Effects of a 12-month moderate weight loss intervention on insulin sensitivity and inflammation status in nondiabetic overweight and obese subjects.

Weight loss intervention is the principal non-pharmacological method for prevention and treatment of type 2 diabetes. However, little is known whether it influences insulin sensitivity directly or via its anti-inflammatory effect. The aim of this study was to assess the independent role of changes in inflammation status and weight loss on insulin sensitivity in this population.Overweight and obese nondiabetic participants without co-morbidities underwent a one-year weight loss intervention focused on caloric restriction and behavioral support. Markers of inflammation, body composition, anthropometric para-meters, and insulin sensitivity were recorded at baseline, 6, and 12 months. Insulin sensitivity was assessed with frequently sampled intravenous glucose tolerance test and Minimal Model. Twenty-eight participants (F: 15, M: 13, age 39±5 years, BMI 33.2±4.6 kg/m(2)) completed the study, achieving 9.4±6.9% weight loss, which was predominantly fat mass (7.7±5.6 kg, p<0.0001). Dietary intervention resulted in significant decrease in leptin, leptin-to-adiponectin ratio, hs-CRP, and IL-6 (all p<0.02), and improvement in HOMA-IR and Insulin Sensitivity Index (SI) (both p<0.001). In response to weight loss IL-1ß, IL-2, leptin, and resistin were significantly associated with insulin, sensitivity, whereas sICAM-1 had only marginal additive effect. Moderate weight loss in otherwise healthy overweight and obese individuals resulted in an improvement in insulin sensitivity and in the overall inflammation state; the latter played only a minimal independent role in modulating insulin sensitivity.

Functional thermogenic beige adipogenesis is inducible in human neck fat.

BACKGROUND: Recent studies suggest human neck brown adipose tissue (BAT) to consist of 'brown adipocyte (BA)-like' or beige adipocytes. However, little is known about their thermogenic function. Within the beige adipocyte transcriptome, fibroblast growth factor-21 (FGF21) is a gene whose protein product acts as an adipokine, regulating cold-induced thermogenesis in animals. Here, we explored (i) the adipogenic potential, thermogenic function and FGF21 secretory capacity of beige adipocytes derived from human neck fat and (ii) the role of FGF21 in modulating adipose bioenergetics. METHODS: Progenitors isolated from human cervical fat were differentiated into adipocytes with either a BA-like or white adipocyte (WA) phenotype. FGF21 secretion was measured by enzyme-linked immuosorbent assay. Real-time PCR/western blotting was used to determine cellular mRNA/protein levels. Extracellular flux bioanalyzer was used to quantify adipocyte oxygen consumption and fatty acid oxidation. Adipocyte heat production was measured by infrared thermography. RESULTS: Under hormonal manipulation, primary human neck pre-adipocytes differentiated into adipocytes with either BA-like or WA phenotypes, on gene/protein and functional levels. BA-like cells expressed beige but not classic BA markers. During BA differentiation, FGF21 gene expression and secretion were increased, and were augmented following norepinephrine exposure (a cold mimic in vitro). Differentiated WA expressed ß-klotho, a critical co-factor mediating FGF21 action. Treatment of WA with FGF21-induced UCP1 expression and increased oxygen consumption, respiratory uncoupling, norepinephrine-mediated thermogenesis, fatty acid oxidation and heat production, thus recapitulating the association between cold-induced FGF21 secretion and cold-induced thermogenesis in vivo. CONCLUSION: Beige adipocytes are thermogenic in humans. FGF21 is a beige adipokine capable of promoting a brown fat-like thermogenic program in WAs. SIGNIFICANCE: This study provides first evidence of inducible functional thermogenic beige adipogenesis in human neck fat. FGF21 holds promise as a cold-induced beige adipokine with metabolic benefits of therapeutic relevance through browning of white adipose tissue.

Fibroblast growth factor 21 (FGF21) and bone: is there a relationship in humans?

