Acute disruption of leptin signaling in vivo leads to increased insulin levels and insulin resistance

Leptin, an adipocyte-derived hormone, plays an essential role in the maintenance of normal body weight and energy expenditure, as well as glucose homeostasis. Indeed, leptin-deficient ob/ob mice are obese with profound hyperinsulinemia, insulin resistance, and often hyperglycemia. Interestingly, low doses of exogenous leptin can reverse the hyperinsulinemia and hyperglycemia in these animals without altering body weight. The hyperinsulinemia in ob/ob mice may result directly from the absence of leptin signaling in pancreatic β-cells and, in turn, contribute to both obesity and insulin resistance. Here, we acutely attenuated endogenous leptin signaling in normal mice with a polyethylene glycol (PEG)ylated mouse leptin antagonist (PEG-MLA) to determine the contribution of leptin signaling in the regulation of glucose homeostasis. PEG-MLA was either injected or continuously administered via osmotic minipumps for several days, and various metabolic parameters were assessed. PEG-MLA-treated mice had increased fasting and glucose-stimulated plasma insulin levels, decreased whole-body insulin sensitivity, elevated hepatic glucose production, and impaired insulin-mediated suppression of hepatic glucose production. Moreover, PEG-MLA treatment resulted in increased food intake and increased respiratory quotient without significantly altering energy expenditure or body composition as assessed by the lean:lipid ratio. Our findings indicate that alterations in insulin sensitivity occur before changes in the lean:lipid ratio and energy expenditure during the acute disruption of endogenous leptin signaling.     

Acute elevation of free fatty acid levels leads to hepatic insulin resistance in obese subjects

Raised levels of free fatty acids (FFA) compete with glucose for utilization by insulin-sensitive tissues, and, therefore, they may induce insulin resistance in the normal subject. The influence of experimental elevations in FFA levels on glucose metabolism in native insulin-resistant states is not known. We studied seven women with moderate obesity (63% above their ideal body weight) but normal glucose tolerance with the use of the insulin clamp technique with or without an infusion of Intralipid + heparin. Upon raising plasma insulin levels to approximately 60 microU/mL while maintaining euglycemia, whole body glucose utilization (3H-3-glucose) rose similarly without (from 66 +/- 7 to 113 +/- 11 mg/min m2, P less than .02) or with (from 70 +/- 7 to 137 +/- 19 mg/min m2, P less than .02) concomitant lipid infusion. In contrast, endogenous glucose production was considerably (73%) suppressed (from 66 +/- 7 to 15 +/- 8 mg/min m2, P less than .001) during the clamp without lipid, but declined only marginally (from 70 +/- 7 to 48 +/- 7 mg/min m2, NS) with lipid administration. The difference between the control and the lipid study was highly significant (P less than .02), and amounted to an average of 3.8 g of relative glucose overproduction during the second hour of the clamp. Blood levels of lactate rose by 34 +/- 15% (.1 greater than P greater than .05) in the control study but only by 17 +/- 10% (NS) during lipid infusion. Blood pyruvate concentrations fell in both sets of experiments (by approximately 45% at the end of the study) with similar time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic caffeine intake reverses age-induced insulin resistance in the rat: effect on skeletal muscle Glut4 transporters and AMPK activity

The role of caffeine consumption on insulin action is still under debate. The hypothesis that chronic caffeine intake reverses aging-induced insulin resistance in the rat was tested in this work. The mechanism by which caffeine restores insulin sensitivity was also investigated. Six groups of rats were used: 3 months old (3 M), 3 months old caffeine-treated (3MCaf), 12 months old (12 M), 12 months old caffeine-treated (12MCaf), 24 months old (24 M), and 24 months old caffeine-treated (24MCaf). Caffeine was administered in drinking water (1 g/l) during 15 days. Insulin sensitivity was assessed by means of the insulin tolerance test. Blood pressure, body weight, visceral and total fat, fasting glycemia and insulinemia, plasma nonesterified fatty acids (NEFA), total antioxidant capacity (TAC), cortisol, nitric oxide, and catecholamines were monitored. Skeletal muscle Glut4 and 5′-AMP activated protein kinase (AMPK) protein expression and activity were also assessed. Aged rats exhibited diminished insulin sensitivity accompanied by hyperinsulinemia and normoglycemia, increased visceral and total fat, decreased TAC and plasma catecholamines, and also decreased skeletal muscle Glut4 and AMPK protein expression. Chronic caffeine intake restored insulin sensitivity and regularized circulating insulin and NEFA in both aging models. Caffeine neither modified skeletal muscle AMPK expression nor activity in aged rats; however, it decreased visceral and total fat in 12 M rats and it restored skeletal muscle Glut4 expression to control values in 24 M rats. We concluded that chronic caffeine intake reverses aging-induced insulin resistance in rats by decreasing NEFA production and also by increasing Glut4 expression in skeletal muscle.     

