A concise review of glycerol derivatives for use as fuel additives.

Due to renewable fuel mandates worldwide, the increase in biodiesel production has caused an oversupply of low-cost glycerol on the markets, which can negatively affect the sustainability of the biodiesel industry as a whole. In order to avoid that scenario, the transformation of glycerol into value-added products has been investigated, and the production of additives for internal combustion engine fuels is one good example of glycerol valorization. The present work presents a summary of the literature describing the most important chemical pathways through which glycerol can be converted into fuel additives, to be subsequently mixed with either gasoline, biodiesel, or diesel fuel. The focus is on the three major categories, namely glycerol acetals/ketals, ethers, and esters (acetates). Moreover, the effectiveness of the different glycerol-derived compounds is illustrated through several examples from the literature. Finally, a few research gaps on the topic are identified and suggestions for future work are described.

Soil texture and climate limit cultivation of the arsenic hyperaccumulator Pteris vittata for phytoextraction in a long-term field study.

Long term field studies are required to bridge gaps between research and practical application of arsenic phytoextraction with the arsenic-hyperaccumulating fern Pteris vittata. In a 4-year field study, we investigated the effects of nutrient application (compost, inorganic or organic nitrogen, inorganic or organic phosphorus) and soil texture (13 % and 35 % clay) on arsenic phytoextraction with P. vittata in moderately contaminated soils (74-79 mg As/kg in the 0-15 cm depth interval). We found the highest phytoextraction rates, 5 ± 1 kg As/ha/y, in a coarse-textured compost-amended soil after 2 years of phytoextraction. Phytoextraction rates decreased over time, likely due to decreased root growth in mature stands, indicating plants should be replaced every 2-3 years to maintain phytoextraction efficiency. Across soil textures, nitrogen or phosphorus application led to a 60 % decrease in mean frond arsenic concentrations, leading to mean phytoextraction rates 54 % lower than in control ferns. In the fine-textured soil, frond arsenic concentrations were 54 % lower than in the coarse-textured soil, and fewer ferns survived from year 3 to 4. Across soil textures, compost application increased fern survival. We show that phytoextraction with P. vittata is limited to specific soil and climate conditions, narrower than those under which P. vittata grows in the wild.

Umbilical cord blood graft engineering: challenges and opportunities.

We are entering a very exciting era in umbilical cord blood transplantation (UCBT), where many of the associated formidable challenges may become treatable by ex vivo graft manipulation and/or adoptive immunotherapy utilizing specific cellular products. We envisage the use of double UCBT rather than single UCBT for most patients; this allows for greater ability to treat larger patients as well as to manipulate the graft. Ex vivo expansion and/or fucosylation of one cord will achieve more rapid engraftment, minimize the period of neutropenia and also give certainty that the other cord will provide long-term engraftment/immune reconstitution. The non-expanded (and future dominant) cord could be chosen for characteristics such as better HLA matching to minimize GvHD, or larger cell counts to enable part of the unit to be utilized for the development of specific cellular therapies such as the production of virus-specific T-cells or chimeric-antigen receptor T-cells which are reviewed in this study.

Clinical trials and tribulations--lessons from pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a dreadful disease that lacks adequate therapy. A number of treatment trials have been performed and have utilized a variety of primary efficacy endpoints. Endpoints that provide the most useful efficacy information are clinical endpoints that are directly related to how a patient feels, functions or survives. Unfortunately, there are no properly established patient-reported outcome measures or measures of functional status in IPF, making survival the most robust primary efficacy endpoint. Clinically meaningful events such as hospitalization can also provide important efficacy information. The use of non-validated surrogate endpoints as primary outcome measures often leads to uncertainty when interpreting trial results.

Pulmonary fibrosis is associated with an elevated risk of thromboembolic disease.

