Amanda R. Crisma

Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice

The aim of this study was to investigate whether treatment with tributyrin (Tb; a butyrate prodrug) results in protection against diet-induced obesity and associated insulin resistance. C57BL/6 male mice fed a standard chow or high-fat diet were treated with Tb (2 g/kg body wt, 10 wk) and evaluated for glucose homeostasis, plasma lipid profile, and inflammatory status. Tb protected mice against obesity and obesity-associated insulin resistance and dyslipidemia without food consumption being affected. Tb attenuated the production of TNFα and IL-1β by peritoneal macrophages and their expression in adipose tissue. Furthermore, in the adipose tissue, Tb reduced the expression of MCP-1 and infiltration by leukocytes and restored the production of adiponectin. These effects were associated with a partial reversion of hepatic steatosis, reduction in liver and skeletal muscle content of phosphorylated JNK, and an improvement in muscle insulin-stimulated glucose uptake and Akt signaling. Although part of the beneficial effects of Tb are likely to be secondary to the reduction in body weight, we also found direct protective actions of butyrate reducing TNFα production after LPS injection and in vitro by LPS- or palmitic acid-stimulated macrophages and attenuating lipolysis in vitro and in vivo. The results, reported herein, suggest that Tb may be useful for the treatment and prevention of obesity-related metabolic disorders.

Both GLS silencing and GLS2 overexpression synergize with oxidative stress against proliferation of glioma cells.

Mitochondrial glutaminase (GA) plays an essential role in cancer cell metabolism, contributing to biosynthesis, bioenergetics, and redox balance. Humans contain several GA isozymes encoded by the GLS and GLS2 genes, but the specific roles of each in cancer metabolism are still unclear. In this study, glioma SFxL and LN229 cells with silenced isoenzyme glutaminase KGA (encoded by GLS) showed lower survival ratios and a reduced GSH-dependent antioxidant capacity. These GLS-silenced cells also demonstrated induction of apoptosis indicated by enhanced annexin V binding capacity and caspase 3 activity. GLS silencing was associated with decreased mitochondrial membrane potential (ΔΨm) (JC-1 dye test), indicating that apoptosis was mediated by mitochondrial dysfunction. Similar observations were made in T98 glioma cells overexpressing glutaminase isoenzyme GAB, encoded by GLS2, though some characteristics (GSH/GSSG ratio) were different in the differently treated cell lines. Thus, control of GA isoenzyme expression may prove to be a key tool to alter both metabolic and oxidative stress in cancer therapy. Interestingly, reactive oxygen species (ROS) generation by treatment with oxidizing agents: arsenic trioxide or hydrogen peroxide, synergizes with either KGA silencing or GAB overexpression to suppress malignant properties of glioma cells, including the reduction of cellular motility. Of note, negative modulation of GLS isoforms or GAB overexpression evoked lower c-myc and bcl-2 expression, as well as higher pro-apoptotic bid expression. Combination of modulation of GA expression and treatment with oxidizing agents may become a therapeutic strategy for intractable cancers and provides a multi-angle evaluation system for anti-glioma pre-clinical investigations.Key messageSilencing GLS or overexpressing GLS2 induces growth inhibition in glioma cell lines.Inhibition is synergistically enhanced after arsenic trioxide (ATO) or H2O2 treatment.Glutatione levels decrease in GLS-silenced cells but augment if GLS2 is overexpressed.ROS synergistically inhibit cell migration by GLS silencing or GLS2 overexpression.c-myc, bid, and bcl-2 mediate apoptosis resulting from GLS silencing or GLS2 overexpression.

Sunflower Oil Supplementation Has Proinflammatory Effects and Does Not Reverse Insulin Resistance in Obesity Induced by High-Fat Diet in C57BL/6 Mice

High consumption of polyunsaturated fatty acids, such as sunflower oil has been associated to beneficial effects in plasma lipid profile, but its role on inflammation and insulin resistance is not fully elucidated yet. We evaluated the effect of sunflower oil supplementation on inflammatory state and insulin resistance condition in HFD-induced obese mice. C57BL/6 male mice (8 weeks) were divided in four groups: (a) control diet (CD), (b) HFD, (c) CD supplemented with n-6 (CD + n-6), and (d) HFD supplemented with n-6 (HFD + n-6). CD + n-6 and HFD + n-6 were supplemented with sunflower oil by oral gavage at 2 g/Kg of body weight, three times per week. CD and HFD were supplemented with water instead at the same dose. HFD induced whole and muscle-specific insulin resistance associated with increased inflammatory markers in insulin-sensitive tissues and macrophage cells. Sunflower oil supplementation was not efficient in preventing or reducing these parameters. In addition, the supplementation increased pro-inflammatory cytokine production by macrophages and tissues. Lipid profile, on the other hand, was improved with the sunflower oil supplementation in animals fed HFD. In conclusion, sunflower oil supplementation improves lipid profile, but it does not prevent or attenuate insulin resistance and inflammation induced by HFD in C57BL/6 mice.

