Endoplasmic Reticulum Stress and Autophagy Markers in Soleus Muscle Disuse-Induced Atrophy of Rats Treated with Fish Oil.

Endoplasmic reticulum stress (ERS) and autophagy pathways are implicated in disuse muscle atrophy. The effects of high eicosapentaenoic (EPA) or high docosahexaenoic (DHA) fish oils on soleus muscle ERS and autophagy markers were investigated in a rat hindlimb suspension (HS) atrophy model. Adult Wistar male rats received daily by gavage supplementation (0.3 mL per 100 g b.w.) of mineral oil or high EPA or high DHA fish oils (FOs) for two weeks. Afterward, the rats were subjected to HS and the respective treatments concomitantly for an additional two-week period. After four weeks, we evaluated ERS and autophagy markers in the soleus muscle. Results were analyzed using two-way analysis of variance (ANOVA) and Bonferroni post hoc test. Gastrocnemius muscle ω-6/ω-3 fatty acids (FAs) ratio was decreased by both FOs indicating the tissue incorporation of omega-3 fatty acids. HS altered (p < 0.05) the protein content (decreasing total p38 and BiP and increasing p-JNK2/total JNK2 ratio, and caspase 3) and gene expressions (decreasing BiP and increasing IRE1 and PERK) of ERS and autophagy (decreasing Beclin and increasing LC3 and ATG14) markers in soleus. Both FOs attenuated (p < 0.05) the increase in PERK and ATG14 expressions induced by HS. Thus, both FOs could potentially attenuate ERS and autophagy in skeletal muscles undergoing atrophy.

Impaired brown adipose tissue is differentially modulated in insulin-resistant obese wistar and type 2 diabetic Goto-Kakizaki rats.

Brown adipose tissue (BAT) is a potential target to treat obesity and diabetes, dissipating energy as heat. Type 2 diabetes (T2D) has been associated with obesogenic diets; however, T2D was also reported in lean individuals to be associated with genetic factors. We aimed to investigate the differences between obese and lean models of insulin resistance (IR) and elucidate the mechanism associated with BAT metabolism and dysfunction in different IR animal models: a genetic model (lean GK rats) and obese models (diet-induced obese Wistar rats) at 8 weeks of age fed a high-carbohydrate (HC), high-fat (HF) diet, or high-fat and high-sugar (HFHS) diet for 8 weeks. At 15 weeks of age, BAT glucose uptake was evaluated by 18F-FDG PET under basal (saline administration) or stimulated condition (CL316,243, a selective ß3-AR agonist). After CL316, 243 administrations, GK animals showed decreased glucose uptake compared to HC animals. At 16 weeks of age, the animals were euthanized, and the interscapular BAT was dissected for analysis. Histological analyses showed lower cell density in GK rats and higher adipocyte area compared to all groups, followed by HFHS and HF compared to HC. HFHS showed a decreased batokine FGF21 protein level compared to all groups. However, GK animals showed increased expression of genes involved in fatty acid oxidation (CPT1 and CPT2), BAT metabolism (Sirt1 and Pgc1-α), and obesogenic genes (leptin and PAI-1) but decreased gene expression of glucose transporter 1 (GLUT-1) compared to other groups. Our data suggest impaired BAT function in obese Wistar and GK rats, with evidence of a whitening process in these animals.

Effects of glutamine supplementation on kidney of diabetic rat.

Glutamine is the most important donor of NH(3) in kidney playing an important role in acid-base buffering system. Besides this effect, glutamine presents many other relevant functions in the whole body, such as a precursor of arginine in adult and neonates. In addition to these effects, some studies have shown that glutamine can potentiate renal disease. In the present study, the effect of short-term treatment (15 days) with glutamine on control and diabetic rats was investigated. Using biochemical, histological and molecular biology analysis from control and diabetic rats we verified that glutamine supplementation increase in pro-inflammatory interleukins (IL)-1beta and IL-6 content in renal cortex and induce alteration in glomerular characteristics. This study showed that short-term treatment with glutamine in association with increased glucose levels could cause important alterations in glomerular morphology that may result in fast progression of kidney failure.

