Rebeca García-Macedo

Glycine increases mRNA adiponectin and diminishes pro-inflammatory adipokines expression in 3T3-L1 cells

Obesity and type 2 diabetes course with chronic low-grade inflammation, where adiponectin is down-regulated and pro-inflammatory markers, like interleukin (IL)-6, tumor necrosis factor alpha (TNF-alpha), and C-reactive protein (CRP), are up-regulated. A treatment option to improve the micro- and macro-complications in type 2 diabetes is the use of glycine, which has been demonstrated previously to increase the expression of anti-inflammatory cytokine IL-10 in monocytes and down-regulate the expression of TNF-alpha in monocytes and Kupffer cells. Recently, our group demonstrated that glycine decreases the pro-inflammatory plasmatic cytokines in type 2 diabetes. The aim of this study was to test the effect of glycine on adipokines expression in 3T3-L1 cells. Cells were grown and differentiated in the presence of 10 mM glycine. After 2 days of confluence, cells were differentiated to adipocytes in the same medium supplemented with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The RNA was extracted at days 0 and 8 of differentiation (fibroblasts and mature adipocyte phenotypes, respectively). The expression of PPAR-gamma (peroxisome proliferator-activated receptor-gamma), adiponectin, resistin, IL-6 and TNF-alpha were analyzed by real-time PCR. We demonstrated that when 3T3-L1 cells were treated with glycine, IL-6, resistin and TNF-alpha mRNA expression was decreased, but surprisingly adiponectin and PPAR-gamma were up-regulated. In all cases the values were statistically significant (P<0.05) between glycine treatment and controls. These results show that glycine improves the pro-inflammatory profile and up-regulates adiponectin gene expression. Therefore, glycine could be useful as a modulator of the pro-inflammatory state observed in obesity and type 2 diabetes.

Glycine regulates the production of pro-inflammatory cytokines in lean and monosodium glutamate-obese mice

Fat tissue plays an important role in the regulation of inflammatory processes. Increased visceral fat has been associated with a higher production of cytokines that triggers a low-grade inflammatory response, which eventually may contribute to the development of insulin resistance. In the present study, we investigated whether glycine, an amino acid that represses the expression in vitro of pro-inflammatory cytokines in Kupffer and 3T3-L1 cells, can affect in vivo cytokine production in lean and monosodium glutamate-induced obese mice (MSG/Ob mice). Our data demonstrate that glycine treatment in lean mice suppressed TNF-alpha transcriptional expression in fat tissue, and serum protein levels of IL-6 were suppressed, while adiponectin levels were increased. In MSG/Ob mice, glycine suppressed TNF-alpha and IL-6 gene expression in fat tissue and significantly reduced protein levels of IL-6, resistin and leptin. To determine the role of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in the modulation of this inflammatory response evoked by glycine, we examined its expression levels in fat tissue. Glycine clearly increased PPAR-gamma expression in lean mice but not in MSG/Ob mice. Finally, to identify alterations in glucose metabolism by glycine, we also examined insulin levels and other biochemical parameters during an oral glucose tolerance test. Glycine significantly reduced glucose tolerance and raised insulin levels in lean but not in obese mice. In conclusion, our findings suggest that glycine suppresses the pro-inflammatory cytokines production and increases adiponectin secretion in vivo through the activation of PPAR-gamma. Glycine might prevent insulin resistance and associated inflammatory diseases.

Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells.

The mechanisms related to hyperglycemia-induced pancreatic β-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Δ < eqid1 > m), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Δ < eqid2 > m, lead to an important pancreatic β-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia.

Multiparametric study of atresia in ewe antral follicles: Histology, flow cytometry, internucleosomal DNA fragmentation, and lysosomal enzyme activities in granulosa cells and follicular fluid

The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI−), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow citometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3–6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size‐differential behavior was found in free or membrane‐bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles. Mol. Reprod. Dev. 55:270–281, 2000.

Adiponectin in eutrophic and obese children as a biomarker to predict metabolic syndrome and each of its components

