Dietary methionine restriction (MR) improves metabolic health in various animals. Beneficial effects on mice include reduced obesity and improved metabolism associated with the appearance of multilocular uncoupling protein 1 (UCP1) positive adipocytes in white fat. This suggests that MR-induced UCP1 thermogenesis contributes significantly to metabolic health. To test this, we evaluated MR effects using unique UCP1-deficient mice on a hybrid (F1) background that is resistant to developing hypothermia upon acute cold exposure. F1.Ucp1HET and F1.Ucp1KO mice were fed high fat control diet (HF-CD) and then switched to a high fat MR diet (HF-MRD). Indirect calorimetry was used to monitor energy metabolism; and, serum hormones, metabolites, tissue gene expression, and histology were evaluated. While both genotypes showed improved metabolic parameters when fed HF-MRD, F1.Ucp1KO mice exhibited further changes in iWAT morphology and more pronounced metabolic improvements in glucose tolerance, serum metabolic biomarker profiles and hepatic steatosis compared to F1.Ucp1HET. Strikingly, F1.Ucp1KO mice showed elevated fatty acid oxidation compared with F1.Ucp1HET when fed high fat control diet (HF-CD) that was sustained after the switch to HF-MRD. We posit that F1.Ucp1KO mice employ fatty acid oxidation for body temperature maintenance as at least a part of a mechanism that protects them from acute exposure to cold temperatures in addition to development of diet-induced obesity. Furthermore, our studies suggest that many of the metabolic benefits of MR in mice do not require functional UCP1 thermogenesis.

IntroductionMesoderm-specific transcript (Mest), a paternally expressed imprinted gene, is involved in the modulation of adipose tissue expansion. Mest is also highly expressed in the developing and adult brain, suggesting a role in behavioral phenotypes. Previously, we showed that female mice with paternal Mest inactivation (MestpKO) exhibit no discernible behavioral impairments compared to wild-type mice. In this study, we performed metabolic phenotyping of female MestpKO mice in response to a dietary challenge.MethodsEight-week-old female and male wild-type and MestpKO mice were fed a control or Western diet (40 kcal% fat) until 24 weeks of age. Body weight, body composition, and metabolic parameters were measured during the course of the feeding regimens, and gene expression and type-2-deiodinase (DIO2) activity were examined in white adipose tissue and brain at the end of the study.ResultsMestpKO female mice fed a Western diet were protected against diet-induced obesity. Strikingly, these mice showed increased ambulatory activity and speed, coupled with reduced resting parameters, suggesting a role for MEST in the regulation of spontaneous physical activity, a form of nonexercise activity thermogenesis. When considering body mass (control diet) and lean mass (Western diet), energy expenditure was increased in the female MestpKO mice. Male MestpKO mice did not exhibit these changes. Analyses of hypothalamic gene expression revealed upregulation of Dio2, and RNA-seq highlighted differential expression of numerous thyroid hormone-responsive genes in MestpKO female mice.ConclusionMechanistically, our results suggest that MEST directly or indirectly regulates thyroid hormone-responsive genes in the hypothalamus, thereby modulating the neurobiological control of nonexercise activity thermogenesis in Western diet-fed female mice.

Adipogenesis is regulated by the coordinated activity of adipogenic transcription factors including PPAR-gamma and C/EBP alpha, while dysregulated adipogenesis can predispose adipose tissues to adipocyte hypertrophy and hyperplasia. We have previously reported that Cthrc1-null mice have increased adiposity compared to wildtype mice, supporting the notion that CTHRC1 regulates body composition. Herein, we derived conditioned medium from 3T3-L1 cells expressing human CTHRC1 and investigated its anti-adipogenic activity. This constituent significantly reduced 3T3-L1 cell adipogenic differentiation commensurate to the marked suppression of Cebpa and Pparg gene expression. It also increased the expression of the anti-adipogenic transcription factor SOX9 and promoted its nuclear translocation. Importantly, Sox9 gene knockdown demonstrated that the anti-adipogenic effect produced by this conditioned medium is dependent on SOX9 expression, while its ability to positively regulate SOX9 was attenuated by the application of Rho and Rac1 signaling pathway inhibitors. We also identified the selective expression of CTHRC1 in PDGFRA-expressing cell populations in human white adipose tissue, but not brown or perivascular adipose tissues. Congruently, flow cytometry revealed CTHRC1 expression in PDGFR-alpha+ stromal cells of mouse white adipose tissue, thus defining a novel stromal cell population that could underpin the ability of CTHRC1 to regulate adiposity.

