Ultrasound-assisted encapsulation of curcumin and fisetin into Saccharomyces cerevisiae cells: a multistage batch process protocol.

Some of the challenges of yeast encapsulation protocols are low phytochemical internalization rates and limited intracellular compartments of yeasts. This study uses an ultrasound-assisted batch encapsulation (UABE) protocol to optimize the encapsulation of curcumin and fisetin by recovering nonencapsulated biomaterial and further incorporating it into nonloaded yeasts in three encapsulation stages (1ES, 2ES and 3ES). The effect of selected acoustic energies (166·7 and 333·3 W l-1 ) on the encapsulation efficiency (EE), yield (EY) and antioxidant activity retention were evaluated, and then, compared with a control process (without ultrasound treatment). Compared to the control, enhanced EEs were achieved for both curcumin (10·9% control to 58·5% UABE) and fisetin (18·6% control to 76·6% UABE) after 3ES and the use of 333·3 W l-1 . Similarly, the yeast maximum loading capacity was improved from 6·6 to 13·4 mg g-1 for curcumin and from 11·1 to 26·4 mg g-1 for fisetin after UABE protocol. The antioxidant activity of produced biocapsules was positively correlated with the bioactive-loaded content of yeasts when ultrasound treatment was applied. Overall, results from this study provide valuable information regarding UABE processes, and moreover, bring new and creative perspectives for ultrasound technology in the food industry.

Curcumin and fisetin internalization into Saccharomyces cerevisiae cells via osmoporation: impact of multiple osmotic treatments on the process efficiency.

Cell osmoporation is a simple and straightforward procedure of creating food-grade biocapsules. This study proposes a new protocol of sequential cell osmoporation stages and evaluates its impact on the efficiency of curcumin and fisetin internalization into Saccharomyces cerevisiae cells. To the best of our knowledge, this is the first report in the literature regarding the subject. To assess how multiple osmoporation stages influence the encapsulation efficiency (% EE), encapsulated amount of curcumin (IC) and fisetin (IF) into S. cerevisiae cells and cell viability, the residual supernatant was used for the subsequent encapsulation stages and viability was assessed by the CFU method. Quantification was carried through direct extraction, using an ultrasonic bath and UV-Vis spectrophotometry. Experimental data demonstrated that the addition of a second osmoporation stage increases both the EE (% EE) and the amount of encapsulated curcumin and fisetin (IC and IF). As a result, the EE was considerably improved and the obtained microcapsules contained a higher amount of the targeted bioactive compounds in its internal structure. However, adding a third osmoporation stage proved to less beneficial to the process efficiency due to its lower yield and the significant negative impact to cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time in the literature, a protocol of serial osmoporation stages to enhance the encapsulation efficiency of hydrophobic low molecular weight molecules (curcumin and fisetin) into Saccharomyces cerevisiae cells was determined. By increasing overall efficiency, this protocol empowers the encapsulation process and creates a rational way to reduce waste for future industrial osmoporation applications.

Lymphocyte reconstitution following autologous stem cell transplantation for progressive MS.

BACKGROUND: Autologous stem cell transplantation (ASCT) for progressive multiple sclerosis (MS) may reset the immune repertoire. OBJECTIVE: The objective of this paper is to analyse lymphocyte recovery in patients with progressive MS treated with ASCT. METHODS: Patients with progressive MS not responding to conventional treatment underwent ASCT following conditioning with high-dose cyclophosphamide and antithymocyte globulin. Lymphocyte subset analysis was performed before ASCT and for two years following ASCT. Neurological function was assessed by the EDSS before ASCT and for three years post-ASCT. RESULTS: CD4+ T-cells fell significantly post-transplant and did not return to baseline levels. Recent thymic emigrants and naïve T-cells fell sharply post-transplant but returned to baseline by nine months and twelve months, respectively. T-regulatory cells declined post-transplant and did not return to baseline levels. Th1 and Th2 cells did not change significantly while Th17 cells fell post-transplant but recovered to baseline by six months. Neurological function remained stable in the majority of patients. Progression-free survival was 69% at three years. CONCLUSION: This study demonstrates major changes in the composition of lymphocyte subsets following ASCT for progressive MS. In particular, ablation and subsequent recovery of thymic output is consistent with the concept that ASCT can reset the immune repertoire in MS patients.

Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis.

The data presented in this article are related to the research article entitled "The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis" Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6.

The autoimmune risk gene ZMIZ1 is a vitamin D responsive marker of a molecular phenotype of multiple sclerosis.

Multiple Sclerosis (MS) is a neurological condition driven in part by immune cells from the peripheral circulation, the targets for current successful therapies. The autoimmune and MS risk gene ZMIZ1 is underexpressed in blood in people with MS. We show that, from three independent sets of transcriptomic data, expression of ZMIZ1 is tightly correlated with that of hundreds of other genes. Further we show expression is partially heritable (heritability 0.26), relatively stable over time, predominantly in plasmacytoid dendritic cells and non-classical monocytes, and that levels of ZMIZ1 protein expression are reduced in MS. ZMIZ1 gene expression is increased in response to calcipotriol (1,25 Vitamin D3) (p < 0.0003) and associated with Epstein Barr Virus (EBV) EBNA-1 antibody titre (p < 0.004). MS therapies fingolimod and dimethyl fumarate altered blood ZMIZ1 gene expression compared to untreated MS. The phenotype indicates susceptibility to MS, and may correspond with clinical response and represent a novel clinical target.

Response to interferon-beta treatment in multiple sclerosis patients: a genome-wide association study.

Up to 50% of multiple sclerosis (MS) patients do not respond to interferon-beta (IFN-ß) treatment and determination of response requires lengthy clinical follow-up of up to 2 years. Response predictive genetic markers would significantly improve disease management. We aimed to identify IFN-ß treatment response genetic marker(s) by performing a two-stage genome-wide association study (GWAS). The GWAS was carried out using data from 151 Australian MS patients from the ANZgene/WTCCC2 MS susceptibility GWAS (responder (R)=51, intermediate responders=24 and non-responders (NR)=76). Of the single-nucleotide polymorphisms (SNP) that were validated in an independent group of 479 IFN-ß-treated MS patients from Australia, Spain and Italy (R=273 and NR=206), eight showed evidence of association with treatment response. Among the replicated associations, the strongest was observed for FHIT (Fragile Histidine Triad; combined P-value 6.74 × 10-6) and followed by variants in GAPVD1 (GTPase activating protein and VPS9 domains 1; combined P-value 5.83 × 10-5) and near ZNF697 (combined P-value 8.15 × 10-5).

Combining HLA-DR risk alleles and anti-Epstein-Barr virus antibody profiles to stratify multiple sclerosis risk.

BACKGROUND: Risk factors for multiple sclerosis (MS) include human leukocyte antigen (HLA)-DR and Epstein-Barr virus (EBV)-specific antibody responses, including an epitope within EBV nuclear antigen 1 (EBNA-1) that is of recent interest. OBJECTIVE: The objective of this paper is to assess case-control associations between MS risk and anti-EBV antibody levels as well as HLA-DR profiles, gender and age in a population-based cohort. METHODS: Serological responses to EBV were measured in 426 MS patients and 186 healthy controls. HLA-DR typing was performed using sequence-based methods. RESULTS: MS patients had significantly higher levels of antibodies against epitope-specific and polyspecific EBNA-1 and viral capsid antigen (VCA), compared with controls (all p < 10(-15)). In regression analyses, anti-EBNA-1 and anti-VCA antibody levels, protective HLA-DR*04/07/09 alleles and gender (all p < 0.003) contributed independently to a model that classified cases and controls with an odds ratio > 20 (sensitivity 92%, specificity 64%). Notably, the strong influence of high-risk HLA-DR alleles was abrogated after inclusion of EBV serology results. CONCLUSIONS: The ability to discriminate MS cases and controls can be substantially enhanced by including anti-EBV serology as well as HLA-DR risk profiles. These findings support the relevance of EBV-specific immunity in MS pathogenesis, and implicate both HLA-dependent and HLA-independent immune responses against EBNA-1 as prominent disease risk factors.

Quantitative analysis of elastography images in the detection of breast cancer.