Fish oil prevents changes induced by a high-fat diet on metabolism and adipokine secretion in mice subcutaneous and visceral adipocytes

Fish oil (FO), rich in omega‐3 polyunsaturated fatty acids, has beneficial effects on changes induced by obesity and partially prevents associated comorbidities. The effects of FO on adipocytes from different adipose tissue depots in high‐fat (HF) diet induced obese mice have not been uninvestigated. This is the first study to examine the effects of FO on changes in metabolism and adipokine production in adipocytes from s.c. (inguinal; ING) or visceral (retroperitoneal; RP) white adipose depots in a HF diet‐induced obese mice. Unlike most studies performed previously, FO supplementation was initiated 4 weeks before the induction of obesity. HF diet caused marked changes in ING (glucose uptake and secretion of adiponectin, tumour necrosis factor‐α and interleukin‐6 in ING) and RP (lipolysis, de novo lipogenesis and secretion of pro‐inflammatory cytokines) adipose depots. Previous and concomitant FO administration prevented the changes in ING and RP adipocytes induced by the HF diet.

High-fat diet blunts activation of the nuclear factor-κB signaling pathway in lipopolysaccharide-stimulated peritoneal macrophages of Wistar rats

OBJECTIVE The present study was designed to investigate the effect of a high-fat diet (HFD) on the inflammatory response of peritoneal macrophages. METHODS Male Wistar rats were fed a control diet (n = 12) or an HFD (n = 12) for 12 wk. After euthanasia, peritoneal macrophages were collected and stimulated (or not) with lipopolysaccharide (LPS). Results from the assays using peritoneal macrophages were analyzed with one-way analysis of variance or an equivalent non-parametric test. The level of significance adopted was 0.05. RESULTS Consumption of the HFD was associated with significant increases in weight gain and fat depots (P < 0.05). Despite having no influence in systemic markers of inflammation, such as interleukin (IL)-6, tumor necrosis factor-α, and plasminogen activator inhibitor-1, the HFD intake significantly decreased insulin sensitivity, as evaluated by the homeostasis model assessment index (P < 0.05). A decreased production of IL-1β, IL-6, IL-10, and nitric oxide in response to the LPS stimulation was observed in peritoneal macrophages from the HFD group (P < 0.05). Also, in HFD-fed animals, LPS incubation did not increase IL-1β and IL-6 mRNA expression (P < 0.05). These effects were associated with an attenuation of IκB inhibitor kinase-β phosphorylation and nuclear factor-κB activation in response to LPS and with a failure to decrease IκB inhibitor-α expression (P < 0.05). CONCLUSION Chronic consumption of an HFD decreased the LPS-induced inflammatory response of peritoneal macrophages, which was associated with a downregulation of the nuclear factor-κB signaling pathway.

Malnourished mice display an impaired hematologic response to granulocyte colony-stimulating factor administration

The aim of this study was to determine if protein-energy malnutrition could affect the hematologic response to granulocyte colony-stimulating factor (G-CSF). Swiss mice were fed a low-protein diet containing 4% protein, whereas control mice were fed a 20% protein-containing diet. After the malnourished group lost 20% of their original body weight, the mice were subdivided in 2 treatment groups, and hematopoietic parameters were studied. Mice were injected with either 8 microg/kg per day of G-CSF or saline twice daily for 4 days. Malnourished mice developed anemia with reticulopenia and leukopenia with depletion of granulocytes and lymphocytes. Both malnourished and control mice treated with G-CSF showed a significant increase in neutrophils; however, in the control group, this increase was more pronounced compared to the malnourished group (4.5-fold and 3.4-fold, respectively). Granulocyte colony-stimulating factor administration increased bone marrow blastic (P < .001) and granulocytic (P < .01) compartments in the controls but had no significant effect on these hematopoietic compartments in the malnourished animals (P = .08 and P = .62, respectively). We report that malnourished mice display an impaired response to G-CSF, which contributes to the decreased production of leukocytes in protein-energy malnutrition.

DNA Methylation Changes Induced by a High-Fat Diet and Fish Oil Supplementation in the Skeletal Muscle of Mice

Background/Aims: To investigate the global changes in DNA methylation and methylation of the promoter region of the peroxisome proliferator-activated receptor gamma transcript variant 2 (Pparg2) gene resulting from a high-fat diet (HFD) and/or fish oil supplementation. Methods: Fish oil, rich in omega-3 polyunsaturated fatty acids, or water was orally administered to male mice for 12 weeks. After the first 4 weeks, the animals were fed a control diet or an HFD until the end of the experimental protocol, when the epididymal fat, gastrocnemius muscle and liver were excised. Results:Pparg2 mRNA expression was upregulated by obesity and downregulated by fish oil supplementation in the liver. In the gastrocnemius muscle, diet-induced obesity increased global DNA methylation. Fish oil prevented the decrease in Pparg2 promoter methylation induced by obesity in the gastrocnemius muscle. Regardless of the diet given, fish oil supplementation increased Pparg2 promoter methylation at CpG-263 in muscle and adipose tissue. Conclusion: HFD and fish oil modified global and Pparg2 promoter DNA methylation in a tissue-specific manner. Fish oil supplementation attenuated body weight gain, abolished the increase in Pparg2 expression in the liver and prevented the decrease in Pparg2 promoter methylation in the muscle induced by the HFD.