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1^-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1^-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1^-/- islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1^-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1^-/- islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1^-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.     

Low-grade hypothalamic inflammation leads to defective thermogenesis, insulin resistance, and impaired insulin secretion

Hypothalamic inflammation is present in animal models of obesity, and the intracerebroventricular injection of TNFα can reproduce a number of features of the hypothalamus of obese animals. Because obesity is a risk factor for type 2 diabetes (DM2) we hypothesized that, by inducing hypothalamic inflammation, we could reproduce some clinical features of DM2. Lean Wistar rats and TNF receptor 1-knockout mice were employed to determine the effects of hypothalamic actions of TNFα on thermogenesis and metabolic parameters. Signal transduction and protein expression were evaluated by immunoblot and real-time PCR. Thermogenesis was evaluated in living rats, and respirometry was determined in isolated muscle fiber. In Wistar rats, hypothalamic TNFα blunts the anorexigenic effect of leptin, which is accompanied by reduced leptin signaling and increased expression of suppressor of cytokine signaling 3. In addition, hypothalamic TNFα reduces O(2) consumption and the expression of thermogenic proteins in brown adipose tissue and skeletal muscle. Furthermore, hypothalamic inflammation increases base-line plasma insulin and insulin secretion by isolated pancreatic islets, which is accompanied by an impaired insulin signal transduction in liver and skeletal muscle. Hypothalamic inflammation induced by stearic acid also reduces O(2) consumption and blunts peripheral insulin signal transduction. The use of intracerebroventricular infliximab restores O(2) consumption in obese rats, whereas TNF receptor 1-knockout mice are protected from diet-induced reduced thermogenesis and defective insulin signal transduction. Thus, low-grade inflammation of the hypothalamus is sufficient to induce changes in a number of parameters commonly impaired in obesity and DM2, and TNFα is an important mediator of this process.     

Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene

We have been using the human multiple drug resistance (MDR) gene to transduce murine hematopoietic cells via retroviruses as a model system for potential human gene therapy. In this paper, we show that transplantation of MDR-transduced midgestational fetal liver cells (FLCs) into lethally irradiated mice leads to the continued presence and expression of the human MDR gene in the short-lived granulocyte- macrophages of recipients' peripheral blood (PB) for up to 12 months. We have also shown the ability of this retroviral system to efficiently transduce several murine FLC subpopulations enriched for hematopoietic stem cells (FL-HSCs) both (1) short-term by MDR-polymerase chain reaction analysis of individual day 12 colony-forming unit-spleen and (2) long-term by in vivo maintenance of MDR and expression of its product, p-glycoprotein, up to 1 year in PB. More highly enriched FL- HSC subpopulations show the greatest number of circulating granulocyte- macrophage cells expressing MDR long-term. These studies also show that preselection by fluorescence-activated cell sorting of MDR-transduced and -expressing cells before transplant significantly increases the percentage of circulating granulocyte-macrophage cells that express MDR at all time points analyzed posttransplant as compared with unsorted cells transduced in the same manner (P < .01). These results have potentially significant implications for future human gene therapy trials.     

