Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice

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

UCP1 is an essential mediator of the effects of methionine restriction on energy balance but not insulin sensitivity

© FASEB. Dietary methionine restriction (MR) by 80%increases energy expenditure (EE), reduces adiposity, and improves insulin sensitivity. We propose that the MRinduced 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\u27s effects on EE and insulin sensitivity, EE did not differ between wild-type (WT) and Ucp1-/- mice on the control diet, butMR 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

PULSATILE LUTEINIZING HORMONE SECRETION IN THE CASTRATE MALE BOVINE: EFFECTS OF TESTOSTERONE OR ESTRADIOL REPLACEMENT THERAPY

Luteinizing hormone (LH) secretory profiles have been determined for the male bovine following castration and steroid replacement therapy. Serum LH concentrations increased approximately threefold during the first week following castration and thereafter remained elevated (6.6 ± .7 ng/ml). Castrates not receiving steroid replacement showed a rhythmic pattern of LH release that was of high frequency (mean pulse interval; 85 ± 5 min) and high amplitude (mean peak concentration, 11.2 ± 1.4 ng/ml). Chronic administration of estradiol-17β via subdermal Silastic implants reduced mean serum LH concentrations (2.1 ± .3 ng/ml) and blocked the pulsatile pattern of LH release in all steers. Similar administration of testosterone suppressed mean serum LH and blocked pulsatile LH release in two of four animals. The number of implants used in this study provided physiological concentrations of estradiol (9.8 ± 1.5 pg/ml) and testosterone (4.1 + .2 ng/ml) in systemic blood for the two respective treatment groups. Differences in the LH secretory profiles among testosterone;implanted steers may have been related, in part, to differences in the amounts of steroid not bound to serum proteins. These findings demonstrate that estradiol is a particularly potent inhibitor of pulsatile LH secretion in the male bovine and suggest that gonadal steroid feedback on LH secretion may, in part, be imposed at the level of the hypothalamus. The mechanism for pulsatile LH release is discussed relative to a centrally-located luteinizing hormone releasing hormone pulse generator

Alteration of cAMP-mediated hormonal responsiveness by bile acids in cells of nonhepatic origin

The present study was undertaken to determine whether bile acids could inhibit hormone-induced adenosine 3\u27,5\u27-cyclic monophosphate (cAMP) production in cells of nonhepatic origin, as previously reported in the liver [Bouscarel et al., Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G300-G310, 1995]. The bile acids, ursodeoxycholic acid (UDCA), chenodeoxycholic acid, and deoxycholic acid inhibited prostaglandin E1 (PGE1)- and isoproterenol-induced cAMP production by 40-60% in human skin fibroblasts and human umbilical vein endothelial cells, respectively, to a similar extent as that observed in the liver. However, in both models, the taurine conjugates of these respective dihydroxy bite acids were without effect. After permeabilization of fibroblasts with saponin, UDCA, and its taurine conjugates inhibited hormone-induced cAMP production in a similar manner with a maximum inhibition of ~ 55%. The other taurine-conjugated dihydroxy bile acids were also able to inhibit PGE1-induced cAMP production. Furthermore, in human fibroblasts, UDCA was taken up in a dose- and time- dependent manner, whereas there was no uptake of taurocholic acid, even after 30 min of incubation. Therefore these results suggest that plasma membrane crossing of bile acids is a requirement for their inhibition of hormone- induced cAMP production. The ability of certain bile acids to affect hormone- induced cAMP production in extrahepatic tissues may be of pathophysiological significance in certain cholestatic liver diseases