Insulin resistance, recently recognized as a strong predictor of disease in adults, has become the leading element of the metabolic syndrome and renewed as a focus of research. The condition exists when insulin levels are higher than expected relative to the level of glucose. Thus, insulin resistance is by definition tethered to hyperinsulinemia. The rising prevalence of medical conditions where insulin resistance is common has energized research into the causes. Many causes and consequences have been identified, but the direct contributions of insulin itself in causing or sustaining insulin resistance have received little sustained attention. We examine situations where insulin itself appears to be a proximate and important quantitative contributor to insulin resistance. 1) Mice transfected with extra copies of the insulin gene produce basal and stimulated insulin levels that are two to four times elevated. The mice are of normal weight but show insulin resistance, hyperglycemia, and hypertriglyceridemia. 2) Somogyi described patients with unusually high doses of insulin and hyperglycemia. Episodes of hypoglycemia with release of glucose-raising hormones, postulated as the culprits in early studies, have largely been excluded by studies including continuous glucose monitoring. 3) Rats and humans treated with escalating doses of insulin show both hyperinsulinemia and insulin resistance. 4) The pulsatile administration of insulin (rather than continuous) results in reduced requirements for insulin. 5) Many patients with insulinoma who have elevated basal levels of insulin have reduced (but not absent) responsiveness to administered insulin. In summary, hyperinsulinemia is often both a result and a driver of insulin resistance. Diabetes Care 31 (Suppl. 2):S262-S268, 2008

Diabetes leads to vascular leakage, glial dysfunction, and neuronal apoptosis within the retina. The goal of the studies reported here was to determine the role that retinal microglial cells play in diabetic retinopathy and assess whether minocycline can decrease microglial activation and alleviate retinal complications. Immunohistochemical analyses showed that retinal microglia are activated early in diabetes. Furthermore, mRNAs for interleukin-1␤ and tumor necrosis factor-␣, proinflammatory mediators known to be released from microglia, are also increased in the retina early in the course of diabetes. Using an in vitro bioassay, we demonstrated that cytokine-activated microglia release cytotoxins that kill retinal neurons. Furthermore, we showed that neuronal apoptosis is increased in the diabetic retina, as measured by caspase-3 activity. Minocycline represses diabetes-induced inflammatory cytokine production, reduces the release of cytotoxins from activated microglia, and significantly reduces measurable caspase-3 activity within the retina. These results indicate that inhibiting microglial activity may be an important strategy in the treatment of diabetic retinopathy and that drugs such as minocycline hold promise in delaying or preventing the loss of vision associated with this disease. Diabetes 54:1559 -1565, 2005

Glial cells are thought to supply energy for neurotransmission by increasing nonoxidative glycolysis; however, oxidative metabolism in glia may also contribute to increased brain activity. To study glial contribution to cerebral energy metabolism in the unanesthetized state, we measured neuronal and glial metabolic fluxes in the awake rat brain by using a double isotopic-labeling technique and a twocompartment mathematical model of neurotransmitter metabolism. Rats (n ϭ 23) were infused simultaneously with ). The glial TCA cycle rate was ϳ30% of total TCA cycle activity. A high pyruvate carboxylase rate (V PC , ϳ0.14 -0.18 mol ⅐ gm Ϫ1 ⅐ min Ϫ1) contributed to the glial TCA cycle flux. This anaplerotic rate in the awake rat brain was severalfold higher than under deep pentobarbital anesthesia, measured previously in our laboratory using the same 13 C-labeling technique. We postulate that the high rate of anaplerosis in awake brain is linked to brain activity by maintaining glial glutamine concentrations during increased neurotransmission.