UNLABELLED: In animals, high fibroblast growth factor 21 (FGF21) states improve insulin resistance but induce bone loss. Whether FGF21 relates to bone mineral density (BMD) is unknown in humans. Contrary to prediction from animal findings, we found higher FGF21 levels associating with greater BMD in women, independent of age and body composition. INTRODUCTION: Recent laboratory studies suggest that FGF21 is involved in reciprocal regulation of bone and energy homeostasis. Systemic administration of FGF21 protects animals from obesity and diabetes but causes severe bone loss, smothering the enthusiasm over FGF21 as a potential antiobesity therapeutic. To date, there is no information on whether FGF21 relates to BMD in humans. We thus studied the relationship between plasma FGF21 levels and BMD in healthy adults. METHODS: Fasting plasma FGF21 levels were measured by enzyme-linked immunosorbent assay and body composition by dual-energy X-ray absorptiometry. RESULTS: Among 40 healthy volunteers (age 32 ± 10 year, 16 women), men had significantly higher lean body mass (p < 0.01) and total BMD (p < 0.05), and lower percent body fat than women (p < 0.01). Median plasma FGF21 levels were not different between the sexes. While there was no association between FGF21 concentrations and body composition in men, FGF21 levels correlated positively with fat mass (p < 0.01) in women. In men, no significant correlation between FGF21 with BMD was observed. However, in women, FGF21 correlated positively with total BMD (R (2) = 0.69, p = 0.003) and spine BMD (R (2) = 0.76, p = 0.001); the correlation remained significant after adjusting for age, ethnicity, and body composition. CONCLUSIONS: This study reveals for the first time a strong positive association between plasma FGF21 levels and BMD in healthy women, suggesting the association between bone loss and high FGF21 states in animals may not be directly translated to humans in physiologic states. We hypothesize that FGF21 may increase bone mass particularly in women through paracrine mechanisms in the bone-adipose interface.

Cold-activated brown adipose tissue is an independent predictor of higher bone mineral density in women.

UNLABELLED: In animals, defective brown adipogenesis leads to bone loss. Whether brown adipose tissue (BAT) mass relates to bone mineral density (BMD) in humans is unclear. We determined the relationship between BAT mass and BMD by cold-stimulated positron-emission tomography (PET) and dual-energy X-ray absorptiometry (DXA) in healthy volunteers. Higher BAT mass was associated with higher BMD in healthy women, but not in men, independent of age and body composition. INTRODUCTION: Contrary to the traditional belief that BAT is present only in infants, recent studies revealed significant depots of BAT present in adult humans. In animals, defective brown adipogenesis leads to bone loss. While white adipose tissue mass is a known determinant of BMD in humans, the relationship between BAT and BMD in humans is unclear. We thus examined the relationship between BAT and BMD in healthy adults. METHODS: BAT volume (ml) and activity (standard uptake value) were determined by 18F-fluorodeoxyglucose PET after overnight mild cold exposure at 19 °C, and BMD was determined by DXA. RESULTS: Among 24 healthy adults (age 28±1 years, F=10), BAT volumes were 82.4±99.5 ml in women and 49.7±54.5 ml in men. Women manifested significantly higher BAT activity, by 9.4±8.1% (p=0.03), than men. BAT volume correlated positively with total and spine BMD (r2=0.40 and 0.49, respectively, p<0.02) in women and remained a significant predictor after adjustment for age, fat, and lean body mass (p<0.05). Total and spine BMD were higher in women who harbored visually detectable BAT on PET images than those without by 11±2% (p=0.02) and 22±2% (p<0.01), respectively. No associations were observed between BAT parameters and BMD in men. CONCLUSIONS: This study demonstrated higher BMD among healthy women with more abundant BAT, independent of age and other body compositional parameters. This was not observed in men. The data suggest that brown adipogenesis may be physiologically related to modulation of bone density.

The IGF axis in prostate cancer.

Prostate cancer, the most frequent non-cutaneous malignancy in men from industrialized countries, is a growing medical problem, representing the second leading cause of male cancer deaths. In the last decade, converging evidence from epidemiological and biological studies suggests that the Insulin-like Growth Factor (IGF) axis is involved in the tumorigenesis and neoplastic growth of prostate cancer. Epidemiological observations indicated that circulating IGF-I levels are positively associated with the increased risk of prostate cancer. The activation of type I IGF receptor (IGF-IR) by IGF-I and/or IGF-II, has mitogenic and antiapoptotic effects on normal and malignant prostate cells. Altered expression of IGF axis components has also been reported in vitro and in animal models of prostate cancer, as well as in human prostate cancer tissue samples. In this review we address and analyze epidemiological studies, in vitro and in vivo cancer models, and human ex vivo prostate cancer researches performed to date supporting the role of IGF axis in prostate cancer.