High fat diet modulates the protein content of nutrient transporters in the small intestine of mice: possible involvement of PKA and PKC activity.

AIMS: Chronic high fat consumption has been shown to modulate nutrient transporter content in the intestine of obese mice; however it is unclear if this regulation occurs before or after the establishment of obesity, and the underlying molecular mechanism requires elucidation. MAIN METHODS: Towards this goal C57BL/6 mice were fed a low fat diet (LFD) or high fat diet (HFD), and specific protein and gene expression levels were assessed for up to 12 weeks. Similar experiments were also performed with leptin-deficient (Ob/Ob) mice. KEY FINDINGS: The results showed that the HFD group presented decreased GLUT2, PEPT1, FAT/CD36 and NPC1L1, and increased NHE3, MTTP and L-FABP content. Animals fed an HFD also presented enhanced lipid transporter gene expression of Slc27a4, Npc1l1, Cd36, Mttp and L-Fabp. Additionally, FAT/CD36 and NPC1L1 protein levels were reduced in both HFD-induced obese and Ob/Ob mice. Ob/Ob mice also exhibited increased Slc2a2 and Slc15a1 mRNAs expression, but the protein expression levels remained unchanged. The HFD also attenuated PKA and PKC activities. The inhibition of PKA was associated with decreased FAT/CD36 content, whereas increased L-FABP levels likely depend on CREB activation, independent of PKA. It is plausible that the HFD-induced changes in NPC1L1, MTTP and L-FABP protein content involve regulation at the level of transcription. Moreover, the changes in GLUT2 and PEPT1 content might be associated with low PKC activity. SIGNIFICANCE: The results indicated that an HFD is capable of reducing nutrient transporter content, possibly attenuating nutrient uptake into the intestine, and may represent a feedback mechanism for regulating body weight. Furthermore, the elevated levels of NHE3, L-FABP and MTTP may account for the increased prevalence of hypertension and dyslipidemia in obese individuals. All of these changes are potentially linked to reduced PKA or PKC activities.

New insights on the regulation of cancer cachexia by N-3 polyunsaturated fatty acids.

Cancer cachexia is a multifactorial syndrome that develops during malignant tumor growth. Changes in plasma levels of several hormones and inflammatory factors result in an intense catabolic state, decreased activity of anabolic pathways, anorexia, and marked weight loss, leading to cachexia development and/or accentuation. Inflammatory mediators appear to be related to the control of a highly regulated process of muscle protein degradation that accelerates the process of cachexia. Several mediators have been postulated to participate in this process, including TNF-α, myostatin, and activated protein degradation pathways. Some interventional therapies have been proposed, including nutritional (dietary, omega-3 fatty acid supplementation), hormonal (insulin), pharmacological (clenbuterol), and nonpharmacological (physical exercise) therapies. Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid, are recognized for their anti-inflammatory properties and have been used in therapeutic approaches to treat or attenuate cancer cachexia. In this review, we discuss recent findings on cellular and molecular mechanisms involved in inflammation in the cancer cachexia syndrome and the effectiveness of n-3 PUFAs to attenuate or prevent cancer cachexia.

Obesity and Type 2 Diabetes mellitus induce lipopolysaccharide tolerance in rat neutrophils.