Dietary methionine restriction (MetR) promotes metabolic health, and we tested the impact of short durations of MetR on high fat diet (HFD)‐induced metabolic dysfunction with the maintenance of HFD. Male C57BL/6J mice were fed HFD from 10 to 25 weeks of age, then maintained on HFD or fed HFD with 80% reduced methionine (HFD‐MetR) for 3, 5, or 10 days. Blood, liver, adipose tissue, and aortae underwent phenotypic assessment, proteomics, and metabolomics. HFD‐MetR induced rapid weight loss and robust metabolic improvement within 10 days. Significant reductions in body weight, circulating triglycerides, glucose, insulin, adipokines and hepatokines reflected metabolic health. Proteomics revealed enriched metabolic signatures in perivascular adipose tissue (PVAT) and structural remodeling signatures in aorta. Metabolomics identified a cardioprotective signature in blood plasma, and activated mitochondrial activity and energy production in liver and brown adipose tissue. HFD‐MetR reversed metabolic dysfunction, and novel proteomic and metabolomic signatures were identified. Multi‐organ molecular changes in lipid metabolism, mitochondrial function, and bioenergetics are predicted to impact adipose tissue and liver function and cardiovascular health. Our identification of rapid changes in protein and metabolite signatures with accelerated restoration of metabolic health can be leveraged to evaluate biomarkers of metabolic health and disease in a translational context.

That uncoupling protein 1 (UCP1) is the sole mediator of adipocyte thermogenesis is a conventional viewpoint that has primarily been inferred from the attenuation of the thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue. To mitigate these potentially confounding ancillary changes, we constructed mice with inducible adipocyte-selective Ucp1 disruption. We find that, although germline Ucp1-/- mice succumb to cold -induced hypothermia with complete penetrance, most mice with the inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co -deletion of Ucp1 and creatine kinase b (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold intolerance. Following UCP1 deletion or UCP1/CKB co -deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature brown adipocytes that coordinately restore UCP1 and CKB expression. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy-through UCP1 and CKB-to promote cold -induced energy dissipation.

Objective: Differences in white adipose tissue (WAT) expression of mesoderm-specific transcript (Mest) in C57BL6/J mice fed a high-fat diet (HFD) are concomitant with and predictive for the development of obesity. However, the basis for differences in WAT Mest among mice is unknown. This study investigated whether HFD-inducible WAT Mest, as well as susceptibility to obesity, is transmissible from parents to offspring. Methods: WAT biopsies of mice fed an HFD for 2 weeks identified parents with low and high WAT Mest for breeding. Obesity phenotypes, WAT Mest, hepatic gene expression, and serum metabolites were determined in offspring fed an HFD for 2 weeks. Results: Offspring showed no heritability of obesity or WAT Mest phenotypes from parents but did show hepatic and serum metabolite changes consistent with their WAT Mest. Importantly, retired male breeders showed WAT Mest expression congruent with initial WAT biopsies even though HFD exposure occurred early in life. Conclusions: Disparity of HFD-induced Mest in mice is not heritable but, rather, is reestablished during each generation and remains fixed from an early age to adulthood. Short-term HFD feeding reveals variation of WAT Mest expression within isogenic mice that is positively associated with the development of obesity.

Objective: Perivascular adipose tissue (PVAT) regulates vascular health. Dietary methionine restriction (MetR) impacts age-related adiposity, and this study addresses its effects in PVAT. Methods: Male C57BL/6 mice at 8, 52, and 102 weeks of age were fed a standard (0.86%) or low-methionine (0.12%) diet for 52 weeks in 8-week-old and 52-week-old mice and for 15 weeks in 102-week-old mice. Results: Mice with dietary MetR were resistant to weight gain and maintained a healthy blood profile. Aging increased lipid accumulation, and MetR reversed this phenotype. Notch signaling in inguinal white adipose tissue (iWAT) was decreased by MetR but increased in gonadal white adipose tissue. However, the Notch phenotype of brown adipose tissue (BAT) was not affected by MetR. Uncoupling protein 1 (UCP1) was increased in PVAT, iWAT, and BAT by MetR when initiated in young mice, but this effect was lost in middle-aged mice. Conclusions: Lipid in mouse PVAT peaked at 1 year of age, consistent with peak body mass. MetR reduced body weight, normalized metabolic parameters, and decreased lipid in PVAT in all age cohorts. Mice fed a MetR diet from early maturity to 1 year of age displayed an increased thermogenic adipocyte phenotype in iWAT, PVAT, and BAT, all tissues with thermogenic capacity.