Peripheral metabolism of thyroid hormone and glucose homeostasis.

Thyroid hormone action has long been recognized as an important determinant of glucose homeostasis. Recent advances in the knowledge of the physiology of the deiodinases indicate that through tissue-specific regulation of thyroid hormone metabolism, leading to local specificity of thyroid hormone action and target gene transcription patterns, they may have an important function in the modulation of carbohydrate metabolism. This review briefly addresses the role of thyroid hormone action on glucose homeostasis with a specific focus on the significance of the peripheral metabolism of thyroid hormone in the regulation of glucose homeostasis and insulin sensitivity.

Increased expression of thyroid hormone receptor isoforms in end-stage human congestive heart failure.

Thyroid hormone plays an important role on myocardial development and function. The local effects of thyroid hormone are mediated by the receptor isoforms ultimately driving the expression of cardiac-specific genes. Although overt and subclinical thyroid dysfunction causes well-known changes in the cardiovascular system, little is known about local thyroid hormone action in normal and failing human myocardium. With a newly developed multiplex competitive RT-PCR method, we evaluated the expression of thyroid hormone receptor (TR) isoforms alpha-1, alpha-2, and beta-1 in normal human hearts and in end-stage congestive heart failure. A statistically significant difference in the expression of all three TR isoforms was observed among samples from normal subjects, ischemic heart disease (IHD), and dilated cardiomyopathy (DCM). In DCM, compared with normal, the studied TR isoforms were significantly increased. In IHD, the increased expression was found significant only for alpha-1 and alpha-2 isoforms. No differences were observed between the pathologic groups. In conclusion, a coordinated increment in the expression of the TR isoforms was observed in both DCM and IHD by multiplex competitive RT-PCR. The observed changes could represent a compensatory mechanism to myocardial failure or to locally altered thyroid hormone action.

Age-related prevalence of platelet-associated immunoglobulins G in nonthrombocytopenic patients with autoimmune thyroid disease and autoimmune polyglandular syndrome.

OBJECTIVE: The prevalence of platelet-associated IgG (paIgG) in nonthrombocytopenic patients with autoimmune thyroid disease (AITD) alone or associated with autoimmune polyglandular syndrome (APS) has been studied. SUBJECTS: A total of 164 individuals were enrolled in this study: 81 patients with AITD alone, 33 patients with APS, and 50 healthy controls. RESULTS: The presence of paIgG was recorded in 41 of 81 patients with AITD (51%) as compared with 2 of 50 control subjects (4%, p < 0.0001). The prevalence of paIgG in patients with APS was higher even when compared with patients with AITD alone (25/33, 76%; p = 0.02). The presence of paIgG was not related to the functional thyroid parameters. The prevalence of paIgG was higher in the older than in the younger patients (75 vs. 47%, p = 0.0037). CONCLUSIONS: The results indicate that the prevalence of paIgG in patients with AITD is higher than previously thought, namely in elderly patients and in patients with APS, and not related to the thyroid function.

Molecular scanning for mutations in the insulin receptor substrate-1 (IRS-1) gene in Mexican Americans with Type 2 diabetes mellitus.

