Methionine restriction does good things

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It seemed from the pdf-availed below paper that restricting methionine is healthy. Perrone CE, Mattocks DA, Hristopoulos G, Plummer JD, Krajcik RA, Orentreich N. Methionine restriction effects on 11beta-HSD1 activity and lipogenic/lipolytic balance in F344 rat adipose tissue.J Lipid Res. 2007 Oct 1; [Epub ahead of print] PMID: 17909224 Methionine restriction (MR) limits age-related adiposity in Fischer344 (F344) rats. To assess the mechanism of adiposity resistance, the effect of MR on adipose tissue11©¬-hydroxysteroid dehydrogenase-1 (11ss-HSD1) was examined. MR induced 11©¬-HSD1 activity in all ATs correlating with increased tissue corticosterone. However, an inverse relationship between 11©¬-HSD1 activity and adipocyte size was observed. Because dietary restriction controls lipogenic and lipolytic rates, MR's effects on lipogenic and lipolytic enzymes were evaluated. MR increased adipose triglyceride lipase (ATGL) and acetyl-CoA carboxylase (ACC) protein levels, but induced ACC phosphorylation at serine residues that render the enzyme inactive, suggesting alterations of basal lipolysis and lipogenesis. In contrast, no changes in basal or phosphorylated hormone-sensitive lipase (HSL)

levels were observed. ACC phosphorylated sites were specific for AMP-activated protein kinase (AMPK); therefore, AMPK activation was evaluated. Significant differences in AMPKa protein, phosphorylation and activity levels were observed only in retroperitoneal fat from MR rats. No differences in protein kinase A (PKA) phosphorylation and intracellular cAMP levels were detected. In vitro studies revealed increased lipid degradation and a trend toward increased lipid synthesis, suggesting the presence of a futile cycle. In conclusion, MR disrupts the lipogenic/lipolytic balance, contributing importantly to adiposity resistance in F344 rats. Supplementary key words: adiposity, glucocorticoid metabolism, lipogenic and lipolytic enzymes, adipose tissue signal transduction pathways. Abbreviations: MR, methionine restriction; CF, control fed; F344, Fischer344; AT, adipose tissue;

11©¬-HSD1, 11©¬-hydroxysteroid dehydrogenase-1; ATGL, adipose triglyceride lipase; HSL, hormone sensitive lipase; ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; pAMPK, phospho-AMPK; PKA, protein kinase A; cAMP, cyclic adenosine 3¡¯,5¡¯-monophosphate; C/EBP, CCAAT enhancer binding protein; AC, adenyl cyclase; PPAR-gamma, peroxisome proliferator-activated receptor gamma; SREBP-1c, sterol regulatory element-binding protein-1c; CPT, carnitine palmitoyl transferase; FAS, fatty acid synthase; TLC, thin layer chromatography; Thr172, threonine 172; Ser79, serine 79; NEFA, non-esterified fatty acids. ... Purina rat chow containing 0.17% and 0.86% methionine ... Four-week old male F344 rats obtained from Taconic Farms (Germantown, NY) were ... fed a standard control diet for 2 weeks. At 6 weeks of age, the rats were randomly assigned to control or MR diets. Food and water were provided ad libitum. ... RESULTS Serum Chemistry Profile of MR Rats Long-term MR induces hormonal and metabolite changes that correlate with decreased adiposity and improved insulin sensitivity in F344 rats (5); therefore, we examined early MR effects on serum chemistry. Although 1 month MR caused no significant changes in serum metabolites, insulin levels were significantly decreased by 39% and adiponectin levels were significantly increased by 48% in MR rats compared to CF rats (Table 1). By 3 months of MR, glucose and triglycerides were significantly reduced by 21% and 69%, respectively (Table 1). A marginal yet significant decrease in cholesterol was also observed in 3 month MR rats. Insulin levels were also reduced in 3 month MR rats, but were not significantly different from insulin levels in CF rats (Table 1). Finally, MR caused no significant changes in serum corticosterone levels at 1 and 3 months (Table 1). Table 1. Serum chemistry profile in CF and MR rats.============================================Parameter 1 month CF 1 month MR 3 months CF3 months MR============================================Insulin (ng/ml) 1.48¡¾0.26 0.91¡¾0.18**** 0.92¡¾0.26 0.43¡¾0.07Adiponectin (ng/ml) 5.18¡¾0.26 9.92¡¾0.40* 3.71¡¾0.35 12.35¡¾0.46*Corticosterone (ng/ml) 367¡¾110 597¡¾77 252¡¾64 301¡¾86Glucose (mg/dl) 188¡¾13 175¡¾21 207¡¾5 163¡¾3*Triglycerides (mg/dl) 149¡¾31 47¡¾16 106¡¾12.7 32¡¾2.9**Free Fatty Acids (mEq/dl) 0.2¡¾0.1 0.3¡¾0.2 14¡¾1 16¡¾2Cholesterol (mg/dl) 47¡¾4 43¡¾4 46¡¾1.6 40¡¾0.9***============================================ Values are expressed as the mean¡¾SEM of 5-7 samples and analyzed by ANOVA. *p <0.001; **p = 0.002; ***p = 0.026; ****p = 0.041. Effects of MR on 11©¬-HSD1 Activity To determine