Recent epidemiological studies have suggested an increased risk of venous thromboembolism (VTE) in lung fibrosis. Large-scale epidemiological data regarding the risk of VTE in pulmonary fibrosis-associated mortality have not been published. Using data from the National Center for Health Statistics from 1988-2007, we determined the risk of VTE in decedents with pulmonary fibrosis in the USA. We analysed 46,450,489 records, of which 218,991 met our criteria for idiopathic pulmonary fibrosis. Among these, 3,815 (1.74%) records also contained a diagnostic code for VTE. The risk of VTE in pulmonary fibrosis decedents was 34% higher than in the background population, and 44% and 54% greater than among decedents with chronic obstructive pulmonary disease and lung cancer, respectively. Those with VTE and pulmonary fibrosis died at a younger age than those with pulmonary fibrosis alone (females: 74.3 versus 77.4 yrs (p<0.0001); males: 72.0 versus 74.4 yrs (p<0.0001)). Decedents with pulmonary fibrosis had a significantly greater risk of VTE. Those with VTE and pulmonary fibrosis died at a younger age than those with pulmonary fibrosis alone. These data suggest a link between a pro-fibrotic and a pro-coagulant state.

Differential glucose uptake in retina- and brain-derived endothelial cells.

Microangiopathy is a systemic complication of diabetes that is especially severe in the retinal microcirculation. The objective of this study was to compare glucose uptake and glucose transporter expression between retinal endothelial cells and the closely related endothelial cells derived from the cerebral microcirculation. Endothelial cells isolated from bovine brain, bovine retinal, and rat heart microvessels were cultured in the presence of control (5 mM) and high levels of (30 mM) d-glucose for 1-5 days. Glucose uptake by cultured endothelial cells was determined by measuring the uptake of [(3)H]deoxy-d-glucose and glucose transporter protein expression was assessed by Western blot. Our results showed that glucose uptake was significantly (P < 0.001) higher in brain- and heart-derived endothelial cells than in retinal endothelial cells at both physiologic and high concentrations of glucose. High levels of glucose caused a significant (P < 0.05) decrease in glucose uptake in brain-derived and heart endothelial cells but had no effect on retinal endothelial cells. Similarly, in response to high glucose levels there was a significant (P < 0.01) down regulation of GLUT-1 in brain-derived endothelial cells but not in retinal endothelial cells. These results suggest that despite a low basal level of glucose uptake the inability of retinal endothelial cells to down regulate glucose uptake in the presence of high glucose levels could make them especially sensitive to the deleterious effects of hyperglycemia in diabetes.

Regulation of muscle GLUT-4 transcription by AMP-activated protein kinase.

Skeletal muscle GLUT-4 transcription in response to treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMP-activated protein kinase (AMPK), was studied in rats and mice. The increase in GLUT-4 mRNA levels in response to a single subcutaneous injection of AICAR, peaked at 13 h in white and red quadriceps muscles but not in the soleus muscle. The mRNA level of chloramphenicol acyltransferase reporter gene which is driven by 1,154 or 895 bp of the human GLUT-4 proximal promoter was increased in AICAR-treated transgenic mice, demonstrating the transcriptional upregulation of the GLUT-4 gene by AICAR. However, this induction of transcription was not apparent with 730 bp of the promoter. In addition, nuclear extracts from AICAR-treated mice bound to the consensus sequence of myocyte enhancer factor-2 (from -473 to -464) to a greater extent than from saline-injected mice. Thus AMP-activated protein kinase activation by AICAR increases GLUT-4 transcription by a mechanism that requires response elements within 895 bp of human GLUT-4 proximal promoter and that may be cooperatively mediated by myocyte enhancer factor-2.

Identifying physicians likely to benefit from depression education: a challenge for health care organizations.

BACKGROUND: Few methods exist to identify physicians who might benefit from depression education. OBJECTIVES: To develop a measure of physicians' confidence or self-efficacy in caring for depressed patients and assess it's reliability and validity. RESEARCH DESIGN: A national sample of primary care physicians were surveyed and exploratory factor analysis (EFA) was used to identify factors underlying physicians' responses to 26 items. We named the factors, selected items with factor loadings > or = 0.50 for final scales, and tested a priori hypotheses about self-efficacy. SUBJECTS: 1) Random cross-sectional sample of family physicians, internists, obstetrician-gynecologists, and pediatricians (n = 5,369) and 2) 49 general internists and family physicians participating in a prepost evaluation of a depression workshop. RESULTS: In the national sample, 3,712 physicians were eligible and 2,104 responded. Forty-six percent were female, and 51% were family physicians and general internists. EFA identified 5 factors, the first of which was called Self-Efficacy (4 items, alpha = 0.86). More family physicians (64%) had confidence (self-efficacy) in caring for depressed patients compared with general internists (33%), obstetrician-gynecologists (16%), and pediatricians (6%) (P < 0.001). Few physicians intended to change their care of depressed patients (10%) or take CME on depression (24%). Of the 49 physicians attending a depression workshop, 76% reported high self-efficacy after the workshop versus 50% before it (P = 0.013). CONCLUSIONS: This study supports the reliability and validity of the Self-Efficacy scale as one method to identify physicians who might benefit from interventions. New approaches are needed because physicians are unlikely to change.

