoA desaturase decreased with ECP.Conclusions: Combined activation of peroxisome proliferator-activated receptor-gamma and beta-adrenergic receptors has beneficial effects on body weight, plasma triglycerides, and lipid metabolism in subcutaneous fat by increasing the expression of genes required for fatty acid catabolism. [ABSTRACT FROM AUTHOR]

Chimeric G proteins made by replacing the COOH-terminal heptapeptide of Gαq with the COOH-terminal heptapeptide of Gαs or Gαi were used to assess the relative coupling of β3-adrenergic receptor (β3-AR) splice variants (β3A and β3B) to G&s and Gαi. The Gαq/s and Gαq/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium (Ca2+i). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat β3-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, β3-AR-induced coupling to Gαq/s produced a rapid eightfold increase in Ca2+i followed by a slow decay to levels 25% above baseline. Gαq/i also linked rat β3-AR to mobilization of Ca2+i in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in Ca2+i was only 30% of the response obtained with Gαq/s. Activation of the rat β3-AR also increased GTP binding to endogenous Gαi threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse β3A- and β3B-AR to Gαi under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to Gαq/s and to increases in intracellular cAMP through endogenous Gαs. The β3A- and β3B-AR coupled equivalently to Gαq/i, but the temporal patterns of Ca2+i mobilization indicated that coupling was significantly less efficient than coupling to Gαq/s. Collectively, these findings indicate less efficient but equivalent coupling of β3A- and β3B-AR to Gαi vs. Gαs and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to β3-AR activation. [ABSTRACT FROM AUTHOR]

Mouse lines with targeted disruption of the cocaine amphetamine-related transcript (CART), melanocortin receptor 3 (MCR3), or melanocortin receptor 4 (MCR4) were used to assess the role of each component in mediating the anorectic and metabolic effects of leptin, and in regulating the partitioning of nutrient energy between fat and protein deposition. Leptin was administered over a 3 day period using either intraperitoneal or intracerebroventricular routes of injection. The absence of MCR4 blocked leptin's ability to increase UCP1 mRNA in both brown and white adipose tissue, but not its ability to reduce food consumption. In contrast, deletion of MCR3 compromised leptin's ability to reduce food consumption, but not its ability to reduce fat deposition or increase UCP1 expression in adipose tissue. Leptin-dependent effects on food consumption and adipocyte gene expression were unaffected by the absence of CART. Repeated measures of body composition over time indicate that the absence of either MCR3 or MCR4, but not CART, increased lipid deposition and produced comparable degrees of adiposity in both lines. Moreover, modest increases in fat content of the diet (4 to 11%) accentuated fat deposition and produced a rapid and comparable 10-12% increase in % body fat in both genotypes. The results indicate that nutrient partitioning, as well as the anorectic and metabolic responses to leptin, are dependent on integrated but separable inputs from the melanocortin 3 and 4 receptor subtypes. [ABSTRACT FROM AUTHOR]

Abstract: Polyunsaturated fatty acids (PUFA) and a number of drugs (metformin, thiazolidinediones) and hormones (leptin, adiponectin) that activate AMP-activated protein kinase (AMPK) have been reported to improve insulin sensitivity. To determine whether PUFA activate AMPK, Sprague–Dawley rats were adapted to a 3h meal-feeding regimen using a fat-free diet (FFD) supplemented with fish oil (n−3) or triolein (n−9) for 7 days. No differences in hepatic AMPK activity were observed between the groups after 21h of fasting. On the other hand, hepatic AMPK phosphorylation was decreased in rats refed the FFD, the FFD+triolein, and the FFD+PUFA by 80%, 75%, and 50%, respectively, when assessed 2h after completion of a meal. In keeping with these changes, decreases in acetyl-CoA carboxylase phosphorylation and carnitine palmitoyl transferase-1 mRNA and increases in fatty acid synthase gene expression were greatest in rats fed the FFD and least in the PUFA-fed rats. The results indicate that dietary PUFA enhance hepatic AMPK activity in vivo, and implicate AMPK as a component of the nutrient-sensing mechanism through which dietary fatty acids and especially PUFA influence the regulation of hepatic lipid metabolism and gene expression. [Copyright &y& Elsevier]