The pathogenesis of human non-alcoholic fatty liver disease (NAFLD) remains unclear, in particular in the context of its relationship to insulin resistance and visceral obesity. Work on the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in mice has resolved some of the related questions. CEACAM1 promotes insulin clearance by enhancing the rate of uptake of the insulin-receptor complex. It also mediates a negative acute effect of insulin on fatty acid synthase activity. This positions CEACAM1 to coordinate the regulation of insulin and lipid metabolism. Fed a regular chow diet, global null mutation of Ceacam1 manifest hyperinsulinemia, insulin resistance, obesity, and steatohepatitis. They also develop spontaneous chicken-wire fibrosis, characteristic of non-alcoholic steatohepatitis. Reduction of hepatic CEACAM1 expression plays a significant role in the pathogenesis of diet-induced metabolic abnormalities, as bolstered by the protective effect of hepatic CEACAM1 gain-of-function against the metabolic response to dietary fat. Together, this emphasizes that loss of hepatic CEACAM1 links NAFLD to insulin resistance and obesity.

Impairment of insulin clearance is being increasingly recognized as a critical step in the development of insulin resistance and metabolic disease. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Null deletion or liver-specific inactivation of Ceacam1 in mice causes a defect in insulin clearance, insulin resistance, steatohepatitis, and visceral obesity. Immunohistological analysis revealed reduction of hepatic CEACAM1 in obese subjects with fatty liver disease. Thus, we aimed to determine whether this occurs at the hepatocyte level in response to systemic extrahepatic factors and whether this holds across species. Northern and Western blot analyses demonstrate that CEACAM1 mRNA and protein levels are reduced in liver tissues of obese individuals compared to their lean age-matched counterparts. Furthermore, Western analysis reveals a comparable reduction of CEACAM1 protein in primary hepatocytes derived from the same obese subjects. Similar to humans, Ceacam1 mRNA level, assessed by quantitative RT-PCR analysis, is significantly reduced in the livers of obese Zucker (fa/fa, ZDF) and Koletsky (f/f) rats relative to their age-matched lean counterparts. These studies demonstrate that the reduction of hepatic CEACAM1 in obesity occurs at the level of hepatocytes and identify the reduction of hepatic CEACAM1 as a common denominator of obesity across multiple species.

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Sirtuins catalyze NAD+-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1–7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

TNFα plays key roles in the regulation of inflammation, cell death, and proliferation and its signaling cascade cross-talks with the insulin signaling cascade. PKCδ, a novel PKC isoform, is known to participate in proximal TNFα signaling events. However, it has remained unclear whether PKCδ plays a role in distal TNFα signaling events. Here we demonstrate that PKCδ is activated by TNFα in a delayed fashion that is temporally associated with JNK activation. To investigate the signaling pathways activating PKCδ and JNK, we used pharmacological and genetic inhibitors of NFκB. We found that inhibition of NFκB attenuated PKCδ and JNK activations. Further analysis revealed that ER stress contributes to TNFα-stimulated PKCδ and JNK activations. To investigate the role of PKCδ in TNFα action, we used 29-mer shRNAs to silence PKCδ expression. A reduction of ~90% in PKCδ protein levels reduced TNFα-stimulated stress kinase activation, including JNK. Further, PKCδ was necessary for thapsigargin-stimulated JNK activation. Because thapsigargin is a potent inducer of ER stress, we determined whether PKCδ was necessary for induction of the UPR. Indeed, a reduction in PKCδ protein levels reduced thapsigargin-stimulated CHOP induction, a hallmark of the UPR, but not BiP/GRP78 induction, suggesting that PKCδ does not globally regulate the UPR. Next, the role of PKCδ in TNFα mediated cross-talk with the insulin signaling pathway was investigated in cells expressing human IRS-1 and a 29-mer shRNA to silence PKCδ expression. We found that a reduction in PKCδ protein levels reversed the TNFα-mediated reduction in insulin-stimulated IRS-1 Tyr phosphorylation, Akt activation, and glycogen synthesis. In addition, TNFα-stimulated IRS protein Ser/Thr phosphorylation and degradation were blocked. Our results indicate that: 1) NFκB and ER stress contribute in part to PKCδ activation; 2) PKCδ plays a key role in the propagation of the TNFα signal; and 3) PKCδ contributes to TNFα-induced inhibition of insulin signaling events.

