Effect of rosiglitazone on capillary density and angiogenesis in adipose tissue of normoglycaemic humans in a randomised controlled trial.

AIMS/HYPOTHESIS: Recent reports of decreased capillary density in the adipose tissue of obese individuals suggest that an imbalance of angiogenesis and adipogenesis may, in part, underlie insulin resistance. This study aimed to determine whether the insulin-sensitising peroxisome proliferator-activated receptor γ (PPARγ) activator rosiglitazone affects adipose tissue vascularisation in normal humans. METHODS: A randomised, parallel-group, investigator-blinded placebo-controlled trial was conducted with normoglycaemic volunteers with BMI 27-43, recruited from the community at the University of Massachusetts Medical School, Worcester, MA, USA. Peri-umbilical adipose tissue biopsies were obtained before and after treatment for 6 weeks with rosiglitazone (8 mg once daily) or placebo, which were randomly allocated from a sequentially numbered list. The primary outcomes were adipocyte size and capillary density measured by immunohistochemistry, and angiogenic potential assessed by capillary sprout formation in Matrigel. Secondary outcomes were serum adiponectin, glycaemic, lipid and liver function variables. RESULTS: A total of 35 individuals fulfilling the inclusion criteria were randomised, and complete before-vs-after analyses were achieved in 30 participants (13 and 17, placebo and rosiglitazone, respectively). Significant differences, assessed by paired two-tailed Student t tests, were seen in response to rosiglitazone for adipocyte size (3,458 ± 202 vs 2,693 ± 223 µm(2), p = 0.0049), capillary density (5.6 ± 0.5 vs 7.5 ± 0.5 lumens/field, p = 0.0098), serum adiponectin (14.3 ± 1.5 vs 28.6 ± 3.0 ng/ml, p < 0.0001) and alkaline phosphatase (1.04 ± 0.07 vs 0.87 ± 0.05 µkat/l, p = 0.001). A difference in angiogenic potential before and after treatment between the placebo and rosiglitazone groups was also seen (-23.88 ± 14 vs 13.42 ± 13, p = 0.029, two-tailed Mann-Whitney test). CONCLUSIONS/INTERPRETATION: Significant effects on adipose tissue vascular architecture occur after a short period of treatment with rosiglitazone in individuals with normal glucose tolerance. Improved adipose tissue vascularisation may, in part, mediate the therapeutic actions of this class of drugs. TRIAL REGISTRATION: ClinicalTrials.gov NCT01150981 FUNDING: The study was funded by National Institutes of Health grant DK089101 to S. Corvera, and by pilot funding from the University of Massachusetts (UMASS) Center for Clinical Translational Sciences (M. Thompson, S. Malkani and S. Corvera). Morphology core services were supported by UMASS Diabetes Endocrine Research Center (DERC) grant DK32520.

Dietary cow's milk protein does not alter the frequency of diabetes in the BB rat.

One theory of the pathogenesis of IDDM proposes that exposure to cow's milk proteins triggers the disease in genetically susceptible individuals. We tested this hypothesis in the BB/Wor rat model of human IDDM. Diabetes-prone (DP) BB/Wor rats spontaneously develop IDDM. Coisogenic diabetes-resistant (DR) BB/Wor rats do not develop diabetes spontaneously, but IDDM can readily be induced by treatment with polyinosinic:polycytidylic acid and depletion of RT6+ T-cells. Pregnant BB/Wor rats were fed one of four experimental diets or a standard Purina commercial rat chow (5010) that was certified to be free of cow's milk protein. Offspring were maintained on the maternal diet after weaning. DP-BB/Wor rats, fed either of two experimental diets based on hydrolyzed casein and free of intact milk protein (Nutramigen or D11236), developed diabetes at only half the rate of animals fed Purina 5010 chow. Neither the addition of bovine serum albumin (BSA) to Nutramigen nor the substitution of total milk protein for the hydrolyzed casein in the D11236 diet increased the frequency of spontaneous diabetes. In contrast, there was no relationship between diet and susceptibility of DR-BB/Wor rats to IDDM induction. However, the methods used to induce IDDM in DR-BB/Wor animals were found to induce antibodies against BSA. We conclude the following: 1) Dietary modification can reduce spontaneous IDDM expression in DP-BB/Wor rats, but the agent of protection is not elimination of cow's milk protein. 2) The addition of BSA or intact milk protein does not abrogate the effectiveness of a protective diet. 3) The genetic susceptibility of the DR-BB/Wor rat to autoimmune diabetes is unaffected by any of the tested diets, but a role of anti-BSA-like autoreactivity in IDDM expression cannot be excluded.

Specific induction of early growth response gene 1 in the lateral nucleus of the amygdala following contextual fear conditioning in rats.