Insulin-mediated GLUT4 translocation is dependent on the microtubule network.

The GLUT4 facilitative glucose transporter is recruited to the plasma membrane by insulin. This process depends primarily on the exocytosis of a specialized pool of vesicles containing GLUT4 in their membranes. The mechanism of GLUT4 vesicle exocytosis in response to insulin is not understood. To determine whether GLUT4 exocytosis is dependent on intact microtubule network, we measured insulin-mediated GLUT4 exocytosis in 3T3-L1 adipocytes in which the microtubule network was depolymerized by pretreatment with nocodazole. Insulin-mediated GLUT4 translocation was inhibited by more than 80% in nocodazole-treated cells. Phosphorylation of insulin receptor substrate 1 (IRS-1), activation of IRS-1 associated phosphatidylinositide 3-kinase, and phosphorylation of protein kinase B/Akt-1 were not inhibited by nocodazole treatment indicating that the microtubule network was not required for proximal insulin signaling. An intact microtubule network is specifically required for insulin-mediated GLUT4 translocation since nocodazole treatment did not affect insulin-mediated GLUT1 translocation or adipsin secretion. By using in vitro microtubule binding, we demonstrated that both GLUT4 vesicles and IRS-1 bind specifically to microtubules, implicating microtubules in both insulin signaling and GLUT4 translocation. Vesicle binding to microtubules was not mediated through direct binding of GLUT4 or insulin-responsive aminopeptidase to microtubules. A model microtubule-dependent translocation of GLUT4 is proposed.

Primary care pediatricians' roles and perceived responsibilities in the identification and management of depression in children and adolescents.

OBJECTIVE: To describe primary care pediatricians' 1) approach to the identification and management of childhood and adolescent depression and 2) perception of their skills, responsibilities, and barriers in recognizing and managing depression in children and adolescents. DESIGN AND METHODS: National cross-sectional survey of randomly selected primary care pediatricians that assessed the management of recalled last case of child or adolescent depression, attitudes, limitations to care from barriers and skills, and willingness to implement new educational or intervention strategies to improve care. RESULTS: There were 280 completed surveys about child and adolescent depression (63% response rate). Pediatricians overwhelmingly reported it was their responsibility to recognize depression in both children and adolescents (90%) but were unlikely to feel responsible for treating children or adolescents (26%-27%). Those with most of their practice in capitated managed care were less likely to feel responsible for recognizing depression in either children or adolescents. Forty-six percent of pediatricians lacked confidence in their skills to recognize depression in children, and few of them (10%-14%) had confidence in their skills in different aspects of treatment with children or adolescents. Diagnostic, assessment, and management details for their last recalled case of depression in a child or adolescent were provided by 248 of these pediatricians. In addition to referring 78%-79% of the cases to mental health care professionals, 77% of pediatricians provided a wide range of brief interventions. Only 19%-20% prescribed medication. Major factors cited that limited their diagnosis or management were time (56%-68%) and training or knowledge of issues (38%-56%). Fewer pediatricians noted limitations due to insurer or financial issues (8%-39%) or patient issues (19%-31%). The 35% of pediatricians who were motivated to change their recognition and management of suspected depression were significantly more interested in implementing in the future a variety of new strategies to improve care. CONCLUSION: Primary care pediatricians felt responsible for recognizing but not for treating child and adolescent depression. Although the lack of confidence and lack of knowledge and/or skills and time issues are major barriers that limit pediatricians in their treatment of childhood and adolescent depression, pediatricians varied in their readiness to change, with some being more willing to implement new strategies to care for depression. Educational and practice interventions need to focus on how to assist all pediatricians in diagnosis and to prepare these motivated pediatricians to manage depression in primary care settings.