BackgroundObesity is associated with the rise of noncommunicable diseases worldwide. The pathophysiology behind this disease involves the increase of adipose tissue, being inversely related to adiponectin, but directly related to insulin resistance and metabolic syndrome (MetS). Therefore, this study aimed to determine the relationship between adiponectin levels with each component of MetS in eutrophic and obese Mexican children.MethodsA cross sectional study was conducted in 190 school-age children classified as obese and 196 classified as eutrophic. Adiponectin, glucose, insulin, high density lipoprotein cholesterol (HDL-C) and triglycerides were determined from a fasting blood sample. Height, weight, waist circumference, systolic and diastolic blood pressures (BP) were measured; MetS was evaluated with the IDF definition. The study groups were divided according to tertiles of adiponectin, using the higher concentration as a reference. Linear regression analysis was used to assess the association between adiponectin and components of the MetS. Finally, stepwise forward multiple logistic regression analysis controlling for age, gender, basal HOMA-IR values and BMI was performed to determine the odds ratio of developing MetS according to adiponectin tertiles.ResultsAnthropometric and metabolic measurements were statistically different between eutrophic and obese children with and without MetS (P <0.001). The prevalence of MetS in obese populations was 13%. Adiponectin concentrations were 15.5 ± 6.1, 12.0 ± 4.8, 12.4 ± 4.9 and 9.4 ± 2.8 μg/mL for eutrophic and obese subjects, obese without MetS, and obese with MetS, respectively (P <0.001). Obese children with low values of adiponectin exhibited a higher frequency of MetS components: abdominal obesity, 49%; high systolic BP, 3%; high diastolic BP, 2%; impaired fasting glucose, 17%; hypertriglyceridemia, 31%; and low HDL-C values, 42%. Adjusted odds ratio of presenting MetS according to adiponectin categories was 10.9 (95% CI 2.05; 48.16) when the first tertile was compared with the third.ConclusionIn this sample of eutrophic and obese Mexican children we found that adiponectin concentrations and MetS components have an inversely proportional relationship, which supports the idea that this hormone could be a biomarker for identifying individuals with risk of developing MetS.

Glycine regulates inflammatory markers modifying the energetic balance through PPAR and UCP-2

Obesity is widely recognized as cause of metabolic syndrome and cardiovascular disease. It is provoked by imbalance between the spending and consumption of energy associated with a chronic inflammatory condition due to excessive storage of fat tissue. Obese patients have an impaired inflammatory profile that contributes to the development of vascular complications, with fat tissue being partially responsible for controlling both processes: energy balance (through PPAR) and inflammatory condition (through inflammatory markers). White adipose tissue produces cytokines (IL-6, TNF-α, resistin, adiponectin, etc.) and participates in a broad spectrum of processes. Recently, glycine has been reported to have anti-inflammatory properties which reduce TNF-α and IL-6 levels and increase adiponectin in 3T3-L1 adipocytes and in fat tissue of obese mice. In this study, the possible regulatory role of glycine on some factors involved in storage and energy burning (PPAR-γ, PPAR-α, PPAR-δ and UCP-2) was analyzed in lean and monosodium glutamate-induced obese mice (MSG/Ob mice). Glycine clearly increased fat tissue PPAR-γ expression in lean but not in MSG/Ob mice. The PPAR-γ and PPAR-α liver expression was repressed in both groups of mice, while the expression of PPAR-δ decreased only in lean mice. Interestingly, glycine treatment also suppressed the expression of UCP-2, TNF-α and IL-6 in lean mice, and increased adiponectin and insulin serum levels. In conclusion, glycine regulates the production of inflammatory cytokines through PPAR-γ. These results provide clues on glycine signaling mechanisms as an anti-inflammatory agent that might be useful for treatment of metabolic and vascular complications associated to inflammation in obesity.

Monosodium Glutamate Neonatal Intoxication Associated with Obesity in Adult Stage is Characterized by Chronic Inflammation and Increased mRNA Expression of Peroxisome Proliferator‐Activated Receptors in Mice

The monosodium glutamate (MSG) neonatal administration in mice provides a model of obesity with impaired glucose tolerance (IGT) and insulin resistance. However, the inflammatory profile of cytokines produced from fat tissue and its relationship to the metabolic dysfunction induced by MSG have not yet been revealed. The aim of this study was to establish the inflammatory profile attributed to MSG by measuring the expression of adipokines in visceral fat and serum of 19-week-old mice as well as the peroxisome proliferator-activated receptors alpha and gamma (PPARα and γ). Some metabolic and biochemical parameters were also quantified. The MSG increased mRNA expression of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNFα), resistin and leptin, but adiponectin did not exhibit any changes. In addition, impaired glucose tolerance, increased levels of insulin, resistin and leptin were observed in serum. Both PPARα and PPARγ were activated in MSG-induced obese mice, which might explain its inflammatory profile. However, liver transaminases were severely depressed, indicating that MSG may also induce liver injury, contributing to inflammation. The MSG neonatal neuro-intoxication in mice may thus provide a model of obesity and inflammation characterized by the dual activation of PPARα and PPARγ, which might offer new insights into the mechanism of inflammatory diabetes in obesity leading to steatohepatitis, as well as a suitable model to study the role of new therapeutic agents to prevent or reduce insulin resistance, the inflammatory state and liver steatosis.