Although the amygdala is known to be crucial for fear conditioning, little is known about the molecular and cellular mechanisms in the amygdala that are important for fear conditioning. One possible mechanism may be the activation of immediate-early genes, which function as regulatory factors of transcriptional processes. To investigate whether one of the major immediate-early gene families is involved in the learning and memory of fear, we examined the effects of fear conditioning on the expression of the four members of the early growth response (EGR) gene family, EGR-1, EGR-2, EGR-3, and EGR-4. Image analysis of in situ hybridization of messenger RNA of the four family members was performed in the amygdala, hippocampus, and neocortex 15, 30 and 60min following one-trial contextual fear conditioning. Rats were either handled, placed within the testing context without receiving the footshock, and received a footshock immediately upon placement within the context, or received a footshock after a 3-min delay (delayed-shock). Of the four groups, only the delayed-shock group exhibited a fear response (freezing). EGR-1 messenger RNA expression in the dorsolateral part of the lateral amygdaloid nucleus was significantly greater in the delayed-shock group compared with the other groups 15 and 30min following the conditioning. The increased expression of EGR-1 was specifically localized to the lateral nucleus of the amygdala; expression in the hippocampus and cortex was not increased by fear conditioning. In contrast, the expression of EGR-2, EGR-3, and EGR-4 messenger RNA was not increased in the amygdala, hippocampus or cortex following fear conditioning. In addition, following a retention test conducted 24h after fear conditioning, no increases were found in the expression of EGR-1 messenger RNA expression in the amygdala, hippocampus or cortex. The results demonstrate that of the four genes of the EGR family of transcription-regulatory factors, only EGR-1 messenger RNA in the dorsolateral portion of the lateral nucleus of the amygdala was specifically increased with contextual fear conditioning. It is suggested that EGR-1 plays a functional role during learning, but not retrieval, of contextual fear within the lateral nucleus of the amygdala.

N-Methyl-D-aspartate receptor antagonism blocks contextual fear conditioning and differentially regulates early growth response-1 messenger RNA expression in the amygdala: implications for a functional amygdaloid circuit of fear.

N-Methyl-D-aspartate receptors in the amygdala are known to be crucial for the learning of conditioned fear, although the molecular cascades that N-methyl-D-aspartate receptors regulate are not well understood. Recent experiments from our laboratory have shown that messenger RNA expression of the immediate-early messenger gene, early growth response gene 1, increases in the lateral nucleus of the amygdala following contextual fear conditioning. However, the regulation of the increase in early growth response gene 1 expression is not known. To determine if N-methyl-D-aspartate receptors regulate both fear conditioning and the increase in early growth response gene 1 expression in the lateral nucleus of the amygdala, rats were infused i.c.v. with 2.5microg of the N-methyl-D-aspartate antagonist, DL-2-amino-5-phosphonovalerate. Most rats were killed 30min following one-trial contextual fear conditioning and their brains were processed for in situ hybridization detection of early growth response gene 1 messenger RNA expression. The remainder of the rats was tested for retention of fear conditioning 24h later. In DL-2-amino-5-phosphonovalerate-treated rats, post-shock freezing remained intact, whereas fear-conditioned freezing during the retention test was abolished. Image analysis of early growth response gene 1 messenger RNA revealed that DL-2-amino-5-phosphonovalerate blocked the fear-conditioning-associated increase in early growth response gene 1 expression in the lateral nucleus of the amygdala. In addition, DL-2-amino-5-phosphonovalerate significantly increased early growth response gene 1 expression in the central nucleus of the amygdala. The results reveal differential regulation of early growth response gene 1 messenger RNA in the amygdala by N-methyl-D-aspartate receptors and argue for a functional role of early growth response gene 1 in the formation of long-term memory for contextual fear. Furthermore, the results indicate a functional neuroanatomical circuit within the amygdala that includes dampening of excitatory and activation of inhibitory processes in distinct amygdala nuclei, resulting in the reduction of fear conditioning.

Induction of NGFI-B mRNA following contextual fear conditioning and its blockade by diazepam.

Expression of the immediate-early gene, NGFI-B (nerve growth factor inducible gene B), was examined in the amygdala, hippocampus, and neocortex following contextual fear conditioning. Rats were either handled, placed within the testing context without receiving the footshock, received a footshock immediately upon placement within the context, or received a footshock after a 3-min delay (delayed-shock). Only the delayed-shock group displayed a fear response (freezing) in the post-shock period and in a retention test 24 h after fear conditioning. Expression of NGFI-B mRNA was increased in the dorsolateral part of the lateral nucleus of the amygdala (LaDL) and the neocortex 30 min following conditioning in the delayed-shock group compared to the other three groups. In addition, following a retention test conducted 24 h after fear conditioning, NGFI-B mRNA expression was increased in the neocortex of the delayed-shock group compared to the handled group. In a subsequent experiment, the effects of pretreatment with the anxiolytic drug, diazepam, on fear conditioning and the concomitant increases in NGFI-B mRNA were investigated. Rats administered a 2.5 mg/kg, i.p. dose of diazepam before fear conditioning did not acquire contextual fear as demonstrated by a lack of freezing in a retention test. Although diazepam blocked fear conditioning while the 40% propylene glycol, 10% ethanol vehicle solution did not, both diazepam and the vehicle reduced the conditioning-induced increase in NGFI-B expression in the LaDL. In contrast, the fear-conditioning-induced NGFI-B increase in the neocortex was blocked by diazepam, but not by the vehicle. The data suggest that the transcriptional factor NGFI-B in the LaDL and neocortex may play a functional role in learning and memory of contextual fear, but blocking the increase in NGFI-B expression in the LaDL is not essential for diazepam to interfere with fear conditioning.