A high-resolution time series of transcript abundance was generated to describe global expression dynamics in response to nutrition in Drosophila. Nonparametric change-point statistics revealed that within 7 h of feeding upon yeast, transcript levels changed significantly for approximately 3,500 genes or 20% of the Drosophila genome. Differences as small as 15% were highly significant, and 80% of the changes were <1.5-fold. Notably, transcript changes reflected rapid downregulation of the nutrient-sensing insulin and target of rapamycin pathways, shifting of fuel metabolism from lipid to glucose oxidation, and increased purine synthesis, TCA-biosynthetic functions and mitochondria biogenesis. To investigate how nutrition coordinates these transcriptional changes, feeding-induced expression changes were compared with those induced by the insulin-regulated transcription factor dFOXO in Drosophila S2 cells. Remarkably, 28% (995) of the nutrient-responsive genes were regulated by activated dFOXO, including genes of mitochondrial biogenesis and a novel homolog of mammalian peroxisome proliferator-gamma coactivator-1 (PGC-1), a transcriptional coactivator implicated in controlling mitochondrial gene expression in mammals. These data implicate dFOXO as a major coordinator of the transcriptional response to nutrients downstream of insulin and suggest that mitochondria biogenesis is linked to insulin signaling via dFOXO-mediated repression of a PGC-1 homolog.

A new and regioselective strategy was developed for the preparation of fluorine-18-labeled insulin as a novel positron emission tomography (PET) tracer. [18F]-4-Fluorobenzoic acid (4-18FBA), which was produced in 83 +/- 8% yield (n = 10), through the use of succinimidyl [18F]-4-fluorobenzoate (4-(18)FSB), was conjugated through a short spacer (6-aminohexanoic acid, AHx) to the PheB1 residue of a protected form of insulin. 18FB-AHx-insulin (8b) was repeatedly prepared in practical quantities (10-20 mCi, 370-740 MBq) in good radiochemical yield (9 +/- 5%, n = 9) and in a specific activity of 7.8 mCi/micromol. The final product was characterized by comparing the radioHPLC and radioTLC of 8b with that of the 19F-analogue (19FB-AHx-insulin, 8a) and by analyzing a carrier-added synthesis by mass spectrometry. Dithiothreitol and endoproteinase Glu-C digestion experiments on 8a confirmed that the prosthetic group was in fact conjugated to the PheB1 residue. An insulin receptor (IR) phosphorylation assay using CHO-hIR cells overexpressing recombinant human insulin receptors indicated no statistical difference in the extent of autophosphorylation stimulated by 8a as compared to that for human insulin (EC50 values of 0.82 nM and 1.0 nM, respectively). The stimulation of 2-deoxyglucose uptake in 3T3-L1 mouse adipocytes utilizing 8a versus unmodified human insulin gave similar EC50 values of 0.68 nM and 0.41 nM, respectively. The IC50 values for 8a versus native insulin for the displacement of 125I-insulin from HEK-293 cells were also the same within experimental error (2.6 nM for 8a versus 2.4 nM for unmodified human insulin). These results support the use of the 18F-insulin analogue as a PET tracer for imaging the distribution of insulin in vivo.

Studies in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans have revealed that components of the insulin signaling pathway have been highly conserved during evolution. Genetic analysis in Drosophila suggests that structural conservation also extends to the functional level. Flies carrying mutations that reduce insulin signaling have a growth deficiency phenotype similar to that seen in mice with disruptions of genes encoding insulin-like growth factors (IGFs) or the IGF-I receptor. Recent studies in flies have demonstrated a role for the insulin signaling pathway in the regulation of metabolism, reproduction and lifespan via modulation of central neuroendocrine pathways. Similarly, mice with loss of brain insulin receptors or insulin receptor substrate 2 deficiency exhibit neuroendocrine defects and female infertility. These parallels suggest that the insulin system has multiple conserved roles, acting directly to modulate growth and indirectly, via the neuroendocrine system, to modulate peripheral physiology in response to changes in nutrient availability.

RATIONALE AND OBJECTIVES:To develop and test an experimental radiology resident conference format designed to incorporate more cases, improve visibility of cases, increase resident participation, decrease resident anxiety, and provide residents with feedback on their oral presentation skills. METHODS:Sixteen radiology residents were paired into eight teams. Each team was given an identical packet of eight chest radiograph cases, assigned a viewbox, and given 16 minutes to review the cases. Each case was then discussed in front of the group, by one resident from a team, such that all teams participated in oral presentation. The conference moderator provided a written handout, discussion of the findings, and diagnosis for each case. Residents anonymously evaluated each other's performance, the experimental conference format, and 15 traditional conferences. RESULTS:Residents subjectively found the experimental conference to be a statistically significant improvement over the traditional conferences in certain areas (P < .05). CONCLUSION:The experimental conference serves as a valuable alternative to the traditional "hot-seat" conference.

