A MULTICENTER STUDY EVALUATING PERCEPTIONS AND KNOWLEDGE OF INPATIENT GLYCEMIC CONTROL AMONG RESIDENT PHYSICIANS: ANALYZING THEMES TO INFORM AND IMPROVE CARE.

Objective: In this descriptive study, we evaluated perceptions and knowledge of inpatient glycemic control among resident physicians. Methods: We performed this study at four academic medical centers: the University of Mississippi Medical Center, University of Virginia Health System, University of Louisville Health Sciences Center, and Emory University. We designed a questionnaire, and Institutional Review Board approval was granted at each institution prior to study initiation. We then administered the questionnaire to Internal Medicine and Medicine-Pediatric resident physicians. Results: A total of 246 of 438 (56.2%) eligible resident physicians completed the Inpatient Glycemic Control Questionnaire (IGCQ). Most respondents (85.4%) reported feeling comfortable treating and managing inpatient hyperglycemia, and a majority (66.3%) agreed they had received adequate education. Despite self-reported comfort with knowledge, only 51.2% of respondents could identify appropriate glycemic targets in critically ill patients. Only 45.5% correctly identified appropriate inpatient random glycemic target values in noncritically ill patients, and only 34.1% of respondents knew appropriate preprandial glycemic targets in noncritically ill patients. A small majority (54.1%) were able to identify the correct fingerstick glucose value that defines hypoglycemia. System issues were the most commonly cited barrier to successful inpatient glycemic control. Conclusion: Most respondents reported feeling comfortable managing inpatient hyperglycemia but had difficulty identifying appropriate inpatient glycemic target values. Future interventions could utilize the IGCQ as a pre- and postassessment tool and focus on early resident education along with improving system environments to aid in successful inpatient glycemic control. Abbreviations: DM = diabetes mellitus; Emory = Emory University Healthcare; IGC = inpatient glycemic control; IGCQ = Inpatient Glycemic Control Questionnaire; IRB = Institutional Review Board; PGY = postgraduate year; UMMC = University of Mississippi Medical Center; UVA = University of Virginia Health System; UL = University of Louisville Health Sciences Center.

Construction and preliminary evaluation of the inpatient glycemic control questionnaire (IGCQ): a survey tool assessing perceptions and knowledge of resident physicians.

BACKGROUND: Uncontrolled hyperglycemia in hospitalized patients, with or without diabetes mellitus, is associated with many adverse outcomes. Resident physicians are the primary managers of inpatient glycemic control (IGC) in many academic and community medical centers; however, no validated survey tools related to their perceptions and knowledge of IGC are currently available. As identification of common barriers to successful IGC amongst resident physicians may help foster better educational interventions (ultimately leading to improvements in IGC and patient care), we sought to construct and preliminarily evaluate such a survey tool. METHODS: We developed the IGC questionnaire (IGCQ) by using previously published but unvalidated survey tools related to physician perspectives on inpatient glycemic control as a framework. We administered the IGCQ to a cohort of resident physicians from the University of Mississippi Medical Center, University of Louisville, Emory University, and the University of Virginia. We then used classical test theory and Rasch Partial Credit Model analyses to preliminarily evaluate and revise the IGCQ. The final survey tool contains 16 total items and three answer-choice categories for most items. RESULTS: Two hundred forty-six of 438 (56.2%) eligible resident physicians completed the IGCQ during various phases of development. CONCLUSIONS: We constructed and preliminarily evaluated the IGCQ, a survey tool that may be useful for future research into resident physician perceptions and knowledge of IGC. Future studies could seek to externally validate the IGCQ and then utilize the survey tool in pre- and post-intervention assessments.

Protective Role for B-1b B Cells and IgM in Obesity-Associated Inflammation, Glucose Intolerance, and Insulin Resistance.

