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Many targeted cancer therapies rely on biomarkers, which are assessed by standard pathologist scoring of immunohistochemically stained tissue. However, this process is subjective, semi-quantitative and does not assess expression heterogeneity. A quantitative method to measure IHC markers might therefore significantly improve patient selection particularly of proteins expressed at low levels. To address these challenges, we have developed the Quantitative Continuous Scoring (QCS) that deploys the power of fully supervised Deep Learning (DL) algorithms to provide objective and continuous data of biomarkers in digitized IHC whole slide images (WSI). The two DL-based algorithms, developed using pathologist input as the ground truth, identify areas of invasive tumor and segment each individual tumor cell across the WSI into pixels that represent cell nuclei, cytoplasm and/or membrane. This allows to compute biomarker expression as mean Optical Density (OD) in each of these subcellular compartments based on the Hue-Saturation-Density (HSD) model. Of note, this also allows computation of the spatial distribution of tumor cells across the WSI. The measured OD for each cell is aggregated as a histogram to quantitative continuous readouts for each patient sample. The method’s ability to accurately detect low expression range facilitates selection of antibody clones for IHC assays, has been successfully used to delineate mode of action and PK/PD mechanisms, has provided surrogate markers of spatial expression heterogeneity to predict potential bystander activity and has facilitated marker co-expression analysis to inform rational combination therapies. In retrospective clinical trials analysis, QCS showed superior performance in identifying a patient population gaining maximum treatment benefit. QCS-based quantification of PD-L1 membrane expression was able to stratify anti-PD-L1 treated late-stage non-small cell lung cancer (NSCLC) patients [NCT01693562] with a higher prevalence and more significant log rank p-value (64%, p=0.0001) for OS compared to pathologist TPS (59%, p=0.01). In summary, we describe a computational pathology-based approach for precise biomarker quantification and superior patient selection with broad applicability and the potential to transform pathology, thus addressing one of the key challenges of precision oncology. Citation Format: Hadassah Sade, Ansh Kapil, Philipp Wortmann, Andreas Spitzmueller, Nicolas Triltsch, Lina Meinecke, Susanne Haneder, Anatoliy Shumilov, Jan Lesniak, Valeria Bertani, Tze-Heng Tan, Ana Hidalgo-Sastre, Simon Christ, Andrea Storti, Regina Alleze, Dasa Medrikova, Jessica Chan, Simon Lanzmich, Markus Schick, Guenter Schmidt, J. Carl Barrett. Quantitative assessment of IHC using computational pathology allows superior patient selection for biomarker-informed patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 468.

Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Anti-inflammatory, hypolipidemic, and insulin-sensitizing effects of DHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated down-regulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R. Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex down-regulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment.

Adaptive thermogenesis is the process of heat generation in response to cold stimulation. It is under the control of the sympathetic nervous system, whose chief effector is the catecholamine norepinephrine (NE). NE enhances thermogenesis through β3-adrenergic receptors to activate brown adipose tissue and by 'browning' white adipose tissue. Recent studies have reported that alternative activation of macrophages in response to interleukin (IL)-4 stimulation induces the expression of tyrosine hydroxylase (TH), a key enzyme in the catecholamine synthesis pathway, and that this activation provides an alternative source of locally produced catecholamines during the thermogenic process. Here we report that the deletion of Th in hematopoietic cells of adult mice neither alters energy expenditure upon cold exposure nor reduces browning in inguinal adipose tissue. Bone marrow-derived macrophages did not release NE in response to stimulation with IL-4, and conditioned media from IL-4-stimulated macrophages failed to induce expression of thermogenic genes, such as uncoupling protein 1 (Ucp1), in adipocytes cultured with the conditioned media. Furthermore, chronic treatment with IL-4 failed to increase energy expenditure in wild-type, Ucp1(-/-) and interleukin-4 receptor-α double-negative (Il4ra(-/-)) mice. In agreement with these findings, adipose-tissue-resident macrophages did not express TH. Thus, we conclude that alternatively activated macrophages do not synthesize relevant amounts of catecholamines, and hence, are not likely to have a direct role in adipocyte metabolism or adaptive thermogenesis.

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg·kg−1 of body weight·day−1, 0.5% in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n=6–12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n=10–90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified ‘free’ fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial 14C-glucose and 14C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2±0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.

OBJECTIVE The induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs. RESEARCH DESIGN AND METHODS Mice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2−/−) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F). RESULTS Feeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2−/− and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment. CONCLUSIONS Our results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions.

The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30 degrees C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4 degrees C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30 degrees C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.

