METHOD DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE LIQUID CHROMATOGRAPHIC METHOD FOR THE SIMULTANEOUS ESTIMATION OF SELECTED ANTIDIABETIC DRUGS IN THE PRESENCE OF THEIR DEGRADATION PRODUCTS

   Objective: This study was designed to conduct forced degradation and validation studies for the simultaneous estimation of metformin, sitagliptin, pioglitazone, and glimepiride.Methods: Analytes were separated on an Agilent XDB-C18, 150 × 4.6 mm, 5 μm column using an isocratic elution mode having mobile phase composition of 20 mM potassium dihydrogen phosphate buffer (pH 4.0):acetonitrile (65:35% v/v). Analytes were detected at a wavelength of 225 nm. The optimized method was validated as per the ICH Q2 guidelines.Results: The retention times of metformin, sitagliptin, pioglitazone, and glimepiride were 3.47, 4.83, 5.83, and 9.44 min, respectively. The linearity was 25–100 μg/ml for metformin, 2.5–10 μg/ml for sitagliptin, 1–4 μg/ml for pioglitazone, and 0.75–3 μg/ml for glimepiride. The correlation coefficient for calibration curves was >0.99, and accuracy was between 98 and 102% for each analyte. Inter- and intra-day precisions were calculated <2% relative standard deviation for each analyte.Conclusion: A significant degradation was observed in the presence of acidic, basic, neutral, oxidative, and photolytic stress conditions. The method is simple, precise, accurate, robust, and reproducible and was able to successfully separate and quantify metformin, sitagliptin, pioglitazone, and glimepiride in the presence of their degradation products

Regulation of Myosin Light Chain Kinase during Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes

Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIId did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Dual Role for Myosin II in GLUT4-Mediated Glucose Uptake in 3T3-L1 Adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity

Relationship of cognitive function in patients with schizophrenia in remission to disability: a cross-sectional study in an Indian sample

Background: Cognitive deficits in various domains have been consistently replicated in patients with schizophrenia. Most studies looking at the relationship between cognitive dysfunction and functional disability are from developed countries. Studies from developing countries are few. The purpose of the present study was to compare the neurocognitive function in patients with schizophrenia who were in remission with that of normal controls and to determine if there is a relationship between measures of cognition and functional disability. <p/>Methods: This study was conducted in the Psychiatric Unit of a General Hospital in Mumbai, India. Cognitive function in 25 patients with schizophrenia in remission was compared to 25 normal controls. Remission was confirmed using the brief psychiatric rating scale (BPRS) and scale for the assessment of negative symptoms (SANS). Subjects were administered a battery of cognitive tests covering aspects of memory, executive function and attention. The results obtained were compared between the groups. Correlation analysis was used to look for relationship between illness factors, cognitive function and disability measured using the Indian disability evaluation and assessment scale. <p/>Results: Patients with schizophrenia showed significant deficits on tests of attention, concentration, verbal and visual memory and tests of frontal lobe/executive function. They fared worse on almost all the tests administered compared to normal controls. No relationship was found between age, duration of illness, number of years of education and cognitive function. In addition, we did not find a statistically significant relationship between cognitive function and scores on the disability scale. <p/>Conclusion: The data suggests that persistent cognitive deficits are seen in patients with schizophrenia under remission. The cognitive deficits were not associated with symptomatology and functional disability. It is possible that various factors such as employment and family support reduce disability due to schizophrenia in developing countries like India. Further studies from developing countries are required to explore the relationship between cognitive deficits, functional outcome and the role of socio-cultural variables as protective factors

Should providers encourage realistic weight expectations and satisfaction with lost weight in commercial weight loss programs? a preliminary study

Background Attrition is a problem among patients who participate in commercial weight loss programs. One possible explanation is that if patients are unable to reach a weight that they expect to achieve, they may be more likely to drop out of treatment. This study investigated variables associated with attrition among 30 obese patients who completed a liquid meal replacement program (LMR) and enrolled in a 52-week Small Changes Maintenance intervention (SCM). Patients lost a median 18% of body weight during LMR and completed assessments about weight expectations and weight satisfaction pre- and post-SCM. Findings Of the 30 patients who started SCM, 8 (27%) were lost to attrition. Odds of SCM attrition were higher in patients who lost ≤ 18.2% of pre-LMR weight (OR: 12.25, P = 0.035), had lower satisfaction (≤7) pre-SCM (OR: 10.11, P = 0.040), and who expected further weight loss of 9.1 kg or more pre-SCM (OR: 10.11, P = 0.040). SCM completers significantly increased weight loss expectations by a median of 2.3 kg from pre-SCM to post-SCM (WSR P = 0.049) that paralleled weight regained post-SCM (2.7 kg). Conclusions After completion of a medically-supervised commercial weight loss program, patients with the greatest expectations for further weight loss and the lowest weight satisfaction were more likely to drop out of SCM. Failure to participate in maintenance treatment may lead to regain of greater than half of lost weight over the next year. Among SCM completers, lower expectations for further weight loss and greater weight satisfaction appeared to be associated with continued engagement in maintenance treatment

Naked mole-rats have distinctive cardiometabolic and genetic adaptations to their underground low-oxygen lifestyles.

The naked mole-rat Heterocephalus glaber is a eusocial mammal exhibiting extreme longevity (37-year lifespan), extraordinary resistance to hypoxia and absence of cardiovascular disease. To identify the mechanisms behind these exceptional traits, metabolomics and RNAseq of cardiac tissue from naked mole-rats was compared to other African mole-rat genera (Cape, Cape dune, Common, Natal, Mahali, Highveld and Damaraland mole-rats) and evolutionarily divergent mammals (Hottentot golden mole and C57/BL6 mouse). We identify metabolic and genetic adaptations unique to naked mole-rats including elevated glycogen, thus enabling glycolytic ATP generation during cardiac ischemia. Elevated normoxic expression of HIF-1α is observed while downstream hypoxia responsive-genes are down-regulated, suggesting adaptation to low oxygen environments. Naked mole-rat hearts show reduced succinate levels during ischemia compared to C57/BL6 mouse and negligible tissue damage following ischemia-reperfusion injury. These evolutionary traits reflect adaptation to a unique hypoxic and eusocial lifestyle that collectively may contribute to their longevity and health span

The role of PPARγ in carbon nanotube-elicited granulomatous lung inflammation

BACKGROUND: Although granulomatous inflammation is a central feature of many disease processes, cellular mechanisms of granuloma formation and persistence are poorly understood. Carbon nanoparticles, which can be products of manufacture or the environment, have been associated with granulomatous disease. This paper utilizes a previously described carbon nanoparticle granuloma model to address the issue of whether peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear transcription factor and negative regulator of inflammatory cytokines might play a role in granulomatous lung disease. PPARγ is constitutively expressed in alveolar macrophages from healthy individuals but is depressed in alveolar macrophages of patients with sarcoidosis, a prototypical granulomatous disease. Our previous study of macrophage-specific PPARγ KO mice had revealed an intrinsically inflammatory pulmonary environment with an elevated pro-inflammatory cytokines profile as compared to wild-type mice. Based on such observations we hypothesized that PPARγ expression would be repressed in alveolar macrophages from animals bearing granulomas induced by MWCNT instillation. METHODS: Wild-type C57Bl/6 and macrophage-specific PPARγ KO mice received oropharyngeal instillations of multiwall carbon nanotubes (MWCNT) (100 μg). Bronchoalveolar lavage (BAL) cells, BAL fluids, and lung tissues were obtained 60 days post-instillation for analysis of granuloma histology and pro-inflammatory cytokines (osteopontin, CCL2, and interferon gamma [IFN-γ] mRNA and protein expression. RESULTS: In wild-type mice, alveolar macrophage PPARγ expression and activity were significantly reduced in granuloma-bearing animals 60 days after MWCNT instillation. In macrophage-specific PPARγ KO mice, granuloma formation was more extensive than in wild-type at 60 days after MWCNT instillation. PPARγ KO mice also demonstrated elevated pro-inflammatory cytokine expression in lung tissue, laser-microdissected lung granulomas, and BAL cells/fluids, at 60 days post MWCNT exposure. CONCLUSIONS: Overall, data indicate that PPARγ deficiency promotes inflammation and granuloma formation, suggesting that PPARγ functions as a negative regulator of chronic granulomatous inflammation

Predictors of perceived fairness among Latinx adolescents

While most research in Latinx populations has documented that endorsing filial obligation values predicts better psychosocial outcomes, some studies have also found potential negative ramifications of the fulfillment of high levels of filial obligations, including worse academic performance and psychological maladjustment (e.g., higher depressive symptoms) (Fuligni et al., 1999). These contradictory findings may be due to the role of perceived fairness of these obligations such that filial responsibility may only lead to positive outcomes when the youth perceives the responsibility to be fair and reciprocated, but it may lead to worse outcomes when perceived to be unfair (Kuperminc et al., 2013). Despite this vital role of perceived fairness, little is known about what contributes to the perceived fairness of filial obligations. The present study examined factors nested within the acculturative process (i.e., behavioral practices, values, identity, and acculturation gap) as predictors of perceived unfairness related to familial obligations among Latinx adolescents. Results indicate that the endorsement of mainstream cultural practices and values as well as greater acculturation gap conflict are all associated with higher perceptions of unfairness. Future studies would benefit from considering the acculturative context of the sample and focusing on familial dynamics to better understand perceptions of unfairness and its impacts on psychological outcomes