whether the MR-mediated adiposity resistance correlates with changes in AT 11¥â-HSD1 activity, enzyme assays were conducted. 11¥â-HSD1 activity was increased in fat depots from MR rats as early as 7 days on the diet and remained elevated at 1 and 3 months on MR (Figure 1). To verify the MR effects on 11¥â-HSD1, corticosterone levels were measured in AT from 1 and 3 month CF and MR rats. AT corticosterone was increased at 1 and 3 months of MR corresponding with increased 11¥â-HSD1 activity (Figure 2). Corticosterone is also metabolized in liver; therefore, the effect of MR on the liver 11¥â-HSD1 enzyme was examined. In contrast to AT, no changes in liver 11¥â-HSD1 activity were observed in MR rats (Figure 3) suggesting that the MR effects were adipose tissue-specific. Reduced Mature Adipocyte Area in Fat Depots from MR Rats Induction of 11¥â-HSD1 activity and elevated tissue

corticosterone levels have been associated with increased adipogenesis and lipogenesis in contrast to observations on adiposity resistance by MR. Because, corticosterone can also induce lipolysis (29,40,41), morphometric analyses were conducted to assess whether there were histological differences in ATs from CF and MR rats. Hematoxylin:eosin staining revealed that mature adipocytes from 1 and 3 month MR rats were significantly smaller than adipocytes from CF rats (Figures 4A and 4B). On average, 46%, 44%, 23%, and 38% reductions in the cross-sectional areas of inguinal, epididymal, mesenteric, and retroperitoneal adipocytes, respectively, were observed at 3 months in MR (Figures 4B and 4C). These results suggest that MR could be inducing changes in the lipogenic/lipolytic balance. MR Effects on the Expression and Phosphorylation Levels of Lipogenic and Lipolytic Enzymes Western blotting analysis was conducted

to evaluate whether decreased mature adipocyte cross-sectional area in response to MR correlated with increased expression of lipolytic enzymes. No significant changes in basal levels of ATGL and HSL were observed at 1 month on MR diet (data not shown). However, by 3 months, a significant increase in ATGL levels was observed (Figure 5A) suggesting upregulation of basal lipolysis. In contrast, no change in HSL expression was observed in AT from 3 month MR rats (Figure 5B). Because HSL activity can be upregulated by phosphorylation (23), the potential phosphorylation of HSL following MR at serine (Ser) 563 (site for PKA phosphorylation) or Ser565 (site for AMPK phosphorylation) was evaluated. Phosphorylation of HSL Ser563 was not detected by Western blotting. In contrast, a trend showing increased HSL Ser565 phosphorylation in AT from MR rats was observed although HSL Ser565 phosphorylation was not significantly different from CF rats (Figure 5C). Another key enzyme involved in the maintenance of the lipogenic and lipolytic balance is ACC (27). ACC is inactivated by phosphorylation at Ser residues 79, 1200, and 1215 after signaling of inhibitory hormones. Also, during fasting, lipolysis is stimulated following the degradation of ACC by ubiquitination promoted by the pseudokinase Tribbles 3 (TRB3) protein (26). Therefore, ACC protein and phosphorylation (using a phospho-specific antibody for Ser79) levels were examined in ATs from CF and MR rats. In contrast to fasting and caloric restriction, in which ACC levels are decreased (42), MR significantly increased levels of both ACC-1 and ACC-2 isoforms in AT, except for mesenteric fat depots (Figure 5D). Moreover, both ACC isoforms were significantly phosphorylated at the Ser79, except for mesenteric AT (Figure 5E). MR Effects on AMPK, PKA, and cAMP Levels in AT AT lipolysis is controlled by hormonal and