Persistent increase in children's sun protection in a randomized controlled community trial.

BACKGROUND: Reducing sun exposure during childhood may prevent skin cancer later in life. Sun protection increased immediately following implementation of the SunSafe multicomponent, community-based intervention delivered in 1996 through schools, day care centers, primary care offices, and beach recreation areas. Whether sun protection levels would remain higher than preintervention levels the following summer was unknown. METHODS: A randomized controlled trial based in 10 New Hampshire towns addressed children's use of protective clothing, shade, and sunscreen at freshwater beach areas. The intervention was provided initially between March and May 1996. A brief project follow-up contact was provided to schools, day care centers, beaches, and primary care offices between March and May 1997 to restock intervention materials and to answer questions. Observations of 1490 children during June through August of 1997 were compared with observations made prior to any intervention between June and August of 1995. RESULTS: In intervention towns, the proportion of children using at least some sun protection increased by 0.15 from 0.58 in 1995 to 0.73 in 1997 while the proportion in control towns increased by 0.03 (P = 0.033). This increase was due to more use of sunscreen, but not more use of protective clothing or shade. In 1997, care-givers of children in intervention towns reported receiving more sun protection information from school and health care sources than control town caregivers (62% versus 33%, P < 0.006). CONCLUSIONS: In intervention communities, a higher proportion of children used sun protection in 1997 than at baseline. Increases from 1995 to 1997 were similar in magnitude to short-term increases between 1995 and 1996 that we have been previously reported.

A national general pediatric clerkship curriculum: the process of development and implementation.

OBJECTIVE: To describe a new national general pediatrics clerkship curriculum, the development process that built national support for its use, and current progress in implementing the curriculum in pediatric clerkships at US allopathic medical schools. CURRICULUM DEVELOPMENT: A curriculum project team of pediatric clerkship directors and an advisory committee representing professional organizations invested in pediatric student education developed the format and content in collaboration with pediatric educators from the Council on Medical Student Education in Pediatrics (COMSEP) and the Ambulatory Pediatric Association (APA). An iterative process or review by clerkship directors, pediatric departmental chairs, and students finalized the content and built support for the final product. The national dissemination process resulted in consensus among pediatric educators that this curriculum should be used as the national curricular guideline for clerkships. MONITORING IMPLEMENTATION: Surveys were mailed to all pediatric clerkship directors before dissemination (November 1994), and in the first and third academic years after national dissemination (March 1996 and September 1997). The 3 surveys assessed schools' implementation of specific components of the curriculum. The final survey also assessed ways the curriculum was used and barriers to implementation. OUTCOMES: The final curriculum provided objectives and competencies for attitudes, skills, and 18 knowledge areas of general pediatrics. A total of 216 short clinical cases were also provided as an alternative learning method. An accompanying resource manual provided suggested strategies for implementation, teaching, and evaluation. A total of 103 schools responded to survey 1; 84 schools to survey 2; and 85 schools responded to survey 3 from the 125 medical schools surveyed. Before dissemination, 16% of schools were already using the clinical cases. In the 1995-1996 academic year, 70% of schools were using some or all of the curricular objectives/competencies, and 45% were using the clinical cases. Two years later, 90% of schools surveyed were using the curricular objectives, 88% were using the competencies, 66% were using the clinical cases. The extent of curriculum use also increased. Schools using 11 or more of the 18 curriculum's knowledge areas increased from 50% (1995-1996) to 73% (1996-1997). CONCLUSION: This new national general pediatric clerkship curriculum developed broad support during its development and has been implemented very rapidly nationwide. During this period the COMSEP and the APA have strongly supported its implementation with a variety of activities. This development and implementation process can be a model for other national curricula.

Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice.