Long-term overexpression of glucokinase in the liver of transgenic mice leads to insulin resistance

Aims/hypothesis Glucokinase overexpression in the liver increases glucose uptake and utilization, and improves glucose tolerance in young transgenic mice. Here, we examined the long-term effects of hepatic overexpression of glucokinase on glucose homeostasis. Moreover, we determined whether glucokinase overexpression counteracted high-fat diet-induced insulin resistance.Methods Transgenic mice overexpressing glucokinase in liver under the control of the phosphoenolpyruvate carboxykinase promoter, fed either a standard diet or a high-fat diet, were studied. We used non-transgenic littermates as controls.Results Transgenic mice over 6 months old developed impaired glucose tolerance. In addition, at 12 months of age, transgenic mice showed mild hyperglycaemia, hyperinsulinaemia and hypertriglyceridaemia. In spite of increased glucokinase activity, the liver of these mice accumulated less glycogen and increased triglyceride deposition. When 2-month-old glucose-tolerant mice were fed a high-fat diet, transgenic mice gained more body weight and became hyperglycaemic and hyperinsulinaemic. This was concomitant to glucose intolerance, liver steatosis and whole-body insulin resistance.Conclusion/interpretation Long-term overexpression of glucokinase increases hepatic lipogenesis and circulating lipids, which lead to insulin resistance. Our results also suggest that the liver plays a key role in the onset of diabetes.Abbreviations GK glucokinase - PEPCK phosphoenolpyruvate carboxykinase     

Artificial induction of intravascular lipolysis by lipid-heparin infusion leads to insulin resistance in man

Summary Although extensive evidence indicates that free fatty acids can decrease glucose utilization in vitro, it is still controversial how an increase in lipolysis affects glucose metabolism in man. To test the hypothesis that an increase in lipolysis is related to insulin resistance, we examined the effect of lipid-heparin infusion on glucose metabolism in ten normal subjects by the euglycaemic glucose clamp technique and isotopic determination of glucose turnover. In the control euglycaemic clamp studies with insulin infusion at 0.2 and 1.0 mU·kg^–1·min^–1, endogenous glucose production was suppressed from the basal rate of 2.0±0.3 mg· kg^–1min^–1 to 1.1±0.7 mg·kg^–1·min^–1 and -0.4±0.7mg· kg^–1min^–1 respectively. Glucose utilization increased from the basal rate of 2.0±0.3 mg·kg^–1min^–1 to 2.3±0.5mg· kg^–1min^–1 and 5.9±1.8 mg·kg^–1min^–1 respectively. When the euglycaemic clamp studies were coupled with lipid-heparin infusion at comparable low and high rates of insulin infusion, endogenous glucose production increased (1.8± 0.7 mg·kg^–1·min^–1, p<0.001, and 0.3±0.6 mg·kg^–1· min^–1, p<0.05, respectively), and glucose utilization decreased (2.1±0.3 mg·kg^–1·min^–1, not significant, and 3.2±0.7 mg·kg^–1·min^–1, p<0.001 respectively). These data suggest that the artificial induction of intravascular lipolysis by lipid-heparin infusion leads to a state of insulin resistance in man.     

Decreased RNF41 expression leads to insulin resistance in skeletal muscle of obese women

ContextToll-like receptor 4 (TLR4) activation contributes to obesity-associated insulin resistance in skeletal muscles (SM). TLR4 signaling involves two pathways: the myeloid differentiation primary response gene 88 (MyD88) leading to inflammatory cytokines production and the toll/interleukin-1 receptor domain-containing adapter-inducing interferon (IFN) I (TRIF)-dependent pathways leading to type 1 interferon (IFNI) and interferon stimulated genes (ISG) expression. The E3 ubiquitin ligase RNF41 allows the preferential activation of the TRIF-IFNI pathway; however, its role in insulin response has not been reported.MethodsWe measured RNF41 level and IFNI pathway activation (ISG expression) in SM biopsies of obese insulin sensitive (OIS) and obese insulin resistant (OIR) women. Then we isolated and differentiated in myotubes, primary human SM cell progenitors from OIS and OIR SM biopsies. We modulated RNF41 and ISG expression in these myotubes and investigated their effects on insulin response.ResultsRNF41 expression is down-regulated in vivo in OIR SM and myotubes compared to OIS SM and myotubes. TLR4 activation with palmitate induces TRIF-IFNI pathway and ISG in OIS myotubes but not in OIR myotubes. Inhibition of RNF41 expression with siRNF41 in OIS myotubes treated with palmitate attenuates insulin response, IFNI pathway activation and ISG induction, mimicking OIR phenotype. Further, overexpression of RNF41 in OIR myotubes increases insulin response and ISG expression. Exposure to IFNI or to its inducer polyinosinic-polycytidylic acid, restores ISG expression and insulin sensitivity in OIR myotubes and OIS myotubes transfected with siRNF41.ConclusionOur results identify RNF41 as essential to IFNI pathway activation in order to maintain muscle insulin sensitivity during human obesity.     