PURPOSE: The aim of this study was to develop a quantitative method for breast cancer diagnosis based on elastosonography images in order to reduce whenever possible unnecessary biopsies. The proposed method was validated by correlating the results of quantitative analysis with the diagnosis assessed by histopathologic exam. MATERIAL AND METHODS: 109 images of breast lesions (50 benign and 59 malignant) were acquired with the traditional B-mode technique and with elastographic modality. Images in Digital Imaging and COmmunications in Medicine format (DICOM) were exported into a software, written in Visual Basic, especially developed to perform this study. The lesion was contoured and the mean grey value and softness inside the region of interest (ROI) were calculated. The correlations between variables were investigated and receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic accuracy of the proposed method. Pathologic results were used as standard reference. RESULTS: Both the mean grey value and the softness inside the ROI resulted statistically different at the t test for the two populations of lesions (i.e., benign versus malignant): p<0.0001. The area under the curve (AUC) was 0.924 (0.834-0.973) and 0.917 (0.826-0.970) for the mean grey value and for the softness respectively. CONCLUSIONS: Quantitative elastosonography is a promising ultrasound technique in the detection of breast cancer but large prospective trials are necessary to determine whether quantitative analysis of images can help to overcome some pitfalls of the methodic.

Oxidative stress and reduced glutamine synthetase activity in the absence of inflammation in the cortex of mice with experimental allergic encephalomyelitis.

Pathological changes occur in areas of CNS tissue remote from inflammatory lesions in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). To determine if oxidative stress is a significant contributor to this non-inflammatory pathology, cortex tissues from mice with clinical signs of EAE were examined for evidence of inflammation and oxidative stress. Histology and gene expression analysis showed little evidence of immune/inflammatory cell invasion but reductions in natural antioxidant levels and increased protein oxidation that paralleled disease severity. Two-dimensional oxyblots and mass-spectrometry-based protein fingerprinting identified glutamine synthetase (GS) as a particular target of oxidation. Oxidation of GS was associated with reductions in enzyme activity and increased glutamate/glutamine levels. The possibility that this may cause neurodegeneration through glutamate excitotoxicity is supported by evidence of increasing cortical Ca(2+) levels in cortex extracts from animals with greater disease severity. These findings indicate that oxidative stress occurs in brain areas that are not actively undergoing inflammation in EAE and that this can lead to a neurodegenerative process due to the susceptibility of GS to oxidative inactivation.

Pioglitazone has direct effects on insulin sensitivity and intracellular lipid content in L6 skeletal muscle cells.

Using L6 skeletal muscle cell line, rendered insulin resistant by incubation with triglyceride-rich lipoproteins (TGRLs), we sought to answer the question whether pioglitazone has direct effects on this cell line. Incubation of L6 cells with TGRLs led to an increase in the intramyocellular triglyceride content. Moreover, TGRLs led to a reduction in insulin-stimulated glycogen content and GSK-3 phosphorylation. All these changes induced by TGRLs could be antagonized by incubation of L6 cells with pioglitazone. The PPAR-γ antagonist GW9662 reversed the pioglitazone effects. We conclude that pioglitazone has direct insulin-sensitizing effects on the L6 skeletal muscle cell line, which are paralleled by a reduction in intramyocellular triglyceride accumulation.

Effect of postprandial lipemia on plasma concentrations of A-FABP, RBP-4 and visfatin.

BACKGROUND AND AIMS: Several studies indicate that changes in the plasma concentrations of adipocyte-fatty acid binding protein (A-FABP), retinol binding protein-4 (RBP-4) and visfatin are associated with chronic states of insulin resistance. Recent studies have shown that postprandial lipemia induces an acute state of insulin resistance. The aim of this study was to investigate the effect of postprandial lipemia on the plasma concentrations of A-FABP, RBP-4 and visfatin. METHODS AND RESULTS: In a within-subject crossover study, we administered a standardized high-fat meal to 24 healthy subjects (12 males and 12 females). Plasma concentrations of adipocytokines were measured in the morning after an overnight fast and during postprandial lipemia, i.e. 2, 4 and 6 hours after meal ingestion (postprandial experiment). To exclude potential confounding factors affecting the adipocytokine plasma concentrations, a control experiment without meal ingestion was performed over the same time period (postabsorptive control experiment). Comparing plasma concentrations of A-FABP, RBP-4 and visfatin between the postprandial and the postabsorptive control experiments, we found no significant differences. Within either of the two experiments, a decrease of A-FABP was noted reaching, however, statistical significance only in the postprandial experiment, i.e. 2 and 4 hours after meal ingestion. CONCLUSION: Postprandial lipemia has no significant effect on the plasma concentrations of visfatin, A-FABP or RBP-4 in relation to their postabsorptive plasma profiles. We conclude that prolonged states of insulin resistance are required to affect plasma concentrations of these adipocytokines.

