Search

Aims Standardized, population-based kinetics of C-peptide distribution and clearance are used to estimate insulin secretion from plasma C-peptide concentrations without direct measurement of C-peptide kinetics. We then compared the performance of population-based kinetics to directly measured C-peptide kinetics when used to calculate β-cell responsivity indices. To ensure that population-based kinetics apply to all conditions where β-cell function is measured, subjects were studied in the presence and absence of acute insulin resistance. Materials and Methods Somatostatin was used to inhibit endogenous insulin secretion in 56 nondiabetic subjects. Subsequently, a C-peptide bolus was administered and the changing concentrations used to calculate individual kinetic parameters of C-peptide clearance. In addition, they were studied on 2 occasions in random order using an oral glucose tolerance test (OGTT). On one occasion, free fatty acid (FFA) elevation to cause insulin resistance, was achieved by infusion of intralipid + heparin. Disposition Index (DI) was then estimated by the oral minimal model using either population-based or individual C-peptide kinetics. Results There were marked differences in the exchange parameters (k12 and k21) of the model describing C-peptide kinetics, but smaller differences in the fractional clearance, i.e. the irreversible loss from the accessible compartment (k01), obtained from population-based estimates compared to experimental measurement. Since it is predominantly influenced by k01, DI estimated using individual kinetics correlated well with those estimated using population-based kinetics. Conclusions These data support the use of population-based measures of C-peptide kinetics to estimate β-cell function during OGTT.

Glutamine is a nonessential amino acid, which under trauma or critical illness can become essential. A number of historic small single-center randomized controlled trials (RCTs) have demonstrated positive treatment effects on clinical outcomes with glutamine supplementation. Meta-analyses based on these trials demonstrated a significant reduction in hospital mortality, intensive care unit (ICU) length of stay (LOS), and hospital LOS with intravenous (IV) glutamine. Similar results were not noted in 2 large multicenter RCTs (REDOXS and MetaPlus) assessing the efficacy of glutamine supplementation in ventilated ICU patients. The REDOXS trial of 40 ICUs randomized 1223 ventilated ICU patients to glutamine (IV and enteral), antioxidants, both glutamine and antioxidants, or placebo. The main conclusions were a trend toward increased 28-day mortality and significant increased hospital and 6-month mortality in those who received glutamine. The MetaPlus trial of 14 ICUs, which randomized 301 ventilated ICU patients to glutamine-enriched enteral vs an isocaloric diet, noted increased 6-month mortality in the glutamine-supplemented group. Newer RCTs have focused on specific populations and have demonstrated benefits in burn and elective surgery patients with glutamine supplementation. Whether larger studies will confirm these findings is yet to be determined. Recent American Society for Parenteral and Enteral Nutrition guidelines recommend that IV and enteral glutamine should not be used in the critical care setting based on the moderate quality of evidence available. We agree with these recommendations and would encourage larger multicenter studies to evaluate the risks and benefits of glutamine in burn and elective surgery patients.

Background Little is known about fertility outcomes after fertility sparing surgery (FSS) for localized ovarian cancers. Methods A random sample of 783 women treated for ovarian cancer were identified from the California Cancer Registry for survey (age 18–40 years at diagnosis; diagnosed from 1993–2007). We evaluated outcomes including post-treatment amenorrhea, infertility, early menopause (age