We have previously demonstrated that the important cis-acting elements regulating transcription of the human GLUT4 gene reside within 895 base pairs (bp) upstream of the transcription initiation site (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998) J. Biol. Chem. 273, 14285-14292). Our studies demonstrated that an MEF2 binding site within this region was necessary, but not sufficient, for GLUT4 promoter function in transgenic mice. We have identified a second regulatory element (Domain I) that functions cooperatively with the MEF2 domain in regulating GLUT4 transcription. Using a yeast-one hybrid screen, we obtained a partial cDNA and generated an antibody directed against a protein binding specifically to Domain I. Sequence analysis of the partial cDNA indicates that the protein binding to Domain I is a novel protein. The antibody specifically labels two proteins of approximately 70 and 50 kDa in Western blot analysis. These molecular masses correspond to Domain I binding proteins identified by UV-cross-linking nuclear extracts to a Domain I probe. The antibody raised against the Domain I binding protein inhibited formation of a Domain I-protein complex in electrophoretic mobility shift assays. We conclude that we have identified an authentic, novel, Domain I binding protein required for transcriptional regulation of the human GLUT4 promoter.

Sun protection counseling for children: primary care practice patterns and effect of an intervention on clinicians.

OBJECTIVES: To describe current primary care sun protection advice for children and assess the effect on clinicians of an intervention to enhance their sun protection advocacy. SETTING: Primary care practices caring for children in New Hampshire with special attention to clinicians serving 10 towns that were involved in a randomized controlled trial of the multicomponent SunSafe intervention involving schools, recreation areas, and primary care practices. DESIGN/INTERVENTION: A statewide survey of all primary care clinicians serving children addressed their self-reported sun protection advocacy practices. Clinicians in 10 systematically selected rural towns were involved in the subsequent intervention study. The primary care intervention provided assistance to practices in establishing an office system that promoted sun protection advice to children and their parents during office visits. MAIN OUTCOME MEASURES: Sun protection promotion activities of primary care clinicians as determined by their self report, research assistant observation, and parent interviews. RESULTS: Of 261 eligible clinicians responding to the statewide survey, about half provide sun protection counseling "most of the time" or "almost always" during summer well care visits. Pediatricians do so more often than family physicians. Clinicians involved in the intervention increased their use of handouts, waiting room educational materials, and sunscreen samples. Compared with control town parents, parents in intervention towns reported an increase in clinician sun protection advice. CONCLUSIONS: The SunSafe primary care intervention increased sun protection counseling activities of participating clinicians. A single-focus preventive service office system is feasible to include in community interventions to promote sun protection.

Expression of a prenylation-deficient Rab4 interferes with propagation of insulin signaling through insulin receptor substrate-1.

Rab proteins are small GTP-binding proteins of the Ras superfamily that function in the regulation of vesicle transport processes. The Rab4 isoform has been implicated in insulin action. For instance, overexpression of a prenylation-deficient form of Rab4 has been shown to inhibit insulin-dependent GLUT4 translocation. Other steps affected by Rab4 in the cascade of events resulting from insulin receptor activation have not been elucidated. In the present studies, we measured effects on insulin-signaling proteins in 3T3-L1 adipocytes transiently expressing cytoplasmic forms of Rab4 and Rab5. Expression of a mutant Rab4 lacking a prenylation site resulted in reduced insulin-dependent phosphorylation ofcytoplasmic and internal membrane-associated insulin receptor substrate-1, leading to decreased insulin receptor substrate-1-associated phosphatidylinositol 3'-OH kinase activation and decreased Akt activation. These effects were not observed upon introduction of a similar mutant form of Rab5. These data indicate that Rab4 or a Rab4-associated protein is involved at one or more steps in propagating the insulin signal, in addition to any role it may play in the regulation of GLUT4 vesicle translocation. Our results support models of insulin signaling in which regulation of internal membrane trafficking plays a role in transduction of the insulin signal.

Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state.