Comparison of dose distributions in IMRT planning using the gamma function.

Intensity-modulated radiotherapy (IMRT) (1) is an advanced form of 3-D conformal radiotherapy. It uses non uniform spatial modifications in the intensity of the beams across the irradiated field. Consequently, it is necessary to develop sophisticated tools to compare measured and calculated dose distributions in order to verify the accuracy of the results of the planned dose distribution. Different methods have been developed to evaluate the accordance between measured and calculated doses, such as the point-to-point dose difference or the evaluation of the distance between two closed points having the same dose value (2-4). The verification method proposed by Low (5-7) seems to be more complete since it takes into account both the dose difference (DD) and the distance to agreement (DTA), allowing to define a "score", the gamma value, at each point of interest. A software tool (DDE: Dose Distribution Evaluator), based on Low's method, to evaluate the agreement between dose distribution matrices has been implemented. In particular, the proposed gamma curve, as a function of the isodose levels, gives real-time information useful for decision making about the treatment plan. The paper describes the software, and reports the obtained results in a simple geometry and in several clinical cases (head-neck and prostate). Comparison between measured data (film and MapCheck) and calculated data (CadPlan) using DDE has shown very good agreements. Thanks to its higher resolution, film dosimetry showed better accuracy than the MapCheck technique. Similar results can be obtained also with the MapCheck technique when proper measurement methods are used.

Postprandial lipaemia induces an acute decrease of insulin sensitivity in healthy men independently of plasma NEFA levels.

AIMS/HYPOTHESIS: Typical Western diets cause postprandial lipaemia for 18 h per day. We tested the hypothesis that postprandial lipaemia decreases insulin sensitivity. SUBJECTS, MATERIALS AND METHODS: Employing a randomised crossover design, we administered two types of virtually isocaloric meals to ten healthy volunteers on two separate occasions. The meals (Meals 1 and 2) were both designed to produce a rise in triglycerides, but only Meal 1 generated a rise in NEFA, too. Insulin sensitivity, as quantified by an IVGTT with minimal model analysis, was calculated postabsorptively at 08.00 h and postprandially at 13.00 h, i.e. 3 h after meal ingestion. RESULTS: Triglycerides rose from 0.91+/-0.31 mmol/l postabsorptively to 2.08+/-0.70 mmol/l postprandially with Meal 1 (p=0.005) and from 0.92+/-0.41 to 1.71+/-0.79 mmol/l with Meal 2 (p=0.005). Neither the triglyceride levels at 13.00 h, nor the post-meal AUCs for triglycerides were statistically different between Meal 1 and Meal 2. NEFA rose from 0.44+/-0.17 mmol/l postabsorptively to 0.69+/-0.16 mmol/l postprandially with Meal 1 (p=0.005) and showed no significant change with Meal 2 (0.46+/-0.31 mmol/l postabsorptively vs 0.36+/-0.32 mmol/l postprandially, p=0.09). Both the NEFA level at 13.00 h and the post-meal AUC for NEFA were significantly higher after Meal 1 than Meal 2. Compared with the postabsorptive state, insulin sensitivity decreased postprandially after each of the two meals to a comparable degree (Meal 1: -53%, p=0.02; Meal 2: -45%, p=0.005). CONCLUSIONS/INTERPRETATION: Our study reveals a drop in insulin sensitivity during postprandial lipaemia and strongly suggests that decreased insulin sensitivity is brought about by elevated plasma levels of triglyceride-rich lipoproteins independently of plasma NEFA levels.

Olanzapine impairs glycogen synthesis and insulin signaling in L6 skeletal muscle cells.