neuronal factors that upregulate the synthesis of cAMP and the downstream phosphorylation of PKA or that activate AMPK (reviewed in 43). AMPK activation by threonine (Thr) 172 phosphorylation was first examined considering that AMPK phosphorylates and inactivates ACC (23,26,28). Although there is a trend showing decreased basal AMPK levels, no significant differences were observed in ATs from CF and MR rats (Figure 6A). Moreover, no significant changes in AMPK Thr172 phosphorylation were observed in AT from MR rats, except for retroperitoneal fat (Figures 6B). Considering that MR reduced basal APMK protein levels, pAMPK/AMPK¥á ratios were estimated. No significant differences in pAMPK/AMPK¥á ratios were observed, except for retroperitoneal fat where it was increased (data not shown). To further confirm the MR effects on AMPK activation, we conducted AMPK enzyme assays. Corresponding with the Western blot data, no significant differences in AMPK activity were observed in AT

from MR rats, except for retroperitoneal fat (Table 2). Table 2. AMPK activity in AT from 3 month CF and MR rats.============================================AT---AMPK activity (nmoles/min x mg protein) --- CF MR============================================Inguinal 6.31¡¾1.19 5.07¡¾1.05Epididymal 18.20¡¾6.59 19¡¾3.92Mesenteric 2.26¡¾1.19 3.62¡¾0.99Retroperitoneal 0.29¡¾0.09 0.87¡¾0.12*============================================ Values are expressed as the mean¡¾SEM of 5 samples and analyzed by ANOVA (*p = 0.015). The classical lipolytic pathway involves G-protein activation and cAMP synthesis, which activates PKA resulting in the downstream phosphorylation of HSL. Therefore, cAMP and PKA phosphorylation levels were assessed in AT extracts. No changes in cAMP levels were detected in MR fat depots (Figure 7A) suggesting that MR had no effect on PKA activity.

These results agreed with Western blotting showing no changes in PKA C-¥á protein and phosphorylation levels (Figures 7B and 7C). Lipid Metabolism in AT from MR Rats Considering the observed MR effects on ATGL expression levels suggesting increased lipid degradation in ATs from MR rats, lipolysis studies in epididymal mature adipocytes were conducted. Epididymal tissue was chosen given that all ATs from MR rats show similar ATGL protein changes. On average, mature adipocytes from CF rats released 18.6 nM NEFA/106 cells into the medium (Figure 8A). A significant 58% increase in fatty acid release was observed in isoproterenol-treated adipocytes from CF rats (Figure 8A). Corresponding with the MR mediated effects on ATGL expression, basal fatty acid release from MR rat adipocytes was 3-fold greater than that observed from CF rat adipocytes (Figure 8A). Moreover, fatty acid release from MR rat adipocytes was

further induced by isoproterenol treatment (Figure 8A). Corresponding with the fatty acid release data, a trend towards an increase in glycerol release from MR adipocytes was observed (Figure 8B). The glycerol release data from MR rat adipocytes correlates with the findings that HSL protein and phosphorylation levels were not significantly changed by MR. Increased ACC phosphorylation in MR rats suggested decreased lipid synthesis; therefore, MR¡¯s effect on lipogenesis was also examined in vitro. Contrary to our hypothesis, a trend towards an increase in fatty acid synthesis was observed in mature adipocytes from MR rats (Figure 8C). These results suggest the possible presence of a futile cycle in ATs from MR rats. ... In conclusion, changes in the lipogenic/lipolytic balance leading to a lipid futile cycle appear to play important roles in MR-mediated adiposity resistance. MR-mediated changes in the lipogenic/lipolytic balance might

result from increased AT corticosterone and adiponectin levels as well as decreased insulin signaling, IGF-1 or methionine itself. This study provides insight into the intracellular responses of AT to MR, but further investigation is required to establish how the physiological changes induced by MR integrate resulting in adiposity resistance. -- Al Pater, alpater@...

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