18F-FPPRGD2, which was approved for clinical study recently, has favorable properties for integrin targeting and showed potential for antiangiogenic therapy and early response monitoring. However, the time-consuming multiple-step synthesis may limit its widespread applications in the clinic. In this study, we developed a simple lyophilized kit for labeling PRGD2 peptide (18F-AlF-NOTA-PRGD2, denoted as 18F-alfatide) using a fluo-ride–aluminum complex that significantly simplified the labeling procedure. Methods Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18F-alfatide, and 1 tuberculosis patient was investigated using both 18F-alfatide and 18F-FDG imaging. Standardized uptake values were measured in tumors and other main organs at 30 min and 1 h after injection. Kinetic parameters were calculated by Logan graphical analysis. Immunohisto-chemistry and staining intensity quantification were performed to confirm the expression of integrin αvβ3. Results Under the optimal conditions, the whole radiosynthesis including purifica-tion was accomplished within 20 min with a decay-corrected yield of 42.1% ± 2.0% and radiochemical purity of more than 95%. 18F-alfatide PET imaging identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. Conclusion 18F-alfatide can be produced with excellent radiochemical yield and purity via a simple, 1-step, lyophilized kit. PET scanning with 18F-alfatide allows specific imaging of αvβ3 expression with good contrast in lung cancer patients. This technique might be used for the assessment of angiogene-sis and for planning and response evaluation of cancer therapies that would affect angiogenesis status and integrin expression levels.

The study provided new insights into the role of miRNAs and their target genes in the pathogenesis of fetal alcohol syndrome.

Fibrotic skin disease represents a major global healthcare burden, characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix. Fibroblasts are found to be heterogeneous in multiple fibrotic diseases, but fibroblast heterogeneity in fibrotic skin diseases is not well characterized. In this study, we explore fibroblast heterogeneity in keloid, a paradigm of fibrotic skin diseases, by using single-cell RNA-seq. Our results indicate that keloid fibroblasts can be divided into 4 subpopulations: secretory-papillary, secretory-reticular, mesenchymal and pro-inflammatory. Interestingly, the percentage of mesenchymal fibroblast subpopulation is significantly increased in keloid compared to normal scar. Functional studies indicate that mesenchymal fibroblasts are crucial for collagen overexpression in keloid. Increased mesenchymal fibroblast subpopulation is also found in another fibrotic skin disease, scleroderma, suggesting this is a broad mechanism for skin fibrosis. These findings will help us better understand skin fibrotic pathogenesis, and provide potential targets for fibrotic disease therapies.

Hepatocellular carcinoma (HCC) ranks third in cancer-related mortality due to late diagnosis and poor treatment options. Autophagy is a lysosome-mediated protein and organelle degradation process which is characterized by the formation of double-membrane vesicles, known as autophagosomes. Increasing evidence reveals that autophagy functions as a survival mechanism in liver cancer cells against drug-induced apoptosis. In this study, we found that autophagy was suppressed by miR-101 in the HCC cell line HepG2. miR-101 inhibited autophagy via targets including RAB5A, STMN1 and ATG4D. Moreover, miR-101 enhanced apoptosis induced by cisplatin in the HepG2 cell line. The possible mechanism of this effect may be through inhibition of autophagy. Our results indicate a novel and critical role for miR-101 and autophagy in the chemoresistance of cisplatin in HCC. We propose that gene therapy targeting miR-101/autophagy should be investigated further as a potential alternative therapeutic strategy for HCC.

The three-dimensional structure of a yeast ubiquitin-conjugating enzyme, encoded by the Saccharomyces cerevisiae UBC4 gene, has been determined at 2.7 A. The structure was solved using molecular replacement techniques and refined by simulated annealing to an R-factor of 0.198. Bond lengths and angles in the molecule have root mean square deviations from ideal values of 0.018 A and 4.0 degrees, respectively. Ubc4 is an alpha/beta protein with four alpha-helices and a four-stranded antiparallel beta-sheet. The ubiquitin-accepting cysteine is located in a cleft between two loops. Comparison with the recently determined structure of a different plant enzyme suggests that class I ubiquitin-conjugating enzymes are highly conserved in their three-dimensional folding. Except for two extra residues at the N- and the C-terminus of the plant enzyme, the C alpha atoms of the two enzymes can be superimposed with a root mean square deviation of only 1.52 A. Greater variations are found between the surfaces of the two molecules, as most of the identical residues between the two enzymes are either buried or clustered on the surface that lies adjacent to the ubiquitin-accepting cysteine. We suggest that this conserved surface functions in protein-protein binding during ubiquitin thiol ester formation.