ObesityVolume 31, Issue 12 p. 2885-2886 IN MEMORIAM Leslie Paul Kozak, PhD (1940–2023) Eric Ravussin, Eric Ravussin orcid.org/0000-0003-2129-547X Pennington Biomedical Research Center, Baton Rouge, Louisiana, USASearch for more papers by this authorSven Enerback, Sven Enerback Department of Medical Biochemistry and Cell Biology, University of Gothenburg Institute of Biomedicine, Gothenburg, SwedenSearch for more papers by this authorRobert Koza, Corresponding Author Robert Koza [email protected] orcid.org/0000-0001-6994-7268 Center for Molecular Medicine, MaineHealth Institute for Research, Scarborough, Maine, USA Correspondence Robert Koza, Center for Molecular Medicine, MaineHealth Institute for Research, Scarborough, ME, USA. Email: [email protected]Search for more papers by this author Eric Ravussin, Eric Ravussin orcid.org/0000-0003-2129-547X Pennington Biomedical Research Center, Baton Rouge, Louisiana, USASearch for more papers by this authorSven Enerback, Sven Enerback Department of Medical Biochemistry and Cell Biology, University of Gothenburg Institute of Biomedicine, Gothenburg, SwedenSearch for more papers by this authorRobert Koza, Corresponding Author Robert Koza [email protected] orcid.org/0000-0001-6994-7268 Center for Molecular Medicine, MaineHealth Institute for Research, Scarborough, Maine, USA Correspondence Robert Koza, Center for Molecular Medicine, MaineHealth Institute for Research, Scarborough, ME, USA. Email: [email protected]Search for more papers by this author First published: 04 October 2023 https://doi.org/10.1002/oby.23919Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1Kozak LP. Play Up! Play Up! And Play the Game!. Friesen Press; 2020. 2Enerback S, Jacobsson A, Simpson EM, et al. Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature. 1997; 387(6628): 90-94. doi:10.1038/387090a0 3Jacobsson A, Stadler U, Glotzer MA, Kozak LP. Mitochondrial uncoupling protein from mouse brown fat. Molecular cloning, genetic mapping, and mRNA expression. J Biol Chem. 1985; 260: 16250-16254. 4Kozak UC, Kopecky J, Teisinger J, Enerback S, Boyer B, Kozak LP. An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene. Mol Cell Biol. 1994; 14: 59-67. 5Guerra C, Koza R, Yamashita H, King KW, Kozak LP. Emergence of brown adipocytes in white fat is under genetic control. Effects on body weight and adiposity. J Clin Invest. 1998; 102: 412-420. 6Koza RA, Nikonova L, Hogan J, et al. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice. PLoS Genet. 2006; 2:e81. Volume31, Issue12December 2023Pages 2885-2886 ReferencesRelatedInformation

Objective Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. Results Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3–4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200–400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9 to 7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

Rapamycin treatment has positive and negative effects on progression of type 2 diabetes (T2D) in a recombinant inbred polygenic mouse model, male NONcNZO10/LtJ (NcZ10). Here, we show that combination treatment with metformin ameliorates negative effects of rapamycin while maintaining its benefits. From 12 to 30 weeks of age, NcZ10 males were fed a control diet or diets supplemented with rapamycin, metformin, or a combination of both. Rapamycin alone reduced weight gain, adiposity, HOMA-IR, and inflammation, and prevented hyperinsulinemia and pre-steatotic hepatic lipidosis, but exacerbated hyperglycemia, hypertriglyceridemia, and pancreatic islet degranulation. Metformin alone reduced hyperinsulinemia and circulating c-reactive protein, but exacerbated nephropathy. Combination treatment retained the benefits of both while preventing many of the deleterious effects. Importantly, the combination treatment reversed effects of rapamycin on markers of hepatic insulin resistance and normalized systemic insulin sensitivity in this inherently insulin-resistant model. In adipose tissue, rapamycin attenuated the expression of genes associated with adipose tissue expansion (Mest, Gpam), inflammation (Itgam, Itgax, Hmox1, Lbp), and cell senescence (Serpine1). In liver, the addition of metformin counteracted rapamycin-induced alterations of G6pc, Ppara, and Ldlr expressions that promote hyperglycemia and hypertriglyceridemia. Both rapamycin and metformin treatment reduced hepatic Fasn expression, potentially preventing lipidosis. These results delineate a state of "insulin signaling restriction" that withdraws endocrine support for further adipogenesis, progression of the metabolic syndrome, and the development of its comorbidities. Our results are relevant for the treatment of T2D, the optimization of current rapamycin-based treatments for posttransplant rejection and various cancers, and for the development of treatments for healthy aging.