Cachexia represents a fatal energy-wasting syndrome in a large number of patients with cancer that mostly results in a pathological loss of skeletal muscle and adipose tissue. Here we show that tumor cell exposure and tumor growth in mice triggered a futile energy-wasting cycle in cultured white adipocytes and white adipose tissue (WAT), respectively. Although uncoupling protein 1 (Ucp1)-dependent thermogenesis was dispensable for tumor-induced body wasting, WAT from cachectic mice and tumor-cell-supernatant-treated adipocytes were consistently characterized by the simultaneous induction of both lipolytic and lipogenic pathways. Paradoxically, this was accompanied by an inactivated AMP-activated protein kinase (Ampk), which is normally activated in peripheral tissues during states of low cellular energy. Ampk inactivation correlated with its degradation and with upregulation of the Ampk-interacting protein Cidea. Therefore, we developed an Ampk-stabilizing peptide, ACIP, which was able to ameliorate WAT wasting in vitro and in vivo by shielding the Cidea-targeted interaction surface on Ampk. Thus, our data establish the Ucp1-independent remodeling of adipocyte lipid homeostasis as a key event in tumor-induced WAT wasting, and we propose the ACIP-dependent preservation of Ampk integrity in the WAT as a concept in future therapies for cachexia.

Definiens and Mosaic Laboratories developed an immuno-oncology (IO) panel consisting of multiplex chromogenic IHC assays and image and data analysis solutions to determine a standardized profile of the tumor microenvironment in non-small cell lung cancer (NSCLC). Expression of PD-L1 on tumor and immune cells, the density of infiltrating immune cell populations, and their spatial pattern have been shown to be predictive for response to immunotherapy. These factors are currently being evaluated to determine whether they can guide treatment across different categories of immunotherapies (e.g., checkpoint inhibition, vaccination, CAR-T cell therapy, or combinations thereof). Multiplex chromogenic IHC assays were developed for PDL1/CD68/CD3 and FoxP3/PD-1/CD8 and single-stain IHC was used to detect cells that were positive for granzyme B. Image analysis were optimized to detect biomarker positive cells (e.g., PD-L1+, CD3-, CD68-), double positive cells (e.g., PD-L1+, CD3-, CD68+), negative cells, or percent of area positive for Granzyme B. Densities for each cell population were quantified and pathologist annotations were used to distinguish between tumor core and invasive margin regions. Machine learning with random forests and decision trees was used to automatically distinguish tumor and stroma compartments to evaluate differences in densities of infiltrating immune cells. Densities of the different cell populations and their spatial relationships were analyzed to assign cases to one of the following immune status categories: acquired immune resistance, immune tolerance, adaptive checkpoint inhibitor resistance (all three are subtypes of high immune cell density), impaired infiltration, immune ignorance, intrinsic checkpoint inhibitor resistance (subtypes of low immune cell density). NSCLC tissues were analyzed and examples of biomarker profiles for each category are shown. The distribution of immune categories between squamous and non-squamous NSCLC subtypes and a comparison of immune cell densities and spatial relationships are shown. We conclude that the IO panel can be used to establish standardized immune profiles in NCCLC. In the future, it can be used to search for novel prognostic and predictive signatures and to establish the prevalence of patients with defined biomarker profiles in different subsets of patients (e.g, different stages, morphologic subtypes, or mutational status). Citation Format: Lisa M. Dauffenbach, Christopher A. Kerfoot, Gela Sia, Anthony Masci, Johannes Zimmermann, Jan Lesniak, Alexei Budco, Svenja Lippok, Katrin Schneider, René Korn, Tobias Wiestler, Dasa Medrikova, Florian Leiß. Characterization of inflammatory cell patterns and densities using multiplex immunohistochemistry immuno-oncology assays [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B069.

Regulatory T (Treg) cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT). Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress.

Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) in the diet protect against insulin resistance and obesity. Fibroblast growth factor-21 (Fgf21) is a hormonal factor released mainly by the liver that has powerful anti-diabetic effects. Here, we tested whether the beneficial metabolic effects of LC n-3 PUFA involve the induction of Fgf21. C57BL/6 J mice were exposed to an obesogenic, corn-oil-based, high-fat diet (cHF), or a diet in which corn oil was replaced with a fish-derived LC n-3 PUFA concentrate (cHF + F) using two experimental settings: short-term (3 weeks) and long-term treatment (8 weeks). CHF + F reduced body weight gain, insulinemia, and triglyceridemia compared to cHF. cHF increased plasma Fgf21 levels and hepatic Fgf21 gene expression compared with controls, but these effects were less pronounced or absent in cHF + F-fed mice. In contrast, hepatic expression of peroxisome proliferator-activated receptor (PPAR)-α target genes were more strongly induced by cHF + F than cHF, especially in the short-term treatment setting. The expression of genes encoding Fgf21, its receptors, and Fgf21 targets was unaltered by short-term LC n-3 PUFA treatment, with the exception of Ucp1 (uncoupling protein 1) and adiponectin genes, which were specifically up-regulated in white fat. In the long-term treatment setting, the expression of Fgf21 target genes and receptors was not differentially affected by LC n-3 PUFA. Collectively, our findings indicate that increased Fgf21 levels do not appear to be a major mechanism through which LC n-3 PUFA ameliorates high-fat-diet-associated metabolic disorders.