OBJECTIVE: Little is known about the role(s) B cells play in obesity-induced metabolic dysfunction. This study used a mouse with B-cell-specific deletion of Id3 (Id3(Bcell KO)) to identify B-cell functions involved in the metabolic consequences of obesity. APPROACH AND RESULTS: Diet-induced obese Id3(Bcell KO) mice demonstrated attenuated inflammation and insulin resistance in visceral adipose tissue (VAT), and improved systemic glucose tolerance. VAT in Id3(Bcell KO) mice had increased B-1b B cells and elevated IgM natural antibodies to oxidation-specific epitopes. B-1b B cells reduced cytokine production in VAT M1 macrophages, and adoptively transferred B-1b B cells trafficked to VAT and produced natural antibodies for the duration of 13-week studies. B-1b B cells null for Id3 demonstrated increased proliferation, established larger populations in Rag1(-/-) VAT, and attenuated diet-induced glucose intolerance and VAT insulin resistance in Rag1(-/-) hosts. However, transfer of B-1b B cells unable to secrete IgM had no effect on glucose tolerance. In an obese human population, results provided the first evidence that B-1 cells are enriched in human VAT and IgM antibodies to oxidation-specific epitopes inversely correlated with inflammation and insulin resistance. CONCLUSIONS: NAb-producing B-1b B cells are increased in Id3(Bcell KO) mice and attenuate adipose tissue inflammation and glucose intolerance in diet-induced obese mice. Additional findings are the first to identify VAT as a reservoir for human B-1 cells and to link anti-inflammatory IgM antibodies with reduced inflammation and improved metabolic phenotype in obese humans.

Type 2 diabetes remission following gastric bypass: does diarem stand the test of time?

OBJECTIVE(S): Roux-en-Y Gastric Bypass (RYGB) is well known to ameliorate type 2 diabetes mellitus (T2DM), and recent work suggests that the preoperative DiaREM model predicts successful remission up to 1 year post-RYGB. However, no data exist for long-term validity. Therefore, we sought to determine the utility of this score on long-term RYGB effectiveness for T2DM resolution at 2 and 10 years, respectively. METHODS: T2DM patients (Age: 48, BMI: 49, HbA1C: 8.1) undergoing RYGB at the University of Virginia between 2004-2006 (n = 42) and 2012-2014 (n = 59) were evaluated prospectively to assess preoperative DiaREM score, defined from insulin use, age, HbA1C, and type of antidiabetic medication. T2DM partial remission status was based on the American Diabetes Association guidelines (HbA1C < 6.5 % and fasting glycemia <125 mg/dL, and no anti-diabetic medications). Chi-square test was used to compare patient's T2DM status to their DiaREM probability of remission. RESULTS: Among RYGB patients with 2-year postoperative data, 2 were lost (n = 1 no follow-up and n = 1 died) resulting in 57 patients for analysis. For the 10-year postoperative data, 11 were lost (n = 6 no follow-up and n = 5 died), thereby resulting in only 31 patients for analysis. Patients were distributed by DiaREM score to correlate with the predicted probability of remission as follows: 0-2 (Predicted 94 %, 2-year 100 % p = 0.61, 10-year 100 % p = 0.72), 3-7 (Predicted 76 %, 2-year 94 % p = 0.08, 10-year 83 % p = 0.57), 8-12 (Predicted 36 %, 2-year 47 % p = 0.38, 10-year 43 % p = 0.72), 13-17 (Predicted 22 %, 2-year 20 % p = 0.92, 10-year 33 % p = 0.64), and 18-22 (Predicted 9 %, 2-year 15 % p = 0.40, 10-year 14 % p = 0.64). CONCLUSIONS: Preoperative DiaREM scores are a good tool for predicting both short- and long-term T2DM remissions following RYGB. This study highlights the need to identify strategies that improve T2DM remission in those at highest risk.

Inhibitor of differentiation-3 mediates high fat diet-induced visceral fat expansion.

OBJECTIVE: Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. METHODS AND RESULTS: Id3(-/-) mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3(-/-) mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. CONCLUSIONS: Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

Impact of a short-term low calorie diet alone or with interval exercise on quality of life and oxidized phospholipids in obese females.

The objective of this study was to test if a low-calorie diet plus interval exercise (LCD+INT) reduced oxidized and non-oxidized phospholipids in relation to improved weight-related quality of life (QoL) to a greater extent than an energy-deficit matched LCD in obese females. Subjects (age: 47.2 ± 2.6 years, body mass index: 37.5 ± 1.3 kg/m2) were randomized to a 13-day LCD (n = 12; mixed meals of ∼1200 kcal/day) or LCD+INT (n = 13; 12 sessions of 60 min/day alternating 3 min at 50% and 90% peak heart rate plus an additional 350 kcal shake fed after exercise to match energy availability between groups). Weight-related QoL (Laval Questionnaire) as well as oxidized (POVPC, HOOA-PC, HPETE-PC, HETE-PC, PEIPC, KOOA-PC) and non-oxidized (PAPC and lysoPC) phospholipids were assessed pre- and post-intervention. Fitness (VO2peak), body composition (BodPod), and clinical bloods were also tested. LCD+INT significantly increased VO2peak (mL/kg/min, P = 0.03) compared to LCD despite similar fat loss, blood glucose, insulin sensitivity, and inflammatory responses. LCD+INT had significantly greater increases in QoL sexual life domain (P = 0.05) and tended to have a greater increase in the emotions domain (P = 0.09) and total score (P = 0.10) compared to LCD. There were no significant differences between treatments for changes in phospholipids despite LCD+INT increasing measured oxidized and non-oxidized phospholipids while LCD decreased POVPC, HOOA-PC, and PEIPC as well as non-oxidized PAPC and lysoPC. Interestingly, the rise in PEIPC correlated with elevated VO2peak (mL/kg/min r = 0.42, P = 0.05). Decreased caloric intake was, however, linked to a decrease in PAPC (r = 0.53, P = 0.01), lysoPC (r = 0.52, P = 0.02), POVPC (r = 0.43, P = 0.05), and HPETE-PC (r = 0.43, P = 0.05). The decrease in HETE-PC also correlated with increases in the QoL domains symptoms (r = -0.46, P = 0.04), hygiene/clothing (r = -0.53, P = 0.01), emotions (r = -0.53, P = 0.01), social interactions (r = -0.49, P = 0.02), and total score (r = -0.52, P = 0.02). In conclusion, although LCD and LCD+INT improved weight related QoL over 13 days in females with obesity, LCD+INT tended to improve sexual life, emotions as well as total QoL score more than LCD. These data suggest caloric restriction and fitness may act through different mechanisms to support QoL.

Electronic Glycemic Management System and Endocrinology Service Improve Value in Cardiac Surgery.

BACKGROUND: Postoperative glycemic control improves cardiac surgery outcomes but insulin protocols are limited by complexity and inflexibility. We sought to evaluate the effect of implementing an electronic glycemic management system (eGMS) in conjunction with a cardiac surgery endocrinology consult service on glycemic control and outcomes after cardiac surgery. METHODS: All patients with a calculated preoperative risk of mortality who underwent cardiac surgery before and after implementation of an eGMS and an endocrinology consult service were identified. Glycemic control and surgical outcomes were compared using univariate analysis, and multivariate regression was used to model the risk-adjusted effects of the interventions on glycemic control, surgical outcomes, and resource utilization. The health care-related value added by the interventions was calculated by dividing risk-adjusted outcomes by total hospital costs. RESULTS: A total of 2612 patients were identified, with 1263 patients in the preimplementation cohort and 1349 in the postimplementation cohort. Multivariate regression demonstrated fewer postoperative hyperglycemic events (odds ratio [OR] 0.8, 95% CI, 0.65-0.99) after protocol implementation without an increase in hypoglycemic events (OR 0.96, 95% CI, 0.71-1.3). Average day-weighted mean glucose decreased from 144 to 138 mg/dL (P < .001). The improved glycemic control correlated with a risk-adjusted decrease in composite morbidity or mortality (OR 0.61, 95% CI, 0.47-0.79). Although hospital costs increased after implementation, the protocol increased health care-related value by 38%. CONCLUSION: Implementation of a protocol consisting of an eGMS paired with a cardiac surgery-specific endocrinology consult service was associated with improved glycemic control and reduced morbidity. Despite higher costs health care-related value increased as a result of eGMS implementation.

Bariatric surgery reduces incidence of atrial fibrillation: a propensity score-matched analysis.

BACKGROUND: Obesity is associated with an increased risk of atrial fibrillation (AF). Bariatric surgery results insubstantial long-term weight loss and the amelioration of several chronic comorbidities. We hypothesized that weightreduction with bariatric surgery would reduce the long-term incidence of AF. OBJECTIVES: To assess the association between bariatric surgery and AF prevention. SETTING: University Hospital, United States. METHODS: All patients who underwent bariatric surgery at a single institution from 1985-2015 (n = 3,572) were propensity score matched 1:1 to a control population of obese patients with outpatient appointments (n = 45,750) in our clinical data repository. Patients with a prior diagnosis of AF were excluded. Demographics, relevant comorbidities, and insurance status were collected and a chart review was performed for all patients with AF. Paired univariate analyses were used to compare the two groups. RESULTS: After propensity score matching, 5,044 total patients were included (2,522 surgical, 2,522 non-surgical). There were no differences in preoperative body mass index (BMI) (47.1 vs 47.7 kg/m2, P = 0.76) or medical comorbidities between groups. The incidence of AF was lower among surgical patients (0.8% vs 2.9%, P = 0.0001). In patients ultimately diagnosed with AF, time from enrollment to development of AF did not differ between groups; however, surgical patients with AF experienced a significantly higher reduction in excess BMI compared to non-surgical patients with AF (57.9% vs -3.8%, P<0.001). CONCLUSION: The incidence of AF was lower among patients who underwent bariatric surgery compared to their medically managed counterparts. Weight reduction with bariatric surgery may reduce the long-term incidence of AF.

Hypoglycemia Following Bariatric Surgery: Our 31-Year Experience.

PURPOSE: The purposes of this study are to identify the cumulative incidence of post-bariatric surgery hypoglycemia (PBSH), describe its symptomatology, and characterize treatment patterns at a large academic institution. MATERIALS AND METHODS: All patients who underwent bariatric surgery at a single institution from 1985 to 2015 were identified using a clinical database, administrative billing data identified patients who were treated for hypoglycemia, and chart reviews were performed to make a diagnosis of PBSH based on Whipple's triad. PBSH cases were reviewed including patient diabetes history, symptomatology, and treatment measures. Univariate analyses were performed to identify correlations based on symptomatology, laboratory values, and treatments utilized. RESULTS: Ninety (2.6%) of 3487 patients were diagnosed with PBSH with preoperative median age of 43 years, mean BMI of 50.0 kg/m2, and median glycated hemoglobin of 6.0%. Median time-to-first hypoglycemic event was 40.6 months. No factors were identified which predict symptom severity or resolution. The 24 (27%) patients who received pharmacotherapy to treat hypoglycemia were younger, had lower nadir blood glucose levels, and more frequent symptoms. Sixty-nine (79%) cases eventually resolved. CONCLUSIONS: PBSH onset and severity are highly variable. Successful management of these patients can prove difficult and should include dietary therapy, the selective use of pharmacotherapy and surgery, and the use of a multidisciplinary team including bariatric surgeons and endocrinologists.

Bariatric Surgery Resistance: Using Preoperative Lifestyle Medicine and/or Pharmacology for Metabolic Responsiveness.

Bariatric surgery is an effective and durable treatment for individuals with obesity and its associated comorbidities. However, not all patients meet weight loss and/or cardiometabolic goals following bariatric surgery, suggesting that some people are bariatric surgery resistant. The reason for this resistance is unclear, but potential factors, such as adiposity-derived inflammation, insulin resistance, hyperglycemia, and aerobic fitness prior to surgery, have been related to blunted surgery responsiveness. Exercise, diet, and/or pharmacology are effective at reducing inflammation and improving insulin action as well as physical function. Herein, we present data that supports the novel hypothesis that intervening prior to surgery can enhance disease resolution in people who are resistant to bariatric surgery.

Bariatric surgery insurance requirements independently predict surgery dropout.

BACKGROUND: Many insurance companies have considerable prebariatric surgery requirements despite a lack of evidence for improved clinical outcomes. The hypothesis of this study is that insurance-specific requirements will be associated with a decreased progression to surgery and increased delay in time to surgery. METHODS: Retrospective data collection was performed for patients undergoing bariatric surgery evaluation from 2010-2015. Patients who underwent surgery (SGY; n = 827; mean body mass index [BMI] 49.1) were compared with those who did not (no-SGY; n = 648; mean BMI: 49.4). Univariate and multivariate analysis were performed to identify specific co-morbidity and insurance specific predictors of surgical dropout and time to surgery. RESULTS: A total of 1475 patients using 12 major insurance payors were included. Univariate analysis found insurance requirements associated with surgical drop out included longer median diet duration (no-SGY = 6 mo; SGY = 3 mo; P<.001); primary care physician letter of necessity (P<.0001); laboratory testing (P = .019); and evaluation by cardiology (P<.001), pulmonology (P<.0001), or psychiatry (P = .0003). Using logistic regression to control for co-morbidities, longer diet requirement (odds ratio [OR] .88, P<.0001), primary care physician letter (OR .33, P<.0001), cardiology evaluation (OR .22, P = .038), and advanced laboratory testing (OR 5.75, P = .019) independently predicted surgery dropout. Additionally, surgical patients had an average interval between initial visit and surgery of 5.8±4.6 months with significant weight gain (2.1 kg, P<.0001). CONCLUSION: Many prebariatric surgery insurance requirements were associated with lack of patient progression to surgery in this study. In addition, delays in surgery were associated with preoperative weight gain. Although prospective and multicenter studies are needed, these findings have major policy implications suggesting insurance requirements may need to be reconsidered to improve medical care.

Postoperative Hypoglycemia Is Associated With Worse Outcomes After Cardiac Operations.

BACKGROUND: Hypoglycemia is a known risk of intensive postoperative glucose control in patients undergoing cardiac operations. However, neither the consequences of hypoglycemia relative to hyperglycemia, nor the possible interaction effects, have been well described. We examined the effects of postoperative hypoglycemia, hyperglycemia, and their interaction on short-term morbidity and mortality. METHODS: Single-institution Society of Thoracic Surgeons (STS) database patient records from 2010 to 2014 were merged with clinical data, including blood glucose values measured in the intensive care unit (ICU). Exclusion criteria included fewer than three glucose measurements and absence of an STS predicted risk of morbidity or mortality score. Primary outcomes were operative mortality and composite major morbidity (permanent stroke, renal failure, prolonged ventilation, pneumonia, or myocardial infarction). Secondary outcomes included ICU and postoperative length of stay. Hypoglycemia was defined as below 70 mg/dL, and hyperglycemia as above 180 mg/dL. Simple and multivariable regression models were used to evaluate the outcomes. RESULTS: A total of 2,285 patient records met the selection criteria for analysis. The mean postoperative glucose level was 140.8 ± 18.8 mg/dL. Overall, 21.4% of patients experienced a hypoglycemic episode (n = 488), and 1.05% (n = 24) had a severe hypoglycemic episode (<40 mg/dL). The unadjusted odds ratio (UOR) for operative mortality for patients with any hypoglycemic episode compared with those without was 5.47 (95% confidence interval [CI] 3.14 to 9.54), and the UOR for major morbidity was 4.66 (95% CI 3.55 to 6.11). After adjustment for predicted risk of morbidity or mortality and other significant covariates, the adjusted odds (AOR) of operative mortality were significant for patients with any hypoglycemia (AOR 4.88, 95% CI 2.67 to 8.92) and patients with both events (AOR 8.29, 95% CI 1.83 to 37.5) but not hyperglycemia alone (AOR 1.62, 95% CI 0.56 to 4.69). The AOR of major morbidity for patients with both hypoglycemic and hyperglycemic events was 14.3 (95% CI 6.50 to 31.4). CONCLUSIONS: Postoperative hypoglycemia is associated with both mortality and major morbidity after cardiac operations. The combination of both hyperglycemia and hypoglycemia represents a substantial increase in risk. Although it remains unclear whether hypoglycemia is a cause, an early warning sign, or a result of adverse events, this study suggests that hypoglycemia may be an important event in the postoperative period after cardiac operations.

Population-Specific Models of Glycemic Control in Intensive Care: Towards a Simulation-Based Methodology for Protocol Optimization.

Stress-induced hyperglycemia is common in critically ill patients, where elevated blood glucose and glycemic variability have been found to contribute to infection, slow wound healing, and short-term mortality. Early clinical studies demonstrated improvement in mortality and morbidity resulting from intensive insulin therapy targeting euglycemia. Follow-up clinical studies have shown mixed results suggesting that the risk of hypoglycemia may outweigh the benefits of aggressive glycemic control. None of the prior studies clarify whether euglycemic targets are in themselves harmful, or if the danger lies in the inadequacy of the available methods for achieving desired glycemic outcomes. In this paper, we use a recently developed simulation model of stress hyperglycemia to demonstrate that given an insulin protocol glycemic outcomes are specific to the patient population under consideration, and that there is a need to optimize insulin therapy at the population level. Next, we use the simulator to demonstrate that the performance of Adaptive Proportional Feedback (APF), a popular format for computerized insulin therapy, is sensitive to its parameters, especially to the parameters that govern the aggressiveness of adaptation. Finally, we propose a framework for simulation-based protocol optimization using an objective function that penalizes below-range deviations more heavily than comparable deviations above.

Hypoxia-inducible factor-1alpha is constitutively expressed in murine Leydig cells and regulates 3beta-hydroxysteroid dehydrogenase type 1 promoter activity.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that plays an essential role in oxygen homeostasis. HIF-1alpha is constitutively made in cells; however, it is ubiquitinated and degraded under normoxic conditions. Hypoxia prevents the ubiquitination of HIF-1alpha, resulting in stabilization of the protein and activation of target genes. Because of its vascular arrangement and the high metabolic demand of spermatogenesis, the testis has been described previously as functioning on the brink of hypoxia; thus, we have hypothesized that HIF-1alpha is constitutively expressed and stabilized in the testis, where it could play a role in testicular homeostasis. Western blot analysis using nuclear proteins from liver, kidney, and testis revealed the presence of HIF-1alpha only in the testis. Immunohistochemistry confirmed this result and revealed that HIF-1alpha was specifically located in interstitial Leydig cells. Electromobility shift assays employing nuclear extracts from the TM3 Leydig cell line revealed that these cells express HIF-1alpha that is capable of binding DNA under normoxic conditions. Furthermore, we found that protein levels can be increased further when the TM3 cells are cultured under hypoxic conditions. Finally, transient transfections of TM3 Leydig cells revealed that the promoter of the mouse 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1) gene, which encodes a key enzyme in testosterone production, is a potential target of HIF-1alpha. In conclusion, HIF-1alpha is constitutively present in the Leydig cells of the murine testis, where it potentially regulates Hsd3b1 transcription, and thus male reproductive function.

Putative regulation of expression of members of the Ets variant 4 transcription factor family and their downstream targets in the rat epididymis.

Several genes expressed in the initial segment of the epididymis depend on factors from the testis that reach the epididymis via the luminal system. These include gamma-glutamyl transpeptidase mRNA IV (Ggt_pr4), steroid 5 alpha reductase (Srd5a1), glutathione peroxidase 5 (Gpx5), and cystatin-related epididymal spermatogenic (Cst8) genes. Promoter analyses indicated that these genes contain several ETS DNA-binding sites. Members of the polyomavirus enhancer activator 3 (ETV4) family bind to ETS sites on the promoter of target genes to regulate transcription. In this study, the role of ETV4 family members (ETV4, ETV5, ETV1) in the transcription of initial segment specific genes was evaluated. All three ETV4 family mRNAs are expressed in the principal cells of the initial segment and depend upon the presence of testicular luminal fluid factors. ETV4 protein was localized to principal cell nuclei and displayed the highest expression in the most proximal region of the initial segment. In addition, ETV4 protein levels were diminished after loss of testicular luminal fluid factors. A dominant-negative construct of ETV5 was in vivo electroporated into the initial segment to determine if ETV4 family members can regulate the transcription of testicular luminal fluid factor-regulated genes. Quantitative PCR indicated that 1 day postelectroporation, all three ETV4 family member mRNAs were significantly decreased. In addition, Ggt_pr4, Srd5a1, and Gpx5 mRNA levels were also significantly decreased. The data suggest that ETV4 family members regulate their own expression, and that they regulate transcription of a subset of genes that are dependent upon testicular luminal fluid factors.

Ischemia-reperfusion of the murine testis stimulates the expression of proinflammatory cytokines and activation of c-jun N-terminal kinase in a pathway to E-selectin expression.

Ischemia-reperfusion (IR) of the testis results in germ cell-specific apoptosis and can lead to aspermatogenesis. Germ cell-specific apoptosis after IR of the testis has been shown to be correlated with and dependent on neutrophil recruitment to the testis after IR. Studies that used E-selectin-deficient mice have demonstrated that E-selectin expression is critical for neutrophil recruitment to subtunical venules in the testis after IR and for the resultant germ cell-specific apoptosis. The present study investigates the in vivo signaling pathway that exists after IR that leads to neutrophil recruitment in the murine testis. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. Results demonstrate that the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta), are stimulated after IR as is the phosphorylation of c-jun N-terminal kinase (JNK). The downstream transcription factors of JNK, ATF-2 and c-jun are also phosphorylated at specific times after IR of the testis. Activation of the JNK stress-related kinase pathway is correlated with an increase in E-selectin expression and neutrophil recruitment to the testis after IR. Intratesticular injection of IL-1beta also caused JNK phosphorylation and neutrophil recruitment to the testis. These results suggest that testicular IR injury stimulates IL-1beta expression, which leads to activation of the JNK signaling pathway and ultimately E-selectin expression and neutrophil recruitment to the testis. This provides the first evidence of a cytokine/stress-related kinase signaling pathway to E-selectin expression in vivo.

Characterization of fibroblast growth factor receptors expressed in principal cells in the initial segment of the rat epididymis.

Studies from our laboratory support a model in which growth factors produced in the testis reach the epididymis via the luminal system and play an important role in maintaining the function of epithelial cells, particularly in the initial segment. Previous work showed that gamma-glutamyl transpeptidase (GGT) mRNA IV, which is highly expressed in the rat initial segment, may be under the control of luminal fibroblast growth factor 2 (FGF-2) from the testis. The current studies were undertaken to identify which fibroblast growth factor receptors (FGFRs) are present in the principal cells of the rat initial segment and to identify other potential ligands for these receptors in rat rete testis fluid (RTF). Immunoblot analysis revealed that FGFRs 1-4 were present, and reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that both the IIIb and IIIc splice variants of FGFRs 1-3 were expressed. However, RT-PCR using RNA isolated from principal cells collected by laser capture microdissection revealed only FGFR-1 IIIc. Additional PCR analysis established that both the alpha and beta forms of FGFR-1 IIIc were expressed in principal cells. Both FGF-4 and FGF-8 were present in rat RTF, as determined by immunoblotting. Thus, FGF-2, -4, and -8, found in RTF, may act upon FGFR-1 IIIc in the principal cells of the initial segment to regulate GGT mRNA IV expression.

Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation.

The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and gamma-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between -530 and -681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.