Mesoderm specific transcript (Mest)/paternally expressed gene-1 (Peg1) is an imprinted gene expressed predominantly from the paternal allele. Aberrations in maternal behavior were previously reported in a Mest global knockout mouse (Mesttm1Masu). In this study, we performed in-depth social and maternal behavioral testing in a mouse model of Mest inactivation developed in our laboratory (Mesttm1.2Rkz). Mice with paternal allele inactivation (MestpKO) did not show anxiety after testing in the elevated plus maze, open field trial, and marble burying; nor depression-like behaviors in the tail suspension test. MestpKO showed normal social behaviors and memory/cognition in the three-chamber box test and the novel object recognition test, respectively. Primiparous MestpKO and MestgKO (biallelic Mest inactivation) female mice exhibited normal nest building and maternal behavior; and, virgin MestpKO and MestgKO female mice showed normal maternal instinct. Analyses of gene expression in adult hypothalamus, embryonic day 14.5 whole brain and adult whole brain demonstrated full abrogation of Mest mRNA in MestpKO and MestgKO mice with no effect on miR-335 expression. Our data indicates no discernible impairments in object recognition memory, social behavior or maternal behavior resulting from loss of Mest. The basis for the differences in maternal phenotypic behaviors between Mesttm1Masu and Mesttm1.2Rkz is not known.

Background Myeloid cells play an important role in a wide variety of cardiovascular disorders, including both ischemic and non-ischemic cardiomyopathies. Neuregulin-1 (NRG-1)/ErbB signaling has recently emerged as an important factor contributing to the control of inflammatory activation of myeloid cells after an ischemic injury. However, the role of ErbB signaling in myeloid cells in non-ischemic cardiomyopathy is not fully understood. This study investigated the role of ErbB3 receptors in the regulation of early adaptive response using a mouse model of transverse aortic constriction (TAC) for non-ischemic cardiomyopathy. Methods and results TAC surgery was performed in groups of age- and sex-matched myeloid cell-specific ErbB3-deficient mice (ErbB3MyeKO) and control animals (ErbB3MyeWT). The number of cardiac CD45 immune cells, CD11b myeloid cells, Ly6G neutrophils, and Ly6C monocytes was determined using flow cytometric analysis. Five days after TAC, survival was dramatically reduced in male but not female ErbB3MyeKO mice or control animals. The examination of lung weight to body weight ratio suggested that acute pulmonary edema was present in ErbB3MyeKO male mice after TAC. To determine the cellular and molecular mechanisms involved in the increased mortality in ErbB3MyeKO male mice, cardiac cell populations were examined at day 3 post-TAC using flow cytometry. Myeloid cells accumulated in control but not in ErbB3MyeKO male mouse hearts. This was accompanied by increased proliferation of Sca-1 positive non-immune cells (endothelial cells and fibroblasts) in control but not ErbB3MyeKO male mice. No significant differences in intramyocardial accumulation of myeloid cells or proliferation of Sca-1 cells were found between the groups of ErbB3MyeKO and ErbB3MyeWT female mice. An antibody-based protein array analysis revealed that IGF-1 expression was significantly downregulated only in ErbB3MyeKO mice hearts compared to control animals after TAC. Conclusion Our data demonstrate the crucial role of myeloid cell-specific ErbB3 signaling in the cardiac accumulation of myeloid cells, which contributes to the activation of cardiac endothelial cells and fibroblasts and development of an early adaptive response to cardiac pressure overload in male mice.