Obesity and diabetes implicate in various health complications and increased mortality caused by infection. Innate immune system is broadly affected by these diseases, leading the patients to an immunosuppressive state. A mechanism that leads innate immune cells to a less capacity of killing microorganism is the impaired TLR4 activation. TLR4 recognizes a component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS), and when activated increases the production of inflammatory substances. Neutrophils are components of the innate immune system and are the first responders to an invading agent. The correct activation of TLR4 in these cells is required for the initiation of the inflammatory process and elimination of the microorganisms. The aim of this study was to evaluate the influence of type 2 diabetes and obesity in the TLR4 pathway in rat neutrophils. Two experimental models were used: Goto-Kakizaki rats and high-fat-diet induced obese Wistar rats. To evaluate neutrophil response to LPS, intratracheal LPS instillation was used. Neutrophils from obese and diabetic animals exhibited tolerance to LPS, mainly by the impaired production of cytokines and chemokines and the low content of phospho-NFκB and phospho-IKBα. Neutrophils from both experimental models had increased cell death, impaired in vivo migration and myeloperoxidase activity.

Autophagy Is Impaired in Neutrophils from Streptozotocin-Induced Diabetic Rats.

We tested the hypothesis that changes reported on functions of neutrophils from streptozotocin-induced diabetic rats involve autophagy impairment. Wistar rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.), and the measurements were carried out 2 weeks afterward. Neutrophils were collected through intraperitoneal cavity lavage after 4 h of i.p. oyster glycogen type 2 injection. Neutrophils cultured with PMA (20 nM) for 1 h were used for analysis of plasma membrane integrity, DNA fragmentation, and mitochondrial depolarization by flow cytometry; expression of Atg5, Atg14, Beclin1, LC3BII, and Rab9 by RT-PCR; the contents of caspase 3, LC3BII/LC3BI, and pS6 by western blotting; ATP content by fluorescence essay; reactive oxygen species production by chemiluminescence (Luminol), and autophagy by immunofluorescence tracking LC3B cleavage. Herein, neutrophils from diabetic rats had high DNA fragmentation, depolarization of mitochondrial membrane, low content of ATP, and high content of cleaved caspase 3 after PMA stimulation. Neutrophils from diabetic rats also had low expression of LC3B, failed to increase the expression of Rab9 and Atg14 induced by PMA stimulation. Neutrophils from diabetic animals also had low cleavage of LC3BI to LC3BII and do not present punctate structures that label autophagosomal membranes after stimulus. The changes of neutrophil function reported in diabetic rats do involve impaired autophagy. The suppression of autophagy in neutrophils from diabetic rats may be associated with the activation of the mTOR signaling as indicated by the high content of pS6.

Comparison of Goto-Kakizaki rats and high fat diet-induced obese rats: Are they reliable models to study Type 2 Diabetes mellitus?

Type 2 Diabetes mellitus (T2DM) is an evident growing disease that affects different cultures throughout the world. T2DM occurs under the influence of three main factors: the genetic background, environmental and behavioral components. Obesity is strongly associated to the development of T2DM in the occident, while in the orient most of the diabetic patients are considered lean. Genetics may be a key factor in the development of T2DM in societies where obesity is not a recurrent public health problem. Herein, two different models of rats were used to understand their differences and reliability as experimental models to study the pathophysiology of T2DM, in two different approaches: the genetic (GK rats) and the environmental (HFD-induced obese rats) influences. GK rats were resistant to weight gain even though food/energy consumption (relative to body weight) was higher in this group. HFD, on the other hand, induced obesity in Wistar rats. White adipose tissue (WAT) expansion in this group was accompanied by immune cells infiltration, inflammation and insulin resistance. GK rats also presented WAT inflammation and insulin resistance; however, no immune cells infiltration was observed in the WAT of this group. Liver of HFD group presented fat accumulation without differences in inflammatory cytokines content, while liver of GK rats didn't present fat accumulation, but showed an increase of IL-6 and IL-10 content and glycogen. Also, GK rats showed increased plasma GOT and GPT. Soleus muscle of HFD presented normal insulin signaling, contrary to GK rats, which presented higher content of basal phosphorylation of GSK-3ß. Our results demonstrated that HFD developed a mild insulin resistance in Wistar rats, but was not sufficient to develop T2DM. In contrast, GK rats presented all the typical hallmarks of T2DM, such as insulin resistance, defective insulin production, fasting hyperglycemia/hyperinsulinemia and lipid plasma alteration. Thus, on the given time point of this study, we may conclude that only GK rats shown to be a reliable model to study T2DM.

Modulation of lipopolysaccharide-induced acute lung inflammation: Role of insulin.

The present study was undertaken to investigate the influence of insulin on lipopolysaccharide (LPS)-induced acute lung injury. Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 30 days) and controls were instilled with saline containing LPS (750 microg/0.4 mL) or saline alone. The following analyses were performed 6 h there after: (a) total and differential cell counts in bronchoalveolar lavage (BAL) fluid, (b) quantification of tumor necrosis factor alpha, interleukin (IL) 1beta, IL-10, and cytokine-induced neutrophil chemoattractant 1 in the BAL (enzyme-linked immunosorbent assay), (c)immunohistochemistry for intercellular adhesion molecule 1 and E-selectin on lung vessels, and (d) quantification of metalloproteinases (MMP) 2 and 9 in the BAL (zymography). Relative to controls, diabetic rats exhibited a reduction in the number of neutrophils (80%) and reduced concentrations of tumor necrosis factor alpha (56%), IL-1beta (66%), and IL-10 (35%) after LPS instillation. Cytokine-induced neutrophil chemoattractant 1 levels did not differ between groups. Increased levels of MMP-2 (90%) and MMP-9 (500%) were observed in diabetic rats compared with controls. Treatment of diabetic rats with neutral protamine Hagedorn insulin (4 IU, s.c.), 2 h before LPS instillation, completely restored the number of neutrophils and concentrations of cytokines in the BAL fluid. Despite no significant differences between diabetic and control groups, there was a remarkable increase in intercellular adhesion molecule 1 and E-selectin expression on lung vessels after insulin treatment. Levels of MMP-2 and MMP-9 did not change after treatment with insulin. Levels of corticosterone were equivalent among groups. Data presented suggest that insulin modulates the production/release of cytokines and the expression of adhesion molecules controlling, therefore, neutrophil migration during the course of LPS-induced acute lung inflammation.

Endoplasmic reticulum stress and autophagy markers in Soleus muscle disuse-induced atrophy of rats treated with fish oil

Endoplasmic reticulum stress (ERS) and autophagy pathways are implicated in disuse muscle atrophy. The effects of high eicosapentaenoic (EPA) or high docosahexaenoic (DHA) fish oils on soleus muscle ERS and autophagy markers were investigated in a rat hindlimb suspension (HS) atrophy model. Adult Wistar male rats received daily by gavage supplementation (0.3 mL per 100 g b.w.) of mineral oil or high EPA or high DHA fish oils (FOs) for two weeks. Afterward, the rats were subjected to HS and the respective treatments concomitantly for an additional two-week period. After four weeks, we evaluated ERS and autophagy markers in the soleus muscle. Results were analyzed using two-way analysis of variance (ANOVA) and Bonferroni post hoc test. Gastrocnemius muscle ω-6/ω-3 fatty acids (FAs) ratio was decreased by both FOs indicating the tissue incorporation of omega-3 fatty acids. HS altered (p < 0.05) the protein content (decreasing total p38 and BiP and increasing p-JNK2/total JNK2 ratio, and caspase 3) and gene expressions (decreasing BiP and increasing IRE1 and PERK) of ERS and autophagy (decreasing Beclin and increasing LC3 and ATG14) markers in soleus. Both FOs attenuated (p < 0.05) the increase in PERK and ATG14 expressions induced by HS. Thus, both FOs could potentially attenuate ERS and autophagy in skeletal muscles undergoing atrophy.

NADPH oxidase-dependent production of reactive oxygen species induces endoplasmatic reticulum stress in neutrophil-like HL60 cells.

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.

Crotoxin-Treated Macrophages Stimulate ROS Production and Killing Activity in co-cultured Neutrophils

Crotoxin (CTX), the predominant toxin in Crotalus durissus terrificus snake venom (CdtV), has anti-inflammatory and immunomodulatory effects. Despite its inhibitory action on neutrophil migration and phagocytosis, CTX does not directly affect the production of reactive oxygen species (ROS) by the neutrophils. In contrast, it enhances the generation of reactive oxygen and nitrogen intermediates by macrophages. Given the importance of macrophage-neutrophil interactions in innate antimicrobial defense, the aim of this study was to investigate the effect of CTX on neutrophil ROS production and killing activity, either through CTX-treated macrophage co-culture or conditioned medium of CTX-treated macrophages. The results showed an important modulatory action of CTX on the neutrophil function as well as neutrophil-macrophage interactions, as demonstrated by the increased production of hydrogen peroxide, hypochlorous acid, nitric oxide and TNF- a , along with the increased fungicidal activity of neutrophils.

Crotoxin-treated macrophages stimulate ROS production and killing activity in co-cultured neutrophils

Crotoxin (CTX), the predominant toxin in Crotalus durissus terrificus snake venom (CdtV), has anti-inflammatory and immunomodulatory effects. Despite its inhibitory action on neutrophil migration and phagocytosis, CTX does not directly affect the production of reactive oxygen species (ROS) by the neutrophils. In contrast, it enhances the generation of reactive oxygen and nitrogen intermediates by macrophages. Given the importance of macrophage-neutrophil interactions in innate antimicrobial defense, the aim of this study was to investigate the effect of CTX on neutrophil ROS production and killing activity, either through CTX-treated macrophage co-culture or conditioned medium of CTX-treated macrophages. The results showed an important modulatory action of CTX on the neutrophil function as well as neutrophil-macrophage interactions, as demonstrated by the increased production of hydrogen peroxide, hypochlorous acid, nitric oxide and TNF- a , along with the increased fungicidal activity of neutrophils.

Reduced cytokine production by glycogen-elicited peritoneal cells from diabetic rats.

IL-1beta, TNF-alpha, cytokine-induced neutrophil chemoattractant-2alpha/beta, and IL-10 measurements were performed in elicited peritoneal cells from control, diabetic, and insulin-treated diabetic rats. Production/liberation of these cytokines was decreased in elicited peritoneal cells from diabetic rats. These changes were abolished by insulin treatment of diabetic rats. The alterations observed might be involved in the impaired inflammatory response and high occurrence of apoptosis observed in neutrophils under diabetic states.

Stress-induced downregulation of macrophage phagocytic function is attenuated by exercise training in rats.

BACKGROUND/AIMS: Acute restraint stress may induce impaired macrophage phagocytic function. Moderate physical training is associated with beneficial effects on immunological functions. We investigated the effects of moderate physical training on phagocytic function of alveolar macrophages in rats submitted to acute restraint stress. METHODS: Thirty male Wistar rats weighing 210-226 g were randomly divided into 4 groups: nontrained rats (n = 7), nontrained rats submitted to stress (n = 8), trained rats (n = 7) and trained rats submitted to stress (n = 8). Trained rats were submitted to a program of moderate running training over a period of 8 weeks. Rats subjected to restraint stress were kept immobilized in glass cylinders (8 cm in diameter and 24 cm long) during 60 min. Phagocytosis capacity of macrophages was evaluated by either Escherichia coli orzymosan stimuli. RESULTS: Restraint stress induced a decrease in phagocytosis of E. coli and zymosan particle stimulation by macrophages. Neither of these alterations was observed in trained animals submitted to acute restraint stress. CONCLUSIONS: Our data confirm that acute restraint stress is associated with impaired function of macrophages. Moreover, moderate physical training attenuates the effects of acute stress by a mechanism that involves an increase in tolerance of macrophages.