Dear Editor, Expression of MITF is often regarded as the key event in melanocyte specification from neural crest cells (Sommer, 2011). The central role of MITF in melanocyte biology and ‘phenotype-switching’ in melanoma depending on the levels of MITF has been documented (Hoek and Goding, 2010), and the importance of the fine-tuning of MITF levels in melanoma was proposed by several groups (Pinner et al., 2009; Shah et al., 2010). Factors such as SOX10, PAX3, CBP, and b-catenin / LEF1 control MITF expression in melanocyte development in mouse model (Hou and Pavan, 2008); however, the mechanisms regulating MITF expression in human melanocyte and melanomas remains obscure. SOX2 is expressed in 45% primary melanomas and 40% metastasis, and SOX2-knockdown inhibited A2058 melanoma growth upon transplantation (Laga et al., 2010). Recent studies suggested SOX2 involvement in melanocyte development in mice (Adameyko et al., 2012) and in human melanoma progression (Girouard et al., 2012). Here, we prvide evidence that SOX2 and MITF expression is correlated with normal human melanocytes and three human melanoma cell lines at a single-cell level. We found that SOX2 regulates the levels of endogenous MITF in these cell lines, suggesting a role for SOX2-MITF axis in biology of normal human melanocyte and human melanomas. We established a human embryonic stem cell (hESC) model of neural crest stem cells and neural crest lineages (Curchoe et al., 2010) and demonstrated a key role of SOX2 in peripheral neurogenesis (Cimadamore et al., 2011). Because MITF marks melanocyte progenitors shortly after the emigration of neural crest cells (Sommer, 2011), we investigated the expression of MITF in hESC-derived neural crest lineage (hESC-NC). MITF is expressed in _25% of hESC-NC (Figure S1A), which are uniformly positive for SOX2 under these culture conditions (Cimadamore et al., 2011). We have previously validated hESC-NC cells engineered with SOX2-specific / scrambled shRNA under the control of Tet-inducible promoter (Cimadamore et al., 2011). Knockdown of SOX2 transcripts decreased MITF mRNA and MITF protein, suggesting that SOX2 is required for endogenous MITF levels in hESC-NC (Figure S1B, C). We investigated the relationship between SOX2 and MITF in normal human melanocytes, which express various levels of both transcription factors at a single-cell level (Figures 1A, S2A). We used immunofluorescence to colocalize both transcription factors and then quantified the coexpression of these factors in the same cell. We found that SOX2 and MITF expression is well correlated at the single-cell level (r = 0.65) (Figure 1B), and the knockdown of SOX2 (using lentiviral delivery of SOX2-specific shRNA) reduced the levels of MITF in these cells (Figure 1C). These results suggest that SOX2 is required for the endogenous levels of MITF in hESC-NC and normal human melanocytes in vitro. Figure 1 SOX2 regulates MITF expression in primary melanocytes and melanoma cell lines. (A, D, G, and J) Costaining for SOX2 and MITF in primary melanocytes (A), MEL501 (D), MeWo (G), and Lu1205 (J) melanoma cell lines. Scale bars = 50lm. (B, E, H, and K) Single-cell ... Next, we analyzed the expression of SOX2 and MITF in three human melanoma cell lines (i.e., MEL501, MeWo, and Lu1205). Although several MITF isoforms have been described (Amae et al., 1998; Hershey and Fisher, 2005), melanoma cells predo- minantly express MITF-M isoform (Amae et al., 1998). Western blot analysis confirmed that MITF-M is a predominant isoform in all three melanoma lines analyzed in the current study (Figure S3). At a single-cell level, the expression of both SOX2 and MITF is highly variable in melanoma lines (Figures 1D, G, J, S2B, C, D). Therefore, we used immunofluorescence to colocalize both transcription factors and then quantified the coexpression of these factors in the same cells. SOX2 and MITF expression was well correlated with MEL501 and MeWo melanoma lines (r = 0.9 and 0.56, respectively) (Figure 1E, H). In Lu1205 melanoma cells, which express lower levels of MITF compared with MEL501 and MeWo lines (Figure S3), SOX2 and MITF expression was moderately correlated (r = 0.52) (Figure 1K). Taken together, these results demonstrate significant correlation of the levels of SOX2 and MITF in normal human melanocytes and melanoma lines, consistent with the role of SOX2 as a modulator of MITF expression. Indeed, we observed no MITF-expressing cells that were negative for SOX2 in these cell lines by immunofluorescence. Next, we investigated functional requirement of SOX2 for MITF expression in three human melanoma cell lines MEL501, MeWo, and Lu1205 expressing high, intermediate, and low levels of MITF as demonstrated by Western blot analysis (Figure S3). All melanoma lines were engineered with lentiviral vectors expressing SOX2-specific shRNA or scrambled control shRNA. The knockdown of SOX2 induced pronounced decrease in the levels of MITF protein as assayed by quantitative immunofluorescence (Figures 1F, I, L, S4). These results suggest that SOX2 function is required for the endogenous levels of MITF in MEL501, MeWo, and Lu1205 melanoma cells expressing very different levels of MITF. Finally, we investigated the effect of SOX2 overexpression (using SOX2-IRES-GFP lentivirus) in MeWo and Lu1205 melanoma cells expressing MITF at variable high and low levels, respectively (Figures 2A, C, S3). The SOX2 overexpression in MeWo cells significantly decreased MITF levels (Figure 2A, B), but not completely eliminated MITF protein as compared to SOX2 knockdown (Figures 2A, B, versus S4B). The SOX2 overexpression in Lu1205 line had no significant effect on MITF levels (Figure 2C, D). These contradictory nonlinear effects of SOX2 on MITF expression would suggest that SOX2 can function as an activator or as a repressor under different conditions of signaling, in vivo versus in vitro, and / or in the context of additional modifiers (e.g., BRAF). Figure 2 Effect of SOX2 overexpression in meWo and Lu1205 cell lines. (A, C) Immunostaining for MITF in MeWo (A) and Lu1205 (C) melanoma cell lines overexpressing GFP (control) or SOX2- IRES-GFP. (B, D) Quantification of MITF expression from A and C. Only GFP-positive ... Recent studies in mice proposed cross-regulatory interactions between SOX2 and MITF (Adameyko et al., 2012). The SOX2 overexpression results in human MeWo melanoma line are consistent with SOX2 suppression of MITF in B16–F10 mouse melanoma line (Adameyko et al., 2012). However, our study also suggests that SOX2 is required to maintain the endogenous levels of MITF in normal human melanocytes and melanoma cell lines. The effect of SOX2 could be indirect, although SOX2 binding to the MITF proximal promoter was observed in mouse model (Adameyko et al., 2012), raising the possibility that SOX2 may interact with other factors to regulate MITF expression (e.g., PAX3, CBP, and b-catenin / LEF1).