Protective role of ErbB3 signaling in myeloid cells during adaptation to cardiac pressure overload.

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.

Mesoderm-specific transcript localization in the ER and ER-lipid droplet interface supports a role in adipocyte hypertrophy.

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.

Lipid Profiling of In Vitro Cell Models of Adipogenic Differentiation: Relationships With Mouse Adipose Tissues.

Our objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MS(ALL) . Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3-L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose-derived BAT-C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl- and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3-L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose-derived cell differentiation models, respectively. MS/MS(ALL) data suggest new lipid biomarkers for tissue-specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo. J. Cell. Biochem. 117: 2182-2193, 2016. © 2016 Wiley Periodicals, Inc.

A High Fat Diet Increases Bone Marrow Adipose Tissue (MAT) But Does Not Alter Trabecular or Cortical Bone Mass in C57BL/6J Mice.

Obesity has been associated with high bone mineral density (BMD) but a greater propensity to fracture. Some obese individuals have increased marrow adipose tissue (MAT), but the impact of MAT on bone turnover remains controversial, as do changes in BMD associated with a high fat diet (HFD). In this study we hypothesized that MAT volume would increase in response to HFD but would be independent of changes in BMD. Hence, we fed C57BL/6J (B6) male mice at 3 weeks of age either a high fat diet (60 kcal %) or regular diet (10 kcal %) for 12 weeks (n = 10/group). We measured MAT volume by osmium staining and micro-CT (µCT) as well as bone parameters by µCT, histomorphometry, and dual-energy X-ray absorptiometry. We also performed a short-term pilot study using 13-week-old B6 males and females fed a HFD (58 kcal %) for 2 weeks (n = 3/sex). Both long- and short-term HFD feedings were associated with high MAT volume, however, femoral trabecular bone volume fraction (BV/TV), bone formation rate and cortical bone mass were not altered in the long-term study. In the short-term pilot study, areal BMD was unchanged after 2 weeks of HFD. We conclude that, for B6 mice fed a HFD starting at wean or 13 weeks of age, MAT increases whereas bone mass is not altered. More studies are needed to define the mechanism responsible for the rapid storage of energy in the marrow and its distinction from other adipose depots.

Interindividual differences of dietary fat-inducible Mest in white adipose tissue of C57BL/6J mice are not heritable.

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.

Parallel control of cold-triggered adipocyte thermogenesis by UCP1 and CKB.

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.

Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation.

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 hours. Percoll purification from 100-200 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 3.9-7.1 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.

Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation.

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.

Inactivation of the mitochondrial carrier SLC25A25 (ATP-Mg2+/Pi transporter) reduces physical endurance and metabolic efficiency in mice.

An ATP-Mg(2+/)P(i) inner mitochondrial membrane solute transporter (SLC25A25), which is induced during adaptation to cold stress in the skeletal muscle of mice with defective UCP1/brown adipose tissue thermogenesis, has been evaluated for its role in metabolic efficiency. SLC25A25 is thought to control ATP homeostasis by functioning as a Ca(2+)-regulated shuttle of ATP-Mg(2+) and P(i) across the inner mitochondrial membrane. Mice with an inactivated Slc25a25 gene have reduced metabolic efficiency as evidenced by enhanced resistance to diet-induced obesity and impaired exercise performance on a treadmill. Mouse embryo fibroblasts from Slc25a25(-/-) mice have reduced Ca(2+) flux across the endoplasmic reticulum, basal mitochondrial respiration, and ATP content. Although Slc25a25(-/-) mice are metabolically inefficient, the source of the inefficiency is not from a primary function in thermogenesis, because Slc25a25(-/-) mice maintain body temperature upon acute exposure to the cold (4 °C). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to muscle function as evidenced from the physical endurance test of mutant mice on a treadmill. Consequently, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity. However, in the absence of UCP1-based thermogenesis, induction of Slc25a25 in mice with an intact gene may contribute to an alternative thermogenic pathway for the maintenance of body temperature during cold stress.

Rapamycin/metformin co-treatment normalizes insulin sensitivity and reduces complications of metabolic syndrome in type 2 diabetic mice.

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.

Social and maternal behavior in mesoderm specific transcript (Mest)-deficient mice.

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.

Sprouty1 regulates gonadal white adipose tissue growth through a PDGFRα/ß-Akt pathway.

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.

Weight Loss and Concomitant Adipose Autophagy in Methionine-Restricted Obese Mice is Not Dependent on Adiponectin or FGF21.