Glycine suppresses TNF-alpha-induced activation of NF-κB in differentiated 3T3-L1 adipocytes

Glycine strongly reduces the serum levels of pro-inflammatory cytokines and increases the levels of anti-inflammatory cytokines. Recently, glycine has been shown to decrease the expression and secretion of pro-inflammatory adipokines in monosodium glutamate-induced obese (MSG/Ob) mice. It has been postulated that these effects may be explained by a reduction in nuclear factor kappa B (NF-κB) activation. NF-κB is a transcription factor, which is crucial to the inflammatory response. Hasegawa et al. (2011 and 2012) recently reported a glycine-dependent reduction in NF-κB levels. Here, we have investigated the role of glycine in the regulation of NF-κB in differentiated 3T3-L1 adipocytes. The results revealed that pretreatment with glycine interfered with the activation of NF-κB, which has been shown to be stimulated by tumor necrosis factor-alpha (TNF-α). Glycine alone stimulated NF-κB activation in an unusual way such that the inhibitor κB-β (IκB-β) degradation was more significant than that of the inhibitor κB-α (IκB-α) and led to NF-κB complexes comprised of p50 and p65 subunits; IκB-ε degradation did not affect by glycine. These findings suggest that glycine could be used as an alternative treatment for chronic inflammation, which is a hallmark of obesity and other comorbidities, and is characterized by an elevated production of pro-inflammatory cytokines.

Diabetogenic Effect of STZ Diminishes with the Loss of Nitric Oxide: Role of Ultraviolet Light and Carboxy-PTIO

Nitric oxide has been demonstrated to participate in β-cell damage during streptozotocin (STZ)-induced diabetes. STZ consists of 2-deoxy-D-glucose substituted by N-methyl-N-nitrosourea at C-2 and therefore can liberate ·NO. However, it has not been proven whether ·NO generation from STZ is responsible for the disease. We found that STZ treated in vitro with ultraviolet (UV) light liberated significantly more ·NO than non-irradiated STZ (1,134.4 ± 104 vs. 256.9 ± 240 nmol). Moreover, the diabetogenic effect of STZ was abolished by UV irradiation before its administration to experimental animals. In these animals the glucose and insulin values were significantly different from those of the diabetic group (151.3 ± 16.6 vs. 364.6 ± 63.4 mg/dl and 36.3 ± 17.9 vs. 0.08 ± 5.5 µIU/ml, respectively) and similar to those of the non-diabetic group (127.2 ± 34.1 mg/dl and 41.7 ± 13.9 µIU/ml, respectively). Carboxy-PTIO treatment returned glycemia to nearly normal levels in 60% of STZ-induced diabetic rats (157.5 ± 11.8 vs. 364.6 ± 63.6 mg/dl of the diabetic group). L-NAME and dexamethasone cannot return either glucose or insulin to normal levels. In conclusion, UV light increased ·NO liberation from STZ and suppressed its diabetogenic activity. It is possible that the diabetogenic activity of STZ is related to the liberation of nitric oxide from STZ, since carboxy-PTIO scavenger had a protective effect, while L-NAME and dexamethasone did not. It is possible that an increase in ·NO concentration into cell, independently of its endogenous or exogenous origin, can induce β-cell damage and diabetes.

Nicotinamide, a glucose-6-phosphate dehydrogenase non-competitive mixed inhibitor, modifies redox balance and lipid accumulation in 3T3-L1 cells.

AIMS Excessive energy uptake of dietary carbohydrates results in their storage as fat and requires glucose-6-phosphate dehydrogenase (G6PD)-mediated NADPH production. We sought to assess whether the nicotinamide-induced reduction of G6PD activity might modulate redox balance and lipid accumulation in 3T3-L1 cells. MAIN METHODS 3T3-L1 preadipocytes (days 4 and 6 of differentiation) and adipocytes were cultured in the presence of 5 or 25 mM glucose. The cells cultured in 25 mM glucose were supplemented with nicotinamide (5-15 mM). Next, we evaluated the following parameters: cell viability, apoptosis, lipid accumulation, lipolysis, reducing power, reactive oxygen species (ROS), NAD(P)H and NAD(P)(+), isocitrate dehydrogenase (IDP), malic enzyme and G6PD, as well as the protein and mRNA levels of G6PD. We also analysed the kinetics of the nicotinamide-induced inhibition of G6PD. KEY FINDINGS G6PD mRNA levels increased at day 4 of adipocyte differentiation, whereas G6PD activity progressively increased at days 4 and 6 of differentiation and was reduced in adipocytes. Concomitantly, ROS, reducing power and lipid accumulation increased gradually as the preadipocytes matured into adipocytes. High glucose increased the activity of G6PD, which coincided with an increase in ROS, reducing power and lipid accumulation. All of these changes are prevented by nicotinamide, with the exception of lipid accumulation in adipocytes. Nicotinamide increased IDP activity without affecting NADPH levels. Lastly, nicotinamide inhibited G6PD in a non-competitive mixed way. SIGNIFICANCE Nicotinamide modulates G6PD via a non-competitive mixed inhibition and decreases high glucose-dependent oxidative stress and lipid accumulation. Nicotinamide maintains NADPH levels by increasing the activity of IDP.