Throughout evolution, effective nutrient sensing and control of systemic energy homeostasis have relied on a close physical and functional interaction between immune and metabolically active cells. However, in today's obesogenic environment, this fine-tuned immunometabolic interface is perturbed. As a consequence, chronic inflammatory conditions and aberrant activation of immune cells have emerged as key features of obesity-related metabolic disorders, including insulin resistance, cardiovascular complications, and type 2 diabetes, whereas a major research focus has been placed on the adipocyte–macrophage interaction in the context of metabolic dysfunction; recent studies have not only expanded the scope of relevant immune cells in this setting but also highlight the impact of distinct metabolic organs, including the liver, on immunometabolic control, metabolic disease development, and potential anti-inflammatory therapeutic options in obesity-driven pathologies. This review will thus summarize recent progress in this emerging area of metabolic research.

Once considered an inert mass of stored energy, the past 2 decades have seen a surge in interest in the complexity of adipose tissue and its role in disease. In addition to serving as a site for energy storage, adipocytes secrete proteins involved in inflammation, appetite regulation, blood pressure control, and energy balance. Adipocytes are unique in their ability to store large quantities of lipids that can be rapidly released and used for energy by other organs, when necessary; however, excessive adipose tissue, particularly in the visceral adipose tissue depot, is associated with increased risk of insulin resistance, cardiovascular disease, and cancer. As such, adipose tissue is capable of extensive expansion or retraction depending on the energy balance or disease state of the host, a plasticity that is unparalleled in other organs. Expansion of adipose tissue is driven by both hypertrophy and hyperplasia of adipocytes, which can renew frequently to compensate for cell death, suggesting the necessity of adipocyte progenitor cells within the adipose tissue depot, that are capable of differentiating into mature and functional adipocytes.

Resolution of low-grade inflammation of white adipose tissue (WAT) is one of the keys for amelioration of obesity-associated metabolic dysfunctions. We focused on the identification of adipokines, which could be involved at the early stages of resolution of WAT inflammation.Male C57BL/6J mice with obesity induced in response to a 22-week feeding corn oil-based high-fat (cHF) diet were divided into four groups and were fed with, for 2 weeks, control cHF diet or cHF-based diets supplemented with: (i) concentrate of n-3 long-chain polyunsaturated fatty acids, mainly eicosapentaenoic and docosahexaenoic acids (cHF+F); (ii) thiazolidinedione drug rosiglitazone (cHF+TZD); and (iii) both compounds (cHF+F+TZD).The short-term combined intervention exerted additive effect in the amelioration of WAT inflammation in obese mice, namely in the epididymal fat, even in the absence of any changes in either adipocyte volume or fat mass. The combined intervention elicited hypolipidaemic effect and induced adiponectin, whereas the responses to single interventions (cHF+F, cHF+TZD) were less pronounced. In addition, analysis in WAT lysates using protein arrays revealed that the levels of a small set of adipose tissue-related proteins, namely macrophage inflammatory protein 1γ, endoglin, vascular cell adhesion molecule 1 and interleukin 1 receptor antagonist, changed in response to the anti-inflammatory interventions and were strongly reduced in the cHF+F+TZD mice. These results were verified using both the analysis of gene expression and enzyme-linked immunosorbent analysis in WAT lysates. In contrast with adiponectin, which showed changing plasma levels in response to dietary interventions, the levels of the above proteins were affected only in WAT.We identified several adipose tissue-related proteins, which are locally involved in resolution of low-grade inflammation and remodelling of WAT.

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.

Background: n-3 polyunsaturated fatty acids, namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), reduce the risk of cardiovascular disease and can ameliorate many of obesity-associated disorders. We hypothesised that the latter effect will be more pronounced when DHA/EPA is supplemented as phospholipids rather than as triglycerides. Methodology/Principal Findings: In a 'prevention study', C57BL/6J mice were fed for 9 weeks on either a corn oil-based high-fat obesogenic diet (cHF; lipids ~35% wt/wt), or cHF-based diets in which corn oil was partially replaced by DHA/EPA, admixed either as phospholipids or triglycerides from marine fish. The reversal of obesity was studied in mice subjected to the preceding cHF-feeding for 4 months. DHA/EPA administered as phospholipids prevented glucose intolerance and tended to reduce obesity better than triglycerides. Lipemia and hepatosteatosis were suppressed more in response to dietary phospholipids, in correlation with better bioavailability of DHA and EPA, and a higher DHA accumulation in the liver, white adipose tissue (WAT), and muscle phospholipids. In dietary obese mice, both DHA/EPA concentrates prevented a further weight gain, reduced plasma lipid levels to a similar extent, and tended to improve glucose tolerance. Importantly, only the phospholipid form reduced plasma insulin and adipocyte hypertrophy, while being more effective in reducing hepatic steatosis and low-grade inflammation of WAT. These beneficial effects were correlated with changes of endocannabinoid metabolome in WAT, where phospholipids reduced 2-arachidonoylglycerol, and were more effective in increasing anti-inflammatory lipids such as N-docosahexaenoylethanolamine. Conclusions/Significance: Compared with triglycerides, dietary DHA/EPA administered as phospholipids are superior in preserving a healthy metabolic profile under obesogenic conditions, possibly reflecting better bioavalability and improved modulation of the endocannabinoid system activity in WAT. © 2012 Rossmeisl et al.