Established diet-induced obesity in female rats leads to offspring hyperphagia, adiposity and insulin resistance

Aims/hypothesis  Accumulating evidence suggests that maternal obesity may increase the risk of metabolic disease in the offspring. We investigated the effects of established maternal diet-induced obesity on male and female offspring appetite, glucose homeostasis and body composition in rats. Methods  Female Wistar rats were fed either a standard chow (3% fat, 7% sugar [wt/wt]) or a palatable obesogenic diet (11% fat, 43% sugar [wt/wt]) for 8 weeks before mating and throughout pregnancy and lactation. Male and female offspring of control and obese dams were weaned on to standard chow and assessed until 12 months of age. Results  At mating, obese dams were heavier than control with associated hyperglycaemia and hyperinsulinaemia. Male and female offspring of obese dams were hyperphagic (p < 0.0001) and heavier than control (p < 0.0001) until 12 months of age. NEFA were raised at 2 months but not at 12 months. At 3 months, OGTT showed more pronounced alteration of glucose homeostasis in male than in female offspring of obese animals. Euglycaemic–hyperinsulinaemic clamps performed at 8 to 9 months in female and 10 to 11 months in male offspring revealed insulin resistance in male offspring of obese dams (p < 0.05 compared with control). Body compositional analysis at 12 months also showed increased fat pad weights in male and female offspring of obese animals. Conclusions/interpretation  Diet-induced obesity in female rats leads to a state of insulin resistance in male offspring, associated with development of obesity and increased adiposity. An increase in food intake may play a role.     

Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming

Maternal obesity is increasingly prevalent and may affect the long-term health of the child. We investigated the effects of maternal diet-induced obesity in mice on offspring metabolic and cardiovascular function. Female C57BL/6J mice were fed either a standard chow (3% fat, 7% sugar) or a palatable obesogenic diet (16% fat, 33% sugar) for 6 weeks before mating and throughout pregnancy and lactation. Offspring of control (OC) and obese dams (OO) were weaned onto standard chow and studied at 3 and 6 months of age. OO were hyperphagic from 4 to 6 weeks of age compared with OC and at 3 months locomotor activity was reduced and adiposity increased (abdominal fat pad mass; P<0.01). OO were heavier than OC at 6 months (body weight, P<0.05). OO abdominal obesity was associated with adipocyte hypertrophy and altered mRNA expression of beta-adrenoceptor 2 and 3, 11 beta HSD-1, and PPAR-gamma 2. OO showed resistance artery endothelial dysfunction at 3 months, and were hypertensive, as assessed by radiotelemetry (nighttime systolic blood pressure at 6 months [mm Hg] mean+/-SEM, male OO, 134+/-1 versus OC, 124+/-2, n=8, P<0.05; female OO, 137+/-2 versus OC, 122+/-4, n=8, P<0.01). OO skeletal muscle mass (tibialis anterior) was significantly reduced (P<0.01) OO fasting insulin was raised at 3 months and by 6 months fasting plasma glucose was elevated. Exposure to the influences of maternal obesity in the developing mouse led to adult offspring adiposity and cardiovascular and metabolic dysfunction. Developmentally programmed hyperphagia, physical inactivity, and altered adipocyte metabolism may play a mechanistic role.     

Short-term in vivo inhibition of insulin receptor substrate-1 expression leads to insulin resistance, hyperinsulinemia, and increased adiposity