Drosophila contain an insulin receptor homologue (im) which is highly conserved relative to its mammalian counterparts. The deduced amino acid sequence (1 2) confirms earlier biochemical work (3) indicating that the receptor is composed of two u subunits and two 3 subunits with the a subunits containing the ligand binding domains and the 3 subunits containing a ligand-activated tyrosine kinase activity. The amino acid sequence of exon 2 of the ¡nr is 45v0 identical to that of the human insulin receptor and the positions of 24 of 25 cysteines in the cysteine-rich domain (exons 2-4) are conserved (I). The high degree of sequence identity in this region is consistent with the ability of the ¡nr to bind mammalian insulin with a reasonably high affinity (15 nM). The subunit is similarly conserved (50V overall sequence identity) with much higher levels of identity in the tyrosine kinase domain (1). However, the Inr differs from receptors in the mammalian insulin receptor family due to the presence of large extensions at the amino and carboxyl termini. The function of the amino terminal extension is not clear, however the structure of the carboxy-terminal extension suggests that it may play a role in signal transduction (I). Nine tyrosine residues are found in the carboxy-terminal extension, four of which are found in a consensus sequence SXNPNYXXM/L which has features common to both SH2domain binding sites and phosphotyrosine-binding (PTB) domains (reviewed in 4). SH2 domain binding specificity is conferred by amino acids carboxy-terminal to the phosphotyrosine residue, and YXXM is a sequence present in high affinity binding sites for the p85 subunit of phosphatidylinositol 3-kinase. Conversely, PTB domain binding specificity is conferred by amino acids that are amino-terminal to the phosphotyrosine residue, and in particular, NPXY or NXXY have been shown to be important determinants of PTB domain binding. The functional role of this carboxyterminal extension is being explored by expression of nearly the entire mr 3 subunit including the transmembrane domain in mammalian cells. The expressed protein is a 180 kDa, constitutively active tyrosine kinase and the interaction of wild-type and mutant forms of this protein with signaling molecules is currently under study. jar mRNA expression is maximal during embryogenesis and widespread throughout the embryo, although nervous system expression is elevated above that in other tissues after midembryogenesis. In larvae, ¡nr mRNA is still expressed in the nervous system and also at high levels in the imaginai discs, actively niitotic tissues which are the precursors of adult structures such as wings, legs, eyes, etc. Genetic analysis has revealed multiple roles for this receptor tyrosine kinase during Drosophila development (2, 5). Loss of function mutations in the ¡nr gene lead to embryonic or early larval lethality in homozygotes, indicating an essential role for ¡nr function. As expected from the high levels of ¡nr expression in developing nervous tissue, abnormal development of the nervous system is observed in homozygous mr mutants (2). However, some mutant mr alleles can complement to yield viable adults with a developmental delay, growth deficiency, female-sterile phenotype (5). The ability of certain mutant alleles in combination to rescue the lethal phenotype is consistent with the multimeric nature of the insulin receptor. When present together in a hybrid holoreceptor these mutations appear to produce a tetramer which is at least partially functional. However, both cases of heteroallelic complementation observed in our studies fail to restore full receptor function as indicated by the persistence of a mutant phenotype. The developmental delay is very pronounced (9lO days), almost doubling the normal developmental time. The delay is due entirely to elongation of the second and third larval stages which are the periods when most imaginai disc cell mitoses occur. Imaginai discs express high levels of ¡nr mRNA. Discs in heteroallelic larvae are reduced in size due to a decrease in cell number indicating that fewer cell divisions have occurred despite the elongated developmental time. The adult heteroallelic flies are about 50% of the size of siblings which carry one wildtype ¡nr allele. Taken together, these results suggest a role for the lnr in the regulation of cell proliferation and ultimately, body size. The resemblance to growth deficiency phenotypes seen in cases of severe insulin resistance in humans and insulin-like growth factor (IGF) and IGF-i receptor mutations in mice suggests that regulation of body size is a function of this hormone/receptor family which has been highly conserved despite widely different modes of assembling an adult organism.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.