Differential expression of EGR-1 mRNA in the amygdala following diazepam in contextual fear conditioning.

The amygdaloid complex is thought to be a major site of action of anxiolytic benzodiazepine agonists. To investigate whether activity in the amygdaloid complex is altered with anxiolytic effects of diazepam, mRNA expression of the immediate-early gene EGR-1 was examined in the amygdala following blockade of fear conditioning by diazepam. It was previously shown that mRNA expression of EGR-1 (also called, NGFI-A, Zif 268, Krox 24) increases in the lateral nucleus of the amygdala (LA) shortly following contextual fear conditioning. It was therefore hypothesized that diazepam would block both contextual fear and the concomitant increase in EGR-1 mRNA expression in the LA induced by fear conditioning. Rats administered systemic diazepam before fear conditioning displayed both anxiolytic effects during the post-shock period and amnesic effects during a retention test 24 h later. Diazepam blocked the fear-conditioning-induced increase in EGR-1 expression in the LA. In addition, diazepam significantly increased EGR-1 mRNA expression in the central nucleus of the amygdala (CeA) in a dose-dependent manner. The results reveal differential regulation of EGR-1 by diazepam in the central and lateral nuclei of the amygdala suggesting that these two amygdala nuclei act in a reciprocal manner during the anxiolytic and amnesic action of the benzodiazepine agonist.

Non-small cell lung cancer cycloxygenase activity and proliferation are inhibited by non-steroidal antiinflammatory drugs.

The effects of non-steroidal antiinflammatory drugs (NSAIDs) on non-small cell lung cancer (NSCLC) were investigated. Arachidonic acid (AA) was metabolized to prostaglandin E2 (PGE2) in NSCLC cells. NSAIDs such as aspirin or indomethacin reduced PGE2 levels in NCI-H157 and H1264 cells, and the decrease caused by PGE2 was reversed by epidermal growth factor (EGF). By RT-PCR, both cyclooxygenase (COX)-1 and COX-2 mRNAs are detected in NCI-H157 and H1264 cells. By Northern analysis, COX-2 mRNA was induced by EGF and phorbol ester. By immunocytochemistry, COX-1 and COX-2 enzymes were localized to NSCLC tumors. Aspirin, indomethacin and ibuprofen decreased NSCLC growth in vitro. Aspirin and indomethacin inhibited proliferation of NSCLC xenografts in nude mice. These data suggest that COX enzymes may be important regulatory components of NSCLC.

Cytokine gene expression in islets and thyroids of BB rats. IFN-gamma and IL-12p40 mRNA increase with age in both diabetic and insulin-treated nondiabetic BB rats.

Inflammatory cytokines, particularly those produced by Th1 type lymphocytes, are hypothesized to play a major role in the pathogenesis of autoimmune diseases. The present studies investigated this hypothesis in the BB rat. Diabetes-prone (DP) BB rats develop spontaneous hyperglycemia and thyroiditis. Coisogenic diabetes-resistant (DR) BB rats do not develop either disorder spontaneously, but both diseases are induced by depletion of RT6+ T cells. Reverse transcriptase-PCR was used to measure mRNA encoding type 1 and type 2 cytokines. In both DP and RT6-depleted DR rats, IFN-gamma mRNA was present in islets before and during disease onset. IL-2 and IL-4 mRNAs were minimal or undetectable in infiltrated islets but present in activated peripheral T cells. IL-10 mRNA was present at low abundance in infiltrating T cells. These observations suggested a Th1 type inflammatory response, and consistent with this interpretation, we observed that mRNA encoding the p40 chain of IL-12 was also present before and during disease onset. Similar cytokine mRNA profiles were observed in the thyroids of RT6-depleted DR rats and in the islets of DP rats treated with prophylactic parenteral insulin to prevent diabetes. We conclude that IFN-gamma and IL-12 may play a major role in the expression of insulitis and thyroiditis in the BB rat, that Th1 lymphocytes may predominate over Th2 lymphocytes in these inflammatory lesions, and that prevention of diabetes by insulin is not associated with an alteration in the cytokine gene profile of islet infiltrating cells.