OBJECTIVE: 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. METHODS: Diet-induced obese (DIO) male C57BL/6J (wild type), Adipoq-deficient (Adipoq knockout [KO]), Fgf21-KO, and Adipoq-Fgf21 double-KO mice were used. Following a switch to high-fat control (DIO-CF, 60% fat/0.86% methionine) or MR (DIO-MR, 60% fat/0.12% methionine) diet, physiological parameters were measured, and inguinal and perigonadal adipose tissues were examined. RESULTS: Obese mice subjected to MR showed loss of body weight and adiposity, increased energy expenditure, and improved glucose tolerance that were independent of the actions of ADIPOQ and FGF21. MR induced reduction of circulating lipids, glucose, insulin, leptin, and insulin like growth factor 1 and increased ß-hydroxybutyrate, ADIPOQ, and FGF21 concentrations. In fat, MR upregulated protein levels of adipose triglyceride lipase, apoptosis-inducing factor, lysosomal-associated membrane proteins 1 and 2, autophagy-related protein 5, beclin-1, and light chain 3B I and II. CONCLUSIONS: MR reduction of adipose tissue mass in obese mice is associated with elevated lipolysis, apoptosis, and autophagy and occurs independently of the actions of ADIPOQ and FGF21.

Mesoderm-specific transcript is associated with fat mass expansion in response to a positive energy balance.

A 50-fold variation in mRNA and protein levels of the mesoderm-specific transcript gene (Mest) in white fat of C57BL/6J (B6) mice fed an obesogenic diet is positively correlated with expansion of fat mass. MEST protein was detected only in adipocytes, in which its induction occurred with both unsaturated and saturated dietary fat. To test the hypothesis that MEST modulates fat mass expansion, its expression was compared to that of stearoyl CoA desaturase (Scd1) in B6 mice exposed to diets and environmental temperatures that generated conditions separating the effects of food intake and adiposity. Under a range of conditions, Mest expression was always associated with variations in adiposity, whereas Scd1 expression was associated with the amount of saturated fat in the diet. Mest mRNA was expressed at its highest levels during early postnatal growth at the onset of the most rapid phase of fat mass expansion. MEST is localized to the endoplasmic reticulum/Golgi apparatus where its putative enzymatic properties as a lipase or acyltransferase, predicted from sequence homology with members of the alpha/beta fold hydrolase superfamily, can enable it to function in lipid accumulation under conditions of positive energy balance. Variations in adiposity and Mest expression in genetically identical mice also provides a model of epigenetic regulation.

Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.

High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.

Transcriptional synergy and the regulation of Ucp1 during brown adipocyte induction in white fat depots.

Induction of brown adipocytes in white fat depots by adrenergic stimulation is a complex genetic trait in mice that affects the ability of the animal to regulate body weight. An 80-fold difference in expression of the mitochondrial uncoupling gene (Ucp1) at the mRNA and protein levels between A/J and C57BL/6J (B6) mice is controlled by allelic interactions among nine quantitative trait loci (QTLs) on eight chromosomes. Overlapping patterns of these QTLs also regulate expression levels of Pgc-1alpha, Pparalpha, and type 2 deiodinase. Independent validation that PPARalpha is associated with Ucp1 induction was obtained by treating mice with the PPARalpha agonist clofibrate, but not from the analysis of PPARalpha knockout mice. The most upstream sites of regulation for Ucp1 that differed between A/J and B6 were the phosphorylation of p38 mitogen-activated protein kinase and CREB and then followed by downstream changes in levels of mRNA for PPARgamma, PPARalpha, PGC-1alpha, and type 2 deiodinase. However, compared to Ucp1 expression, the two- to fourfold differences in the expression of these regulatory components are very modest. It is proposed that small variations in the levels of several transcriptional components of the Ucp1 enhanceosome interact synergistically to achieve large differences in Ucp1 expression.

Cardioprotective effects of dietary rapamycin on adult female C57BLKS/J-Leprdb mice.

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-Leprdb , to determine whether RAPA treatment can mitigate the development of T2D-induced cardiomyopathy in adult mice. Female BKS-Leprdb 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.

Diet-induced adipose tissue expansion is mitigated in mice with a targeted inactivation of mesoderm specific transcript (Mest).