Expansion of visceral white adipose tissue (vWAT) occurs in response to nutrient excess, and is a risk factor for metabolic disease. SPRY1, a feedback inhibitor of receptor tyrosine kinase (RTK) signaling, is expressed in PDGFRa+ adipocyte progenitor cells (APC) in vivo. Global deficiency of Spry1 in mice results in disproportionate postnatal growth of gonadal WAT (gWAT), while iWAT and BAT were similar in size between Spry1KO and WT mice. Spry1 deficiency increased the number of PDGFRa+ stromal vascular fraction (SVF) cells in gWAT and showed increased proliferation and fibrosis. Spry1KO gWAT had increased collagen deposition and elevated expression of markers of inflammation. In vitro, SPRY1 was transiently down regulated during early adipocyte differentiation of SVF cells, with levels increasing at later stages of differentiation. SPRY1 deficiency enhances PDGF-AA and PDGF-BB induced proliferation of SVF cells. Increased proliferation of SVF from Spry1KO gWAT accompanies an increase in AKT activation. PDGF-AA stimulated a transient down regulation of SPRY1 in wild type SVF, whereas PDGF-BB stimulated a sustained down regulation of SPRY1 in wild type SVF. Collectively, our data suggest that SPRY1 is critical for regulating postnatal growth of gWAT by restraining APC proliferation and differentiation in part by regulation of PDGFRa/b-AKT signaling.

in the deterioration of glucose and lipid metabolism in the obese state will require investigating adropin deficiency due to targeted gene deletion.

Identifying novel approaches to combat obesity is important to improve health span. It was hypothesized that methionine restriction (MR) will induce weight loss in obese mice by reducing adipose tissue mass caused by increased energy expenditure and reprogramming of adipose tissue homeostasis. The roles of adiponectin (ADIPOQ) and fibroblast growth factor 21 (FGF21) during weight loss in MR mice were also tested.

Rapamycin (RAPA), an inhibitor of mTORC signaling, has been shown to extend life span in mice and other organisms. Recently, animal and human studies have suggested that inhibition of mTORC signaling can alleviate or prevent the development of cardiomyopathy. In view of this, we used a murine model of type 2 diabetes (T2D), BKS-Lepr(db), to determine whether RAPA treatment can mitigate the development of T2D-induced cardiomyopathy in adult mice. Female BKS-Lepr(db) mice fed diet supplemented with RAPA from 11 to 27 weeks of age showed reduced weight gain and significant reductions of fat and lean mass compared with untreated mice. No differences in plasma glucose or insulin levels were observed between groups; however, RAPA-treated mice were more insulin sensitive (P < 0.01) than untreated mice. Urine albumin/creatinine ratio was lower in RAPA-treated mice, suggesting reduced diabetic nephropathy and improved kidney function. Echocardiography showed significantly reduced left ventricular wall thickness in mice treated with RAPA compared with untreated mice (P = 0.02) that was consistent with reduced heart weight/tibia length ratios, reduced myocyte size and cardiac fibrosis measured by histomorphology, and reduced mRNA expression of Col1a1, a marker for cardiomyopathy. Our results suggest that inhibition of mTORC signaling is a plausible strategy for ameliorating complications of obesity and T2D, including cardiomyopathy.

Highly variable expression of mesoderm-specific transcript (Mest) in adipose tissue among genetically homogeneous mice fed an obesogenic diet, and its positive association with fat mass expansion, suggests that Mest is an epigenetic determinant for the development of obesity. Although the mechanisms by which MEST augments fat accumulation in adipocytes have not been elucidated, it has sequence homology and catalytic peptide motifs which suggests that it functions as an epoxide hydrolase or as a glycerol- or acylglycerol-3-phosphate acyltransferase. To better understand MEST function, detailed studies were performed to precisely define the intracellular organelle localization of MEST using immunofluorescence confocal microscopy. Lentiviral-mediated expression of a C-terminus Myc-DDK-tagged MEST fusion protein expressed in 3T3-L1 preadipocytes/adipocytes, and ear-derived mesenchymal stem cells (EMSC) from mice was observed in the endoplasmic reticulum (ER) membranes and is consistent with previous studies showing endogenous MEST in the membrane fraction of adipose tissue. MEST was not associated with the Golgi apparatus or mitochondria; however, frequent contacts were observed between MEST-positive ER and mitochondria. MEST-positive domains were also shown on the plasma membrane (PM) of non-permeabilized cells but they did not co-localize with ER-PM bridges. Post-adipogenic differentiated 3T3-L1 adipocytes and EMSC showed significant co-localization of MEST with the lipid droplet surface marker perilipin at contact points between the ER and lipid droplet. Identification of MEST as an ER-specific protein that co-localizes with lipid droplets in cells undergoing adipogenic differentiation supports a function for MEST in the facilitation of lipid accumulation and storage in adipocytes.