BACKGROUND: Insulin receptor substrate-1 (IRS-1) is an endogenous substrate for the insulin receptor tyrosine kinase, which plays an important role in insulin signaling. Mutations in the IRS-1 gene are associated in some populations with obesity and Type 2 diabetes. METHODS: To determine whether variation in the IRS-1 gene contributes to genetic susceptibility to insulin resistance and Type 2 diabetes in Mexican Americans, the entire coding region of the IRS-1 gene was screened for variation in 31 unrelated subjects with Type 2 diabetes using single-stranded conformational polymorphism analysis (SSCP) and dideoxy sequence analysis. Variants encoding amino acid substitutions were genotyped in 27 unrelated nondiabetic Mexican Americans and in all family members of subjects containing these variants, and association analyses were performed. To trace the ancestral origins of the variants, Iberian Caucasians and Pima Indians were also genotyped. RESULTS: Eight single base changes were found: four silent polymorphisms and four missense mutations (Ala94Thr, Ala512Pro, Ser892Gly and Gly971Arg). Allele frequencies were 0.009, 0.017, 0.017 and 0.043, respectively. There were no significant associations of any of these variants with diabetes, glucose or insulin levels during an oral glucose tolerance test, or with body mass index (BMI) in Mexican American families except for a modest association between the Ala94Thr variant and decreased BMI (30.4 kg/m(2) vs 24.0 kg/m(2); p=0.035). None of these four missense mutations were detected in Pima Indians. In Iberian Caucasians, neither Ala94Thr nor Ser892Gly were detected, and Ala512Pro was detected in only 0/60 diabetic patients and 1/60 nondiabetic controls. Gly971Arg was relatively more common in Iberian Caucasians with 12/58 diabetic patients and 7/60 nondiabetic controls being heterozygous for this variant (p=0.21 for comparison between diabetic and nondiabetic subjects). CONCLUSIONS: Ala94Thr, Ala512Pro and Ser892Gly mutation are rare in the populations studied. Gly971Arg, is more common in Mexican Americans and Caucasians, but is not a major contributor to genetic susceptibility to Type 2 diabetes.

Structural organization and chromosomal localization of the human type II deiodinase gene.

OBJECTIVE: The selenoenzyme type 2 iodothyronine 5' deiodinase (DII) catalyzes the conversion of thyroxine into its active form tri-iodothyronine (T3), modulating thyroid hormone homeostasis in a local, tissue-specific manner. The amphibian, rodent and human cDNAs encoding this enzyme have been recently cloned and expressed. At present, little information regarding the genomic structure of mammalian DII is available. DESIGN AND METHODS: The complete structure, including intron-exon junctions, of the human DII (hDII) gene was obtained by long PCR and rapid amplification of cDNA ends (RACE). Chromosomal assignment of the hDII gene was performed by fluorescence in situ hybridization using a highly specific probe. RESULTS AND CONCLUSIONS: Our data demonstrated that hDII is a single copy gene located on chromosome 14, position 14q24.3. The gene spans over 15 kb, and the 7 kb transcript is encoded by three exons of 149 bp, 273 bp and 6.6 kb separated respectively by two 274 bp and 7.4 kb introns. A restriction map of the hDII gene is also reported. These data will help in further studies of the role of DII in the maintenance of peripheral thyroid hormone homeostasis.

Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element.

We analyzed the structure and function of the 5' flanking region of the human type 2 deiodinase (hD2) gene. Two major transcription start sites were identified at -470/-474 from the ATG. The 5' flanking region of hD2 gene efficiently directed transcription in transient transfection studies, using luciferase as reporter gene, in HEK 293 cells. Basal transcriptional activity was significantly reduced by deleting the region containing a canonical cAMP-responsive element (CRE) located -766/-759 from ATG. Forskolin treatment significantly increased luciferase activity in cells transfected with CRE-containing constructs. This effect was abolished in constructs that did not contain CRE or contained the mutagenized CRE. Northern blot analysis in JEG-3 cells revealed that the hD2 messenger RNA was markedly increased after stimulation with cAMP agonist. The electrophoretic mobility shift assay with hD2-CRE probe and HEK 293 nuclear extract showed the occurrence of a DNA-protein complex, which was competed by specific unlabeled oligonucleotides and supershifted by the anti-CREB and anti-CRE modulator-1 antibodies. A-CREB, a dominant negative inhibitor of CREB, completely inhibited forskolin induction of the hD2 promoter. CREB protein, once cotransfected with hD2 promoter construct and pKA in F9 teratocarcinoma cells, which are unresponsive to cAMP, was able to stimulate the hD2 gene transcription. These results indicate the existence of a functional promoter within the 5' flanking region of hD2 gene which is characterized by the presence of a CRE. The specific involvement of CREB in the cAMP-mediated hD2 gene promoter induction also has been demonstrated.

Genomic characterization of the coding region of the human type II 5'-deiodinase gene.

Type II 5'-Deiodinase (5'DII) is a key element in the maintenance of peripheral thyroid hormone homeostasis through the regulation of local T4 to T3 conversion in pituitary, brain, brown adipose tissue and placenta. The cDNA containing the coding region of the human 5'DII (HDII) has been recently cloned from infant brain. In the present paper we report the genomic structure, chromosomal localization and restriction map of the coding region of HDII. The presence of a single intron located at codon 75 was demonstrated using a PCR-based strategy; the exon-intron junctions were then cloned and partially sequenced. Chromosomal localization was performed by radiation hybrid mapping. This study demonstrated that the entire coding region of the HDII gene is contained in two exons spliced at codon 75 by a 7.4 Kb intron and that the HDII chromosomal location is 14q24.3. These data will allow further studies of the role of HDII in the pathophysiology of thyroid homeostasis.

Lack of IRS-1 codon 513 and 972 polymorphism in Pima Indians.

Insulin receptor substrate-1 (IRS-1), an endogenous substrate for the insulin receptor tyrosine kinase, mediates many or all of the metabolic actions of insulin. Recently, polymorphism at codons 513 and 972 of the IRS-1 gene resulting in 2 amino acid substitutions that were associated with type II diabetes were found in a Caucasian population. Using allele specific oligonucleotide (ASO) hybridization, we screened 242 diabetic and 190 nondiabetic Pima Indians, a population with a very high prevalence of type II diabetes. Neither of the two mutations was present in either diabetic or nondiabetic subjects. We conclude that polymorphism at codons 513 and 972 of the IRS-1 gene observed in certain Caucasian populations is very rare or absent in Pima Indians.

Time of onset of non-insulin-dependent diabetes mellitus and genetic variation in the beta 3-adrenergic-receptor gene.

BACKGROUND: The beta 3-adrenergic receptor is expressed in visceral adipose tissue and is thought to contribute to the regulation of the resting metabolic rate and lipolysis. METHODS: To investigate whether mutations in the gene for the beta 3-adrenergic receptor predispose patients to obesity and non-insulin-dependent diabetes mellitus (NIDDM), we studied this gene in 10 Pima Indians by analysis of single-stranded conformational polymorphisms and dideoxy sequence analysis. Association studies were performed in 642 Pima subjects (390 with NIDDM and 252 without NIDDM). RESULTS: A missense mutation was identified in the gene for the beta 3-adrenergic receptor that results in the replacement of tryptophan by arginine (Trp64Arg) in the first intracellular loop of the receptor. This mutation was detected with allelic frequencies of 0.31 in Pima Indians, 0.13 in 62 Mexican Americans, 0.12 in 49 blacks, and 0.08 in 48 whites in the United States. Among Pimas, the frequency of the Trp64Arg mutation was similar in nondiabetic and diabetic subjects. However, in subjects homozygous for the mutation the mean (+/- SD) age at the onset of NIDDM was significantly lower (36 +/- 10 years) than in Trp64Arg heterozygotes (40 +/- 10 years) or normal homozygotes (41 +/- 11 years; P = 0.02). Furthermore, subjects with the mutation tended to have a lower adjusted resting metabolic rate (P = 0.14 by analysis of covariance). CONCLUSIONS: Pima subjects homozygous for the Trp64Arg beta 3-adrenergic-receptor mutation have an earlier onset of NIDDM and tend to have a lower resting metabolic rate. This mutation may accelerate the onset of NIDDM by altering the balance of energy metabolism in visceral adipose tissue.

The two nonallelic Xenopus insulin genes are expressed coordinately in the adult pancreas.

We have previously shown that the two nonallelic insulin genes in Xenopus laevis are expressed differentially during neurulation in prepancreatic embryos (Shuldiner et al., 1991, Proc. Natl. Acad. Sci. USA 88, 7679-7683). We now examine pancreatic expression with alterations in ambient temperature, glucose administration, fasting and feeding, somatostatin analog treatment, as well as during postmetamorphic growth. Insulin I and II mRNAs were quantitated by slot blot hybridization with specific probes and were expressed as the number of copies (x 10(8)) per 5 micrograms total RNA +/- SEM. Frogs maintained at 12 degrees showed no significant changes when compared to frogs maintained at 20 degrees. There was a coordinate decrease in insulin I and II mRNA levels in frogs maintained at 29 degrees (Ins I 20, 3.41 +/- 0.34 vs Ins I 29, 2.39 +/- 0.17; Ins II 20, 2.59 +/- 0.36 vs Ins II 29, 1.67 +/- 0.09; P < 0.05). When compared to fasting animals, both insulin I and II mRNA levels decreased slightly in frogs given repeated intraperitoneal injections of glucose and in those fed ad libitum; there were no changes after a single dose of glucose or in frogs given somatostatin. When compared to young frogs (6 to 24 months), older frogs (36 months) had higher insulin I and II mRNA levels (e.g., Ins I 6mo, 2.14 +/- 0.15 vs Ins I 36mo, 3.68 +/- 0.43; Ins II 6mo, 1.21 +/- 0.06 vs Ins II 36mo, 3.26 +/- 0.38; P < 0.05). Further, there was a modest reduction in the percentage of insulin I mRNA with aging (e.g., 6 months 63.6 +/- 3.1% vs 36 months 53.9 +/- 2.7%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of gene dosage by a quantitative adaptation of the polymerase chain reaction (gd-PCR): rapid detection of deletions and duplications of gene sequences.