We have previously reported that insulin and osmotic shock stimulate an increase in glucose transport activity and translocation of the insulin-responsive glucose transporter isoform GLUT4 to the plasma membrane through distinct pathways in 3T3L1 adipocytes (D. Chen, J. S. Elmendorf, A. L. Olson, X. Li, H. S. Earp, and J. E. Pessin, J. Biol. Chem. 272:27401-27410, 1997). In investigations of the relationships between these two signaling pathways, we have now observed that these two stimuli are not additive, and, in fact, osmotic shock pretreatment was found to completely prevent any further insulin stimulation of glucose transport activity and GLUT4 protein translocation. In addition, osmotic shock inhibited the insulin stimulation of lipogenesis and glycogen synthesis. This inhibition of insulin-stimulated downstream signaling occurred without any significant effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, there was no effect on either the insulin-stimulated association of the p85 type I phosphatidylinositol (PI) 3-kinase regulatory subunit with IRS1 or phosphotyrosine antibody-immunoprecipitated PI 3-kinase activity. In contrast, osmotic shock pretreatment markedly inhibited the insulin stimulation of protein kinase B (PKB) and p70S6 kinase activities. In addition, the dephosphorylation of PKB was prevented by pretreatment with the phosphatase inhibitors okadaic acid and calyculin A. These data support a model in which osmotic shock-induced insulin resistance of downstream biological responses results from an inhibition of insulin-stimulated PKB activation.

A community-based randomized trial encouraging sun protection for children.

OBJECTIVE: We evaluated the impact of an intervention promoting sun protection behavior among children 2 to 11 years of age through schools and day care centers, primary care practices, and recreation areas. METHODS: Ten towns in New Hampshire were paired, then assigned randomly to intervention or control status. The multicomponent SunSafe intervention was provided to children and caregivers through primary care practices, day care centers, schools, and beach recreation areas. Training support and materials were provided by the SunSafe project, but project staff had no direct contact with children or parents in providing the intervention. All intervention components promoted the same message: avoid the sun between 11 AM and 3 PM, cover up using hats and protective clothing, use sun block with a sun protection factor >/=15, and encourage sun protection among family and friends. The impact of the intervention was determined by observing children's sun protection behavior at the beach during baseline compared with 1 year later. The primary outcomes of interest were changes in the proportion of children per town using at least some sun protection and changes in the proportion of children fully protected. Children were clustered by town, with the town thus being the unit of analysis. The primary care practice component included one practice meeting for clinicians and staff at which project staff presented background on skin cancer and how to promote its prevention; a sun protection office system manual based on our previous work, which provided specific direction on how to share responsibility among office staff and clinicians in carrying out routines that promote sun protection; and educational posters, pamphlets, and self-adhesive reminder notes designed to enhance sun protection counseling. SunSafe removable tattoos and stickers were offered to children at well-child and illness visits during the summer months. Schools each received three project staff visits: a brief visit with the principal to describe the intervention and to answer questions; an in-service program to educate teachers about skin cancer and to introduce curricular materials; and help with one parent outreach program. Larger day care centers each received one project staff visit. An additional six smaller day care centers received curricular materials through the mail but no visits. Two similar sets of curricular materials were used, one for grade schools and the other for preschools and day care centers. Both emphasized the importance of sun protection rather than the danger of skin cancer. Materials emphasized dynamic activities modeled after the "Slip, Slop, Slap" and "SunSmart" programs and included new material developed to suit regional needs. Both manuals offered structured plans but also provided a variety of activities from which teachers could choose. Teachers agreed to devote a minimum of two class periods to these materials. For recreation areas, lifeguards in each of the intervention communities attended an in-service meeting, during which background about skin cancer prevention was presented by project staff. The project also provided displays about the ultraviolet (UV) light index and about sun protection to be posted at each beach. Subsequently, project staff called beach staff in each community each morning with the predicted UV index for the day to post on the display. Educational pamphlets about the UV index and free sun-block samples were available to beachgoers through the lifeguards. One brief follow-up visit by project staff was made to each beach area to provide reinforcement. RESULTS: We observed 1930 children. Use of some sunscreen on at least one body area increased in all 5 intervention towns compared with paired control towns. In intervention towns, this mean proportion increased from 0.56 of those observed at baseline to 0.76 of those observed postintervention, with a minimal increase among control town children. (ABSTRACT TRUNCATED

Myocyte enhancer factor 2 (MEF2)-binding site is required for GLUT4 gene expression in transgenic mice. Regulation of MEF2 DNA binding activity in insulin-deficient diabetes.

We have previously demonstrated that important regulatory elements responsible for regulated expression of the human GLUT4 promoter are located between -1154 and -412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491-23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription rate of the GLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue.