How Does a Junior Faculty Development Program Affect Burnout? A Mixed Methods Assessment.

OBJECTIVES: Burnout is common among junior faculty. Professional development has been proposed as a method to improve engagement and reduce burnout among academic physicians. The Penn State College of Medicine Junior Faculty Development Program (JFDP) is a well-established, interdisciplinary program. However, an increase in burnout was noted among participants during the program. The authors sought to quantify the change in burnout seen among JFDP participants across 3 cohorts, and to explore sources of well-being and burnout among participants. METHODS: Through a sequential explanatory mixed methods approach, participants in the 2018/19, 2019/20, and 2020/21 cohorts took a survey assessing burnout (Copenhagen Burnout Inventory), quality of life (QoL), job satisfaction, and work-home conflict at the start and end of the course. Descriptive statistics were generated as well as Pearson χ2 test/Fisher exact test for categorical variables and Wilcoxon rank sum tests for continuous variables for group comparisons. To better understand the outcome, past participants were invited to interviews regarding their experience of burnout during the course. Inductive thematic analysis (kappa = 0.86) was used to derive themes. RESULTS: Start- and end-of-course surveys were completed by 84 and 75 participants, respectively (response rates: 95.5% and 85.2%). Burnout associated with patient/learner/client/colleague increased (P = .005) and QoL decreased (P = .02) at the end compared with the start. Nonsignificant trends toward worsening in other burnout categories, work-home conflict, and job satisfaction were also observed. Nineteen interviews yielded themes related to risks and protective factors for burnout including competing demands, benefits of networking, professional growth, and challenges related to diverse faculty roles. CONCLUSION: Junior Faculty Development Program participants demonstrated worsening of burnout and QoL during the program while benefiting from opportunities including skill building and networking. The impact of Junior Faculty Development Programs on the well-being of participants should be considered as an element of their design, evaluation, and refinement over time.

Podocyte-specific chemokine (C-C motif) receptor 2 overexpression mediates diabetic renal injury in mice.

Inflammation is a central pathophysiologic mechanism that contributes to diabetes mellitus and diabetic nephropathy. Recently, we showed that macrophages directly contribute to diabetic renal injury and that pharmacological blockade or genetic deficiency of chemokine (C-C motif) receptor 2 (CCR2) confers kidney protection in diabetic nephropathy. However, the direct role of CCR2 in kidney-derived cells such as podocytes in diabetic nephropathy remains unclear. To study this, we developed a transgenic mouse model expressing CCR2 specifically in podocytes (Tg[NPHS2-Ccr2]) on a nephropathy-prone (DBA/2J) and CCR2-deficient (Ccr2-/-) background with heterozygous Ccr2+/- littermate controls. Diabetes was induced by streptozotocin. As expected, absence of CCR2 conferred kidney protection after nine weeks of diabetes. In contrast, transgenic CCR2 overexpression in the podocytes of Ccr2-/- mice resulted in significantly increased albuminuria, blood urea nitrogen, histopathologic changes, kidney fibronectin and type 1 collagen expression, podocyte loss, and glomerular apoptosis after nine weeks of streptozotocin-induced diabetes. Interestingly, there was no concurrent increase in kidney macrophage recruitment or inflammatory cytokine levels in the mice. These findings support a direct role for CCR2 expression in podocytes to mediate diabetic renal injury, independent of monocyte/macrophage recruitment. Thus, targeting the CCR2 signaling cascade in podocytes could be a novel therapeutic approach for treatment of diabetic nephropathy.

Insulin treatment normalizes retinal neuroinflammation but not markers of synapse loss in diabetic rats.

Diabetic retinopathy is one of the leading causes of blindness in developed countries, and a majority of patients with type I and type II diabetes will develop some degree of vision loss despite blood glucose control regimens. The effects of different insulin therapy regimens on early metabolic, inflammatory and neuronal retinal disease processes such as retinal neuroinflammation and synapse loss have not been extensively investigated. This study compared 3 months non-diabetic and streptozotocin (STZ)-induced diabetic Sprague Dawley rats. Diabetic rats received either no insulin treatment, systemic insulin treatment beginning after 1 week uncontrolled diabetes (early intervention, 11 weeks on insulin), or after 1.5 months uncontrolled diabetes (late intervention, 6 weeks on insulin). Changes in both whole animal metabolic and retinal inflammatory markers were prevented by early initiation of insulin treatment. These metabolic and inflammatory changes were also normalized by the later insulin intervention. Insulin treatment begun 1 week after diabetes induction ameliorated loss of retinal synapse markers. Synapse markers and presumably synapse numbers were equivalent in uncontrolled diabetes and when insulin treatment began at 1.5 months of diabetes. These findings are in agreement with previous demonstrations that retinal synapses are lost within 1 month of uncontrolled diabetes and suggest that synapses are not regained with glycemic control and restoration of insulin signaling. However, increased expression of metabolic and inflammatory markers associated with diabetes was reversed in both groups of insulin treatment. This study also emphasizes the need for insulin treatment groups in diabetic retinopathy studies to provide a more faithful modeling of the human condition.

Nanoliposomal minocycline for ocular drug delivery.

Nanoliposomal technology is a promising drug delivery system that could be employed to improve the pharmacokinetic properties of clearance and distribution in ocular drug delivery to the retina. We developed a nanoscale version of an anionic, cholesterol-fusing liposome that can encapsulate therapeutic levels of minocycline capable of drug delivery. We demonstrate that size extrusion followed by size-exclusion chromatography can form a stable 80-nm liposome that encapsulates minocycline at a concentration of 450 ± 30 µM, which is 2% to 3% of loading material. More importantly, these nontoxic nanoliposomes can then deliver 40% of encapsulated minocycline to the retina after a subconjunctival injection in the STZ model of diabetes. Efficacy of therapeutic drug delivery was assessed via transcriptomic and proteomic biomarker panels. For both the free minocycline and encapsulated minocycline treatments, proinflammatory markers of diabetes were downregulated at both the messenger RNA and protein levels, validating the utility of biomarker panels for the assessment of ocular drug delivery vehicles. FROM THE CLINICAL EDITOR: Authors developed a nano-liposome that can encapsulate minocycline for optimized intraocular drug delivery. These nontoxic nanoliposomes delivered 40% of encapsulated minocycline to the retina after a subconjunctival injection in a diabetes model.

Hyperglycemia-induced O-GlcNAcylation and truncation of 4E-BP1 protein in liver of a mouse model of type 1 diabetes.

4E-BP1 is a protein that, in its hypophosphorylated state, binds the mRNA cap-binding protein eIF4E and represses cap-dependent mRNA translation. By doing so, it plays a major role in the regulation of gene expression by controlling the overall rate of mRNA translation as well as the selection of mRNAs for translation. Phosphorylation of 4E-BP1 causes it to release eIF4E to function in mRNA translation. 4E-BP1 is also subject to covalent addition of N-acetylglucosamine to Ser or Thr residues (O-GlcNAcylation) as well as to truncation. In the truncated form, it is both resistant to phosphorylation and able to bind eIF4E with high affinity. In the present study, Ins2(Akita/+) diabetic mice were used to test the hypothesis that hyperglycemia and elevated flux of glucose through the hexosamine biosynthetic pathway lead to increased O-GlcNAcylation and truncation of 4E-BP1 and consequently decreased eIF4E function in the liver. The amounts of both full-length and truncated 4E-BP1 bound to eIF4E were significantly elevated in the liver of diabetic as compared with non-diabetic mice. In addition, O-GlcNAcylation of both the full-length and truncated proteins was elevated by 2.5- and 5-fold, respectively. Phlorizin treatment of diabetic mice lowered blood glucose concentrations and reduced the expression and O-GlcNAcylation of 4E-BP1. Additionally, when livers were perfused in the absence of insulin, 4E-BP1 phosphorylation in the livers of diabetic mice was normalized to the control value, yet O-GlcNAcylation and the association of 4E-BP1 with eIF4E remained elevated in the liver of diabetic mice. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes.

Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome.

BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia. METHODS: The retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point. RESULTS: Transcriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression. CONCLUSIONS: These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.

Multi-modal proteomic analysis of retinal protein expression alterations in a rat model of diabetic retinopathy.

BACKGROUND: As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies. METHODOLOGY/PRINCIPAL FINDINGS: A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated. CONCLUSIONS/SIGNIFICANCE: These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

Circulating sphingolipid biomarkers in models of type 1 diabetes.

Alterations in lipid metabolism may contribute to diabetic complications. Sphingolipids are essential components of cell membranes and have essential roles in homeostasis and in the initiation and progression of disease. However, the role of sphingolipids in type 1 diabetes remains largely unexplored. Therefore, we sought to quantify sphingolipid metabolites by LC-MS/MS from two animal models of type 1 diabetes (streptozotocin-induced diabetic rats and Ins2(Akita) diabetic mice) to identify putative therapeutic targets and biomarkers. The results reveal that sphingosine-1-phosphate (So1P) is elevated in both diabetic models in comparison to respective control animals. In addition, diabetic animals demonstrated reductions in plasma levels of omega-9 24:1 (nervonic acid)-containing ceramide, sphingomyelin, and cerebrosides. Reduction of 24:1-esterfied sphingolipids was also observed in liver and heart. Nutritional stress via a high-fat diet also reduced 24:1 content in the plasma and liver of mice, exacerbating the decrease in some cases where diabetes was also present. Subcutaneous insulin corrected both circulating So1P and 24:1 levels in the murine diabetic model. Thus, changes in circulating sphingolipids, as evidenced by an increase in bioactive So1P and a reduction in cardio- and neuro-protective omega-9 esterified sphingolipids, may serve as biomarkers for type 1 diabetes and represent novel therapeutic targets.

Skeletal muscle protein balance in mTOR heterozygous mice in response to inflammation and leucine.

Sepsis and lipopolysaccharide (LPS) may decrease skeletal muscle protein synthesis by impairing mTOR (mammalian target of rapamycin) activity. The role of mTOR in regulating muscle protein synthesis was assessed in wild-type (WT) and mTOR heterozygous (+/-) mice under basal conditions and in response to LPS and/or leucine stimulation. No difference in body weight of mTOR(+/-) mice was observed compared with WT mice; whereas whole body lean body mass was reduced. Gastrocnemius weight was decreased in mTOR(+/-) mice, which was attributable in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and mTOR(+/-) mice to the same extent. Reduced muscle protein synthesis in mTOR(+/-) mice under basal and LPS-stimulated conditions was associated with lower 4E-BP1 and S6K1 phosphorylation. LPS also decreased PRAS40 phosphorylation and increased phosphorylation of raptor and IRS-1 (Ser(307)) to the same extent in WT and mTOR(+/-) mice. Muscle atrogin-1 and MuRF1 mRNA content was elevated in mTOR(+/-) mice under basal conditions, implying increased ubiquitin-proteasome-mediated proteolysis, but the LPS-induced increase in these atrogenes was comparable between groups. Plasma insulin and IGF-I as well as tissue expression of TNFalpha, IL-6, or NOS2 did not differ between WT and mTOR(+/-) mice. Finally, whereas LPS impaired the ability of leucine to stimulate muscle protein synthesis and 4E-BP1 phosphorylation in WT mice, this inflammatory state rendered mTOR(+/-) mice leucine unresponsive. These data support the idea that the LPS-induced reduction in mTOR activity is relatively more important in regulating skeletal muscle mass in response to nutrient stimulation than under basal conditions.

Transcriptomic comparison of the retina in two mouse models of diabetes.

Mouse models of type I diabetes offer the potential to combine genetic approaches with other pharmacological or physiological manipulations to investigate the pathophysiology and treatment of diabetic retinopathy. Type I diabetes is induced in mice through chemical toxins or can arise spontaneously from genetic mutations. Both models are associated with retinal vascular and neuronal changes. Retinal transcriptomic responses in C57BL/6J mice treated with streptozotocin and Ins2(Akita/+) were compared after 3 months of hyperglycemia. Specific gene expression changes suggest a neurovascular inflammatory response in diabetic retinopathy. Genes common to the two models may represent the response of the retina to hyperglycemia, while changes unique to each model may represent time-dependent disease progression differences in the various models. Further investigation of the commonalities and differences between mouse models of type I diabetes may define cause and effect events in early diabetic retinopathy disease progression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9045-3) contains supplementary material, which is available to authorized users.

Dendrite remodeling and other abnormalities in the retinal ganglion cells of Ins2 Akita diabetic mice.

PURPOSE: To determine the extent of retinal ganglion cell loss and morphologic abnormalities in surviving ganglion cells in Ins2 Akita/+ diabetic mice. METHODS: Mice that expressed cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) reporter genes under the transcriptional control of the Thy1 promoter were crossed with Ins2 Akita/+ mice. After 3 months of diabetes, the number and morphology of retinal ganglion cells was analyzed by confocal microscopy. The number of CFP-positive retinal ganglion cells was quantified in retinas of Ins2(Akita/+) Thy1-CFP mice. The morphology of surviving cells was examined, and dendritic density was quantified in Ins2 Akita/+ Thy1-YFP mice by using the Sholl analysis. RESULTS: Thy1-CFP expression was limited to retinal ganglion cell bodies. There was a 16.4% reduction in the density of CFP-positive ganglion cells in the peripheral retina of Ins2 Akita/+ mice compared with wild-type control retinas (P < 0.017), but no significant change in the central retina. Thy1-YFP expression occurred throughout the entire structure of a smaller number of cells, including their soma, axons, and dendrites. Six different morphologic clusters of cells were identified in the mouse retinas. The structure of dendrites of ON-type retinal ganglion cells was affected by diabetes, having 32.4% more dendritic terminals (P < 0.05), 18.6% increase in total dendrite length (P < 0.05), and 15.3% greater dendritic density compared with control retinas, measured by Scholl analysis. Abnormal swelling on somas, axons, and dendrites were noted in all subtypes of ganglion cells including those expressing melanopsin. CONCLUSIONS: The data show that retinal ganglion cells are lost from the peripheral retina of mice within the first 3 months of diabetes and that the dendrites of surviving large ON-type cells undergo morphologic changes. These abnormalities may explain some of the early anomalies in visual function induced by diabetes.

Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response.

BACKGROUND: Despite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes. RESULTS: Using the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals. CONCLUSION: These changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.

Nonobese, insulin-deficient Ins2Akita mice develop type 2 diabetes phenotypes including insulin resistance and cardiac remodeling.

Although insulin resistance has been traditionally associated with type 2 diabetes, recent evidence in humans and animal models indicates that insulin resistance may also develop in type 1 diabetes. A point mutation of insulin 2 gene in Ins2(Akita) mice leads to pancreatic beta-cell apoptosis and hyperglycemia, and these mice are commonly used to investigate type 1 diabetes and complications. Since insulin resistance plays an important role in diabetic complications, we performed hyperinsulinemic-euglycemic clamps in awake Ins2(Akita) and wild-type mice to measure insulin action and glucose metabolism in vivo. Nonobese Ins2(Akita) mice developed insulin resistance, as indicated by an approximately 80% reduction in glucose infusion rate during clamps. Insulin resistance was due to approximately 50% decreases in glucose uptake in skeletal muscle and brown adipose tissue as well as hepatic insulin action. Skeletal muscle insulin resistance was associated with a 40% reduction in total GLUT4 and a threefold increase in PKCepsilon levels in Ins2(Akita) mice. Chronic phloridzin treatment lowered systemic glucose levels and normalized muscle insulin action, GLUT4 and PKCepsilon levels in Ins2(Akita) mice, indicating that hyperglycemia plays a role in insulin resistance. Echocardiography showed significant cardiac remodeling with ventricular hypertrophy that was ameliorated following chronic phloridzin treatment in Ins2(Akita) mice. Overall, we report for the first time that nonobese, insulin-deficient Ins2(Akita) mice develop type 2 diabetes phenotypes including peripheral and hepatic insulin resistance and cardiac remodeling. Our findings provide important insights into the pathogenesis of metabolic abnormalities and complications affecting type 1 diabetes and lean type 2 diabetes subjects.

Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle.

Leucine is recognized as a nutrient signal; however, the long-term in vivo consequences of leucine signaling and the role of branched-chain amino acid (BCAA) metabolism in this signaling remain unclear. To investigate these questions, we disrupted the BCATm gene, which encodes the enzyme catalyzing the first step in peripheral BCAA metabolism. BCATm(-/-) mice exhibited elevated plasma BCAAs and decreased adiposity and body weight, despite eating more food, along with increased energy expenditure, remarkable improvements in glucose and insulin tolerance, and protection from diet-induced obesity. The increased energy expenditure did not seem to be due to altered locomotor activity, uncoupling proteins, sympathetic activity, or thyroid hormones but was strongly associated with food consumption and an active futile cycle of increased protein degradation and synthesis. These observations suggest that elevated BCAAs and/or loss of BCAA catabolism in peripheral tissues play an important role in regulating insulin sensitivity and energy expenditure.

Artificial chromosome-based transgenes in the study of genome function.

The transfer of large DNA fragments to the mouse genome in the form of bacterial, yeast or phage artificial chromosomes is an important process in the definition of transcription units, the modeling of inherited disease states, the dissection of candidate regions identified by linkage analysis and the construction of in vivo reporter genes. However, as with small recombinant transgenes, the transferred sequences are usually integrated randomly often with accompanying genomic alterations and variable expression of the introduced genes due to the site of integration and/or copy number. Therefore, alternative methods of integrating large genomic transgenes into the genome have been developed to avoid the variables associated with random integration. This review encourages the reader to imagine the large variety of applications where artificial chromosome transgenes can facilitate in vivo and ex vivo studies in the mouse and provides a context for making the necessary decisions regarding the specifics of experimental design.

Diabetic retinopathy: seeing beyond glucose-induced microvascular disease.

Diabetic retinopathy remains a frightening prospect to patients and frustrates physicians. Destruction of damaged retina by photocoagulation remains the primary treatment nearly 50 years after its introduction. The diabetes pandemic requires new approaches to understand the pathophysiology and improve the detection, prevention, and treatment of retinopathy. This perspective considers how the unique anatomy and physiology of the retina may predispose it to the metabolic stresses of diabetes. The roles of neural retinal alterations and impaired retinal insulin action in the pathogenesis of early retinopathy and the mechanisms of vision loss are emphasized. Potential means to overcome limitations of current animal models and diagnostic testing are also presented with the goal of accelerating therapies to manage retinopathy in the face of ongoing diabetes.

Analysis of embryonic stem cell-derived osteogenic cultures.

The process of bone formation can be approximated in vitro in the form of a mineralized nodule. Osteoprogenitors and mesenchymal stem cells, the immediate precursors to the osteoprogenitor, when placed into culture proliferate and differentiate into osteoblasts. These osteoblasts secrete and mineralize a matrix during a period of 3-4 wk. The differentiation potential of embryonic stem cells (ESCs) suggests that ESCs should also have the ability to form bone nodules in vitro. ESCs were allowed to form embryoid bodies, which were disrupted and plated at concentrations as low as 25 cells/cm2. By 7 d postplating, a significant percentage of the colonies were morphologically characteristic of other types of osteogenic cultures. By 3 wk in culture, these colonies go on to form layered nodules. In a typical experiment, approx 60% of the colonies contain mineralized nodules, as revealed by staining of fixed cultures. Quantitative reverse transcriptase polymerase chain reaction analysis for genes characteristic of the osteoblast lineage has been used to confirm the presence of mature osteoblasts. Differentiation of ESCs into the osteoblast lineage will be a valuable tool for addressing pertinent questions about the proliferation, differentiation, survival, and intercellular communication between cells of the bone lineage in vitro.

Osteogenic nodule formation from single embryonic stem cell-derived progenitors.

The process of bone formation can be approximated in vitro in the form of a mineralized nodule. Osteoprogenitors and mesenchymal stem cells (MSCs), the immediate precursors of the osteoprogenitor, proliferate and differentiate into osteoblasts when placed into culture. These osteoblasts secrete and mineralize a matrix during a period of 3-4 weeks. The differentiation potential of embryonic stem (ES) cells suggests that ES cells should also have the ability to form osteogenic nodules in vitro. ES cells were allowed to form embryoid bodies (EBs) and were cultured in suspension for 2 days; EBs were disrupted and plated as single cells at concentrations as low as 25 cells/cm(2). We provide five lines of evidence for osteogenesis in these ES cell-derived cultures: (1) cell and colony morphology as revealed by phase-contrast microscopy, (2) mineralization of extracellular matrix as revealed by von Kossa staining, (3) quantitative real-time PCR (QRT-PCR) analysis of cDNA from entire plates and individual colonies revealing expression of genes characteristic of, and specific for, osteoblasts, (4) confocal microscopy of nodules from osteocalcin-green fluorescent protein (GFP) ES cell lines demonstrating the appropriate stage and position of osteoblasts expressing the reporter, and (5) immunostaining of nodules with a type I collagen antibody. Our method of initiating osteogenesis from ES cell-derived cultures is the only described method that allows for the observation and manipulation of the commitment stage of mesengenesis from single embryonic progenitors.

The Ins2Akita mouse as a model of early retinal complications in diabetes.

PURPOSE: This study tested the Ins2(Akita) mouse as an animal model of retinal complications in diabetes. The Ins2(Akita) mutation results in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein. The mutation arose and is maintained on the C57BL/6J background. Male mice heterozygous for this mutation have progressive loss of beta-cell function, decreased pancreatic beta-cell density, and significant hyperglycemia, as early as 4 weeks of age. METHODS: Heterozygous Ins2(Akita) mice were bred to C57BL/6J mice, and male offspring were monitored for hyperglycemia, beginning at 4.5 weeks of age. After 4 to 36 weeks of hyperglycemia, the retinas were analyzed for vascular permeability, vascular lesions, leukostasis, morphologic changes of micro- and macroglia, apoptosis, retinal degeneration, and insulin receptor kinase activity. RESULTS: The mean blood glucose of Ins2(Akita) mice was significantly elevated, whereas the body weight at death was reduced compared with that of control animals. Compared with sibling control mice, the Ins2(Akita) mice had increased retinal vascular permeability after 12 weeks of hyperglycemia (P < 0.005), a modest increase in acellular capillaries after 36 weeks of hyperglycemia (P < 0.0008), and alterations in the morphology of astrocytes and microglia, but no changes in expression of Muller cell glial fibrillary acidic protein. Increased apoptosis was identified by immunoreactivity for active caspase-3 after 4 weeks of hyperglycemia (P < 0.01). After 22 weeks of hyperglycemia, there was a 16.7% central and 27% peripheral reduction in the thickness of the inner plexiform layer, a 15.6% peripheral reduction in the thickness of the inner nuclear layer (P < 0.001), and a 23.4% reduction in the number of cell bodies in the retinal ganglion cell layer (P < 0.005). In vitro insulin receptor kinase activity was reduced (P < 0.05) after 12 weeks of hyperglycemia. CONCLUSIONS: The retinas of heterozygous male Ins2(Akita) mice exhibit vascular, neural, and glial abnormalities generally consistent with clinical observations and other animal models of diabetes. In light of the relatively early, spontaneous onset of the disease and the popularity of the C57BL/6J inbred strain as a background for the generation and study of other genetic alterations, combining the Ins2(Akita) mutation with other engineered mutations will be of great use for studying the molecular basis of retinal complications of diabetes.