Interindividual variation of white adipose tissue (WAT) expression of mesoderm specific transcript (Mest), a paternally-expressed imprinted gene belonging to the α/ß-hydrolase fold protein family, becomes apparent among genetically inbred mice fed high fat diet (HFD) and is positively associated with adipose tissue expansion (ATE). To elucidate a role for MEST in ATE, mice were developed with global and adipose tissue inactivation of Mest. Mice with homozygous (MestgKO) and paternal allelic (MestpKO) inactivation of Mest were born at expected Mendelian frequencies, showed no behavioral or physical abnormalities, and did not perturb expression of the Mest locus-derived microRNA miR-335. MestpKO mice fed HFD showed reduced ATE and adipocyte hypertrophy, improved glucose tolerance, and reduced WAT expression of genes associated with hypoxia and inflammation compared to littermate controls. Remarkably, caloric intake and energy expenditure were unchanged between genotypes. Mice with adipose tissue inactivation of Mest were phenotypically similar to MestpKO, supporting a role for WAT MEST in ATE. Global profiling of WAT gene expression of HFD-fed control and MestpKO mice detected few differences between genotypes; nevertheless, genes with reduced expression in MestpKO mice were associated with immune processes and consistent with improved glucose homeostasis. Ear-derived mesenchymal stem cells (EMSC) from MestgKO mice showed no differences in adipogenic differentiation compared to control cells unless challenged by shRNA knockdown of Gpat4, an enzyme that mediates lipid accumulation in adipocytes. Reduced adipogenic capacity of EMSC from MestgKO after Gpat4 knockdown suggests that MEST facilitates lipid accumulation in adipocytes. Our data suggests that reduced diet-induced ATE in MEST-deficient mice diminishes hypoxia and inflammation in WAT leading to improved glucose tolerance and insulin sensitivity. Since inactivation of Mest in mice has minimal additional effects aside from reduction of ATE, an intervention that mitigates MEST function in adipocytes is a plausible strategy to obviate obesity and type-2-diabetes.

A high-fat diet coordinately downregulates genes required for mitochondrial oxidative phosphorylation in skeletal muscle.

Obesity and type 2 diabetes have been associated with a high-fat diet (HFD) and reduced mitochondrial mass and function. We hypothesized a HFD may affect expression of genes involved in mitochondrial function and biogenesis. To test this hypothesis, we fed 10 insulin-sensitive males an isoenergetic HFD for 3 days with muscle biopsies before and after intervention. Oligonucleotide microarray analysis revealed 297 genes were differentially regulated by the HFD (Bonferonni adjusted P < 0.001). Six genes involved in oxidative phosphorylation (OXPHOS) decreased. Four were members of mitochondrial complex I: NDUFB3, NDUFB5, NDUFS1, and NDUFV1; one was SDHB in complex II and a mitochondrial carrier protein SLC25A12. Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1) alpha and PGC1beta mRNA were decreased by -20%, P < 0.01, and -25%, P < 0.01, respectively. In a separate experiment, we fed C57Bl/6J mice a HFD for 3 weeks and found that the same OXPHOS and PGC1 mRNAs were downregulated by approximately 90%, cytochrome C and PGC1alpha protein by approximately 40%. Combined, these results suggest a mechanism whereby HFD downregulates genes necessary for OXPHOS and mitochondrial biogenesis. These changes mimic those observed in diabetes and insulin resistance and, if sustained, may result in mitochondrial dysfunction in the prediabetic/insulin-resistant state.

High-fat/low-carbohydrate diets regulate glucose metabolism via a long-term transcriptional loop.

Insulin sensitivity is characterized by insulin-stimulated glucose metabolism in skeletal muscle. We hypothesized that carbohydrate metabolism and storage might be under transcriptional control. To test this hypothesis, we fed insulin-sensitive males (glucose disposal rate, 14.7 +/- 4.1 mg/kg fat-free mass [FFM] per minute) an isoenergetic high-fat/low-carbohydrate diet (HF/LCD) for 3 days with muscle biopsies before and after intervention. Oligonucleotide microarrays revealed a total of 369 genes of 18861 genes on the arrays were differentially regulated in response to diet (Bonferonni adjusted P < .01). A similar experiment was conducted in mice with a 3-week intervention using a control group and an HF/LCD group to offset the lack of a control group within the human cohort. As part of an analysis of results previously published from this data set, 7 genes in the carbohydrate metabolism pathway changed in response to the HF/LCD, and 3 genes were confirmed by quantitative reverse transcriptase-polymerase chain reaction: fructose-2,6-biphosphatase 3 (PFKFB3), pyruvate dehydrogenase kinase, isoenzyme 4 (PDK4), and glycogen synthase 1 (muscle). In a separate experiment, we fed C57Bl/6J mice an HF/LCD for 3 weeks and found that the same glucose metabolism genes were changed by approximately 70% on average. Fructose-2,6-biphosphatase 3 and pyruvate dehydrogenase kinase, isoenzyme 4 increased and glycogen synthase 1 (muscle) decreased. Combined, these results suggest a mechanism whereby HF/LCD regulates the genes necessary for glucose utilization and storage vis-á-vis transcriptional control.