Effects of high EPA and high DHA fish oils on changes in signaling associated with protein metabolism induced by hindlimb suspension in rats

The effects of either eicosapentaenoic (EPA)‐ or docosahexaenoic (DHA)‐rich fish oils on hindlimb suspension (HS)‐induced muscle disuse atrophy were compared. Daily oral supplementations (0.3 mL/100 g b.w.) with mineral oil (MO) or high EPA or high DHA fish oils were performed in adult rats. After 2 weeks, the animals were subjected to HS for further 2 weeks. The treatments were maintained alongside HS. At the end of 4 weeks, we evaluated: body weight gain, muscle mass and fat depots, composition of fatty acids, cross‐sectional areas (CSA) of the soleus muscle and soleus muscle fibers, activities of cathepsin L and 26S proteasome, and content of carbonylated proteins in the soleus muscle. Signaling pathway activities associated with protein synthesis (Akt, p70S6K, S6, 4EBP1, and GSK3‐beta) and protein degradation (atrogin‐1/MAFbx, and MuRF1) were evaluated. HS decreased muscle mass, CSA of soleus muscle and soleus muscle fibers, and altered signaling associated with protein synthesis (decreased) and protein degradation (increased). The treatment with either fish oil decreased the ratio of omega‐6/omega‐3 fatty acids and changed protein synthesis‐associated signaling. EPA‐rich fish oil attenuated the changes induced by HS on 26S proteasome activity, CSA of soleus muscle fibers, and levels of p‐Akt, total p70S6K, p‐p70S6K/total p70S6K, p‐4EBP1, p‐GSK3‐beta, p‐ERK2, and total ERK 1/2 proteins. DHA‐rich fish oil attenuated the changes induced by HS on p‐4EBP1 and total ERK1 levels. The effects of EPA‐rich fish oil on protein synthesis signaling were more pronounced. Both EPA‐ and DHA‐rich fish oils did not impact skeletal muscle mass loss induced by non‐inflammatory HS.

Distribution of the P2X2 receptor and chemical coding in ileal enteric neurons of obese male mice (ob/ob)

AIM To investigate the colocalization, density and profile of neuronal areas of enteric neurons in the ileum of male obese mice. METHODS The small intestinal samples of male mice in an obese group (OG) (C57BL/6J ob/ob) and a control group (CG) (+/+) were used. The tissues were analyzed using a double immunostaining technique for immunoreactivity (ir) of the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT) and calretinin (Calr). Also, we investigated the density and profile of neuronal areas of the NOS-, ChAT- and Calr-ir neurons in the myenteric plexus. Myenteric neurons were labeled using an NADH-diaphorase histochemical staining method. RESULTS The analysis demonstrated that the P2X2 receptor was expressed in the cytoplasm and in the nuclear and cytoplasmic membranes only in the CG. Neuronal density values (neuron/cm(2)) decreased 31% (CG: 6579 ± 837; OG: 4556 ± 407) and 16.5% (CG: 7796 ± 528; OG: 6513 ± 610) in the NOS-ir and calretinin-ir neurons in the OG, respectively (P < 0.05). Density of ChAT-ir (CG: 6200 ± 310; OG: 8125 ± 749) neurons significantly increased 31% in the OG (P < 0.05). Neuron size studies demonstrated that NOS, ChAT, and Calr-ir neurons did not differ significantly between the CG and OG groups. The examination of NADH-diaphorase-positive myenteric neurons revealed an overall similarity between the OG and CG. CONCLUSION Obesity may exert its effects by promoting a decrease in P2X2 receptor expression and modifications in the density of the NOS-ir, ChAT-ir and CalR-ir myenteric neurons.

Macadamia Oil Supplementation Attenuates Inflammation and Adipocyte Hypertrophy in Obese Mice

Excess of saturated fatty acids in the diet has been associated with obesity, leading to systemic disruption of insulin signaling, glucose intolerance, and inflammation. Macadamia oil administration has been shown to improve lipid profile in humans. We evaluated the effect of macadamia oil supplementation on insulin sensitivity, inflammation, lipid profile, and adipocyte size in high-fat diet (HF) induced obesity in mice. C57BL/6 male mice (8 weeks) were divided into four groups: (a) control diet (CD), (b) HF, (c) CD supplemented with macadamia oil by gavage at 2 g/Kg of body weight, three times per week, for 12 weeks (CD + MO), and (d) HF diet supplemented with macadamia oil (HF + MO). CD and HF mice were supplemented with water. HF mice showed hypercholesterolemia and decreased insulin sensitivity as also previously shown. HF induced inflammation in adipose tissue and peritoneal macrophages, as well as adipocyte hypertrophy. Macadamia oil supplementation attenuated hypertrophy of adipocytes and inflammation in the adipose tissue and macrophages.