Second-generation antipsychotic agents (SGAs) are increasingly replacing first-generation antipsychotic agents due to their superior activity against the negative symptoms of schizophrenia, decreased extrapyramidal symptoms and better tolerability. However, some SGAs are associated with adverse metabolic effects as significant weight gain, lipid disorders and diabetes mellitus. The pathogenesis of SGA-induced disturbances of glucose homeostasis is unclear. In vivo studies suggest a direct influence of SGAs on peripheral insulin resistance. To this end, we analyzed whether olanzapine might alter glycogen synthesis and the insulin-signaling cascade in L6 myotubes. Glycogen content was diminished in a dose- and time-dependent manner. Within the insulin-signaling cascade IRS-1 tyrosine phosphorylation was induced several fold by insulin and was diminished by preincubation with olanzapine. IRS-1-associated PI3K activity was stimulated by insulin three-fold in L6 myotubes. Olanzapine inhibited insulin-stimulated IRS-1-associated PI3K activity in a dose-dependent manner. Protein mass of AKT, GSK-3 and GS was unaltered, whereas phosphorylation of AKT and GSK-3 was diminished, and pGS was increased. Finally, we compared olanzapine with amisulpride, an SGA clinically not associated with the induction of diabetes mellitus. Glycogen content was diminished in olanzapine-preincubated L6 cells, whereas this effect was not observed under the amisulpride conditions. We conclude that olanzapine impairs glycogen synthesis via inhibition of the classical insulin-signaling cascade and that this inhibitory effect may lead to the induction of insulin resistance in olanzapine-treated patients.

Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels.

AIMS/HYPOTHESIS: Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. METHODS: We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. RESULTS: Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. CONCLUSIONS/INTERPRETATION: Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.

Specific increase in p85alpha expression in response to dexamethasone is associated with inhibition of insulin-like growth factor-I stimulated phosphatidylinositol 3-kinase activity in cultured muscle cells.

The stimulation of phosphatidylinositol (PI) 3-kinase by insulin-like growth factor I (IGF-I) in L6 cultured skeletal muscle cells is inhibited by the glucocorticoid dexamethasone. The objective of this study was to investigate the mechanism of dexamethasone action by determining its effects on the expression of the p85alpha and p85beta regulatory subunit isoforms of PI 3-kinase, their coupling with the p110 catalytic subunit, and their association with insulin receptor substrate 1 (IRS-1) in response to IGF-I stimulation. Dexamethasone induced a 300% increase in p85alpha protein content in the L6 cultured myoblast cell line, whereas it increased p110 content by only 38% and had no effect on p85beta. The increase in p85alpha protein was associated with a coordinate increase in p85alpha mRNA. Stimulation with IGF-I induced the association of p85alpha and p85beta with IRS-1, and this was accompanied by increased amounts of the p110 catalytic subunit and markedly increased PI 3-kinase activity in IRS-1 immunoprecipitates. In cells treated with dexamethasone, greater amounts of p85alpha and lower amounts of p85beta, respectively, were found in IRS-1 immunoprecipitates, such that the alpha/beta ratio was markedly higher than in control cells. In spite of the increase in both total and IRS-1-associated p85alpha following dexamethasone treatment, IRS-1-associated p110 catalytic subunit and PI 3-kinase activity were decreased by approximately 50%. Thus, dexamethasone induces a specific increase in expression of the p85alpha regulatory subunit that is not associated with a coordinate increase in the p110 catalytic subunit of PI 3-kinase. As a consequence, in dexamethasone-treated cells, p85alpha that is not coupled with p110 competes with both p85alpha.p110 and p85beta.p110 complexes for association with IRS-1, leading to increased p85alpha but decreased p85beta, p110, and PI 3-kinase activity in IRS-1 immunoprecipitates.

The effect of dietary protein restriction on the progression of diabetic and nondiabetic renal diseases: a meta-analysis.