Adverse effects of obesity on glucose homeostasis are linked to low-grade adipose tissue inflammation and accumulation of lipids in non-adipose tissues. The goal of this study was to evaluate the role of adipose tissue plasticity in a less severe deterioration of glucose homeostasis in females compared with males during the course of high-fat (HF) feeding in mice. Mice of the C57BL/6N strain were fed either a chow or obesogenic HF diet for up to 35 weeks after weaning. Metabolic markers and hormones in plasma, glucose homeostasis, adipocyte size and inflammatory status of gonadal (gWAT) and subcutaneous (scWAT) adipose depots and liver steatosis were evaluated at 15 and 35 weeks of HF feeding. HF-fed males were heavier than females until week ∼20, after which the body weights stabilized at a similar level (55–58 g) in both sexes. Greater weight gain and fat accumulation in females were associated with larger adipocytes in gWAT and scWAT at week 35. Although adipose tissue macrophage infiltration was in general less frequent in scWAT, it was reduced in both fat depots of female as compared with male mice; however, the expression of inflammatory markers in gWAT was similar in both sexes at week 35. In females, later onset of the impairment of glucose homeostasis and better insulin sensitivity were associated with higher plasma levels of adiponectin (weeks 0, 15 and 35) and reduced hepatosteatosis (weeks 15 and 35). Compared with males, female mice demonstrate increased capacity for adipocyte enlargement in response to a long-term HF feeding, which is associated with reduced adipose tissue macrophage infiltration and lower fat deposition in the liver, and with better insulin sensitivity. Our data suggest that adipose tissue expandability linked to adiponectin secretion might have a role in the sex differences observed in obesity-associated metabolic disorders.

The n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exert hypolipidemic effects and prevent development of obesity and insulin resistance in animals fed high-fat diets. We sought to determine the efficacy of alpha-substituted DHA derivatives as lipid-lowering, antiobesity, and antidiabetic agents. C57BL/6 mice were given a corn oil-based high-fat (35% weight/weight) diet (cHF), or cHF with 1.5% of lipids replaced with alpha-methyl DHA ethyl ester (Substance 1), alpha-ethyl DHA ethyl ester (Substance 2), alpha,alpha-di-methyl DHA ethyl ester (Substance 3), or alpha-thioethyl DHA ethyl ester (Substance 4) for 4 months. Plasma markers of glucose and lipid metabolism, glucose tolerance, morphology, tissue lipid content, and gene regulation were characterized. The cHF induced obesity, hyperlipidemia, impairment of glucose homeostasis, and adipose tissue inflammation. Except for Substance 3, all other substances prevented weight gain and Substance 2 exerted the strongest effect (63% of cHF-controls). Glucose intolerance was significantly prevented (~67% of cHF) by both Substance 1 and Substance 2. Moreover, Substance 2 lowered fasting glycemia, plasma insulin, triacylglycerols, and nonesterified fatty acids (73, 9, 47, and 81% of cHF-controls, respectively). Substance 2 reduced accumulation of lipids in liver and skeletal muscle, as well as adipose tissue inflammation associated with obesity. Substance 2 also induced weight loss in dietary obese mice. In contrast to DHA administered either alone or as a component of the EPA/DHA concentrate (replacing 15% of dietary lipids), Substance 2 also reversed established glucose intolerance in obese mice. Thus, Substance 2 represents a novel compound with a promising potential in the treatment of obesity and associated metabolic disturbances.

Mitochondrial uncoupling protein 2 (UCP2) is abundant in developing monocyte/macrophage cells and may affect hematopoiesis by reducing formation of reactive oxygen species. The aims of this study were to further characterize the involvement of UCP2 in hematopoiesis. In situ hybridization in mouse embryos identified UCP2-positive cells in liver and inside primitive blood vessels from 10.5 days of prenatal development. High UCP2 transcript levels were detected in reticulocytes and other maturating erythroid cells in peripheral blood of mice exposed to hypoxia, and in umbilical cord blood of human neonates and peripheral blood of adults. Our results suggest involvement of UCP2 in erythropoiesis.