Screening methods based on the polymerase chain reaction (PCR), such as denaturing gradient gel electrophoresis, single-stranded conformational polymorphism, and heteroduplex analysis, are powerful tools for the detection of point mutations as well as small deletions and insertions, but are unable to detect heterozygous deletions or duplications of exons, genes, or chromosomes. We now report a PCR-based approach, designated gene dosage-PCR (gd-PCR), that allows rapid screening for heterozygous deletions and duplications of genes or exons. Gene dosage-PCR is a quantitative method in which two in vitro synthesized DNA internal standards are coamplified with the genomic DNA sample, one corresponding to the gene of interest (test sequence) and the other to a reference (disomic) gene (reference sequence). Both internal standards are designed to be amplified with the same primer pairs and with efficiencies similar to those of their genomic DNA counterparts, yielding PCR products slightly smaller than those derived from genomic DNA. Amplification of approximately equimolar amounts of the two internal standards and genomic DNA, in the presence of [32P]dCTP, results in four radiolabeled PCR products; after electrophoresis and quantification of the products, gene dosage is easily calculated. For validation, genomic DNA from 56 subjects, 28 with cytogenetically documented Down syndrome (trisomy 21) and 28 controls that were disomic for chromosome 21, was assayed. Using the beta-amyloid precursor protein gene (APP: chromosome 21q21) as the test sequence, control subjects had an adjusted mean gene dose of 2.00 +/- 0.29, while subjects with Down syndrome had a mean gene dose of 3.05 +/- 0.27.(ABSTRACT TRUNCATED AT 250 WORDS)

D-lysine reduces the non-enzymatic glycation of proteins in experimental diabetes mellitus in rats.

D-Lysine, the non-physiological isomer of L-lysine, can competitively reduce protein non-enzymatic glycation in vitro. To study the effect of D-lysine in vivo, 6-8-week old Sprague-Dawley rats with streptozotocin-induced diabetes mellitus were treated from diagnosis for 45 days with two daily subcutaneous injections of D-lysine (0.5 g.ml-1.day-1). Another group of diabetic rats was only injected with equal volumes of physiological saline (0.9% NaCl). Glycated haemoglobin was measured by ion exchange chromatography, and glycated serum and lens proteins by boronate affinity gel chromatography. Serum and urinary creatinine concentrations were evaluated by the alkaline-picrate reaction. Urinary lysine concentrations at mid- and end-study were evaluated by cation exchange chromatography. Blood glucose concentrations, serum creatinine levels and creatinine clearances, measured at the end of the study, were similar in both diabetic groups (> 22.0 mmol/l, < or = 106 mumol/l and approximately 0.02 ml/s, respectively). Urinary lysine concentration in D-lysine-treated diabetic animals was more than 50-fold higher than in placebo-treated diabetic rats. In D-lysine-treated vs placebo-treated diabetic animals, a statistically significant reduction was found in the levels of glycated haemoglobin (stable HbA1; mean +/- SD = 3.00 +/- 0.74% vs 4.02 +/- 0.46%, p < 0.05; labile HbA1 = 3.92 +/- 0.89% vs 5.84 +/- 0.61%, p < 0.005), glycated serum proteins (1.40 +/- 0.47% vs 2.52 +/- 1.15%, p < 0.05) and glycated lens proteins (4.90 +/- 0.96% vs 5.98 +/- 0.65%, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)