BACKGROUND: Dietary protein has long been thought to play a role in the progression of chronic renal disease, but clinical trials to date have not consistently shown that dietary protein restriction is beneficial. PURPOSE: To use meta-analysis to assess the efficacy of dietary protein restriction in previously published studies of diabetic and nondiabetic renal diseases, including the recently completed Modification of Diet in Renal Disease Study. DATA SOURCES: The English-language medical literature published from January 1966 through December 1994 was searched for studies examining the effect of low-protein diets in humans with chronic renal disease. A total of 1413 patients in five studies on nondiabetic renal disease (mean length of follow-up, 18 to 36 months) and 108 patients in five studies of type I diabetes mellitus (mean length of follow-up, 9 to 35 months) were included. STUDY SELECTION: Randomized, controlled studies were selected for nondiabetic renal disease; randomized, controlled studies or time-controlled studies with nonrandomized crossover design were selected for diabetic nephropathy. DATA EXTRACTION: Data in tables, figures, or text were independently extracted by two of the authors. DATA SYNTHESIS: The relative risk for progression of renal disease in patients receiving a low-protein diet compared with patients receiving a usual-protein diet was calculated by using a random-effects model. In five studies of nondiabetic renal disease, a low-protein diet significantly reduced the risk for renal failure or death (relative risk, 0.67 [95% Cl, 0.50 to 0.89]). In five studies of insulin-dependent diabetes mellitus, a low-protein diet significantly slowed the increase in urinary albumin level or the decline in glomerular filtration rate or creatinine clearance (relative risk, 0.56 [Cl, 0.40 to 0.77]). Tests for heterogeneity showed no significant differences in relative risk among studies of either diabetic or nondiabetic renal disease. No significant differences were seen between diet groups in pooled mean arterial blood pressure (diabetic and nondiabetic patients) or glycosylated hemoglobin level (diabetic patients only). CONCLUSION: Dietary protein restriction effectively slows the progression of both diabetic and nondiabetic renal diseases.

A comparison of rat small intestinal insulin and insulin-like growth factor I receptors during fasting and refeeding.

Insulin-like growth factor I (IGF-I) and insulin may be important regulators of intestinal growth. To investigate small intestinal IGF-I receptors (IGF-IR) and insulin receptors (IR) during intestinal cell atrophy and regeneration, we compared indexes of IGF-IR and IR expression in rat jejunum after 72 h of fasting and 24-72 h of enteral refeeding. Fasting induced intestinal atrophy, reduced plasma insulin and IGF-I concentrations, and markedly decreased jejunal IGF-I messenger RNA (mRNA) levels; these changes were reversed by refeeding. Fasting significantly increased jejunal specific insulin binding, IR content (to 230% of the fed control value), and the 9.6- and 7.4-kilobase IR mRNA transcript levels (to 202% and 218% of control values, respectively). These IR indexes rapidly decreased to control levels with refeeding. Levels of IGF-IR (by Scatchard analysis) and IGF-I-R mRNA were not significantly altered with fasting. The 11-kilobase IGF-IR mRNA transcript increased significantly during the first 24 h of refeeding (to 166% of the control value), and IGF-IR number rose 3-fold. We conclude that rat jejunal IR and IGF-IR are differentially regulated by nutrient availability. Up-regulation of jejunal IGF-I and IGF-IR expression during refeeding suggests a role for the IGF action pathway in gut trophic responses to enteral nutrients.

cDNA cloning of the rat IGF I receptors: structural analysis of rat and human IGF I and insulin receptors reveals differences in alternative splicing and receptor-specific domain conservation.

IGF I and insulin receptors are homologous proteins that function in distinct physiological pathways. To define domains that might contribute to differences between IGF I and insulin receptors, we cloned the rat IGF I receptor cDNA and performed a comparative sequence analysis of specific functional domains in the two receptor types of rats and humans. Since alternative splicing has been shown to alter the activities of both IGF I and insulin receptors, we also examined the mRNA splicing patterns of the two receptors. The C-terminal region exhibits the lowest degree of amino acid homology between rat and human IGF I receptors (85%) and the tyrosine kinase domain the highest homology (98%). In the region corresponding to the CAG+/-alternative splicing site of the human IGF I receptor, a nucleotide change in the rat eliminates the alternative acceptor splice site. The rat IGF I receptor has no equivalent to the alternatively spliced exon 11 of the insulin receptor. The IGF I and insulin receptors are highly homologous in the tyrosine kinase domain (84%), but differ markedly in other specific regions (e.g., 22-26% homology in the transmembrane domain, 45% homology in the C-terminal domain). We speculate that these regions of divergent sequence may have roles in determining distinct signaling properties of IGF I and insulin receptors.