Insulin receptor substrate-1 (IRS-1) has an important role as an early intermediary between the insulin and IGF receptors and downstream molecules that participate in insulin and IGF-I signal transduction. Here we employed an antisense oligonucleotide (IRS-1AS) to inhibit whole-body expression of IRS-1 in vivo and evaluate the consequences of short-term inhibition of IRS-1 in Wistar rats. Four days of treatment with IRS-1AS reduced the expression of IRS-1 by 80, 75, and 65% (P < 0.05) in liver, skeletal muscle, and adipose tissue, respectively. This was accompanied by a 40% (P < 0.05) reduction in the constant of glucose decay during an insulin tolerance test, a 78% (P < 0.05) reduction in glucose consumption during a hyperinsulinemic-euglycemic clamp, and a 90% (P < 0.05) increase in basal plasma insulin level. The metabolic effects produced by IRS-1AS were accompanied by a significant reduction in insulin-induced [Ser (473)] Akt phosphorylation in liver (85%, P < 0.05), skeletal muscle (40%, P < 0.05), and adipose tissue (85%, P < 0.05) and a significant reduction in insulin-induced tyrosine phosphorylation of ERK in liver (20%, P < 0.05) and skeletal muscle (30%, P < 0.05). However, insulin-induced tyrosine phosphorylation of ERK was significantly increased (60%, P < 0.05) in adipose tissue of IRS-1AS-treated rats. In rats treated with IRS-1AS for 8 d, a 100% increase (P < 0.05) in relative epididymal fat weight and a 120% (P < 0.05) increase in nuclear expression of peroxisome proliferator-activated receptor-gamma were observed. Thus, acute inhibition of IRS-1 expression in rats leads to insulin resistance accompanied by activation of a growth-related pathway exclusively in white adipose tissue.     

SSADH deficiency leads to elevated extracellular GABA levels and increased GABAergic neurotransmission in the mouse cerebral cortex

Summary  Succinic semialdehyde dehydrogenase (SSADH) deficiency is an inherited disorder in which patients display neurodevelopmental retardation, ataxia, and epileptic seizures. The recently engineered SSADH knock-out (KO) mouse models the severe form of the human disorder. The SSADH enzyme participates in the breakdown of the inhibitory neurotransmitter GABA, and studies have shown increases in brain GABA and downregulation of GABA_A receptor β₂ subunits in the cerebral cortex of these mice. Here, we used brain slice electrophysiology to investigate the alterations in GABA neurotransmission in SSADH KO mouse cortex. In layer 2/3 pyramidal cells, spontaneous inhibitory postsynaptic currents (IPSCs), reflecting activity of GABAergic synaptic contacts, were normal in SSADH KO mice. Also, IPSCs evoked by electrical single-axon stimulation in KO mice were normal. In contrast, tonic inhibition mediated by presumed extrasynaptic GABA_A receptors was strongly increased, indicating significantly raised extracellular GABA levels. The excessive cortical GABAergic neurotransmission may participate in the seizure activity in SSADH deficiency. Competing interests: None declared References to electronic databases: Succinate semialdehyde dehydrogenase deficiency: ALDH5A1; OMIM 271980, 610045.     

Ethnic differences in the ability of triglyceride levels to identify insulin resistance

The Metabolic Syndrome is used to predict the onset of coronary artery disease and Type 2 diabetes. As the predictive value of the Metabolic Syndrome has been challenged, alternative syndromes have been developed. All of these syndromes were developed in populations that were predominantly non-Hispanic white (NHW). They include the Enlarged Waist Elevated Triglyceride Syndrome, the Overweight-Lipid Syndrome and the Hypertriglyceridemic Waist Syndrome. The first applies to postmenopausal women, the second to overweight individuals (BMI> or =25 kg/m(2)), and the third to men. Each syndrome uses hypertriglyceridemia as a criterion. However, the definition of hypertriglyceridemia varies by syndrome i.e. TG> or =128 mg/dL for the Enlarged Waist Elevated Triglyceride Syndrome, TG> or =130 mg/dL for the Overweight-Lipid Syndrome, > or =150 mg/dL for the Metabolic Syndrome, and TG> or =176 mg/dL for the Hypertriglyceridemic Waist Syndrome. Insulin resistance and hypertriglyceridemia are highly correlated. But as insulin resistant non-Hispanic blacks (NHB) often have triglyceride (TG) levels below the thresholds set by these syndromes, the ability of either TG or these syndromes to identify high risk NHB is unknown. Using the National Health and Nutrition Examination Survey (NHANES) 1999-2002, our goals were to determine by ethnicity: (1) the prevalence of each of these syndromes; (2) the ability of fasting TG concentrations to identify insulin resistance at cut-off levels established by these syndromes, specifically 130, 150 and 176 mg/dL. Participants were 2804 adults from NHANES 1999-2002. The cohort was divided into tertiles of homeostasis model assessment. Insulin resistance was defined as the upper tertile (> or =2.73). The prevalence of each syndrome was lower in NHB than NHW or Mexican Americans (MA) (all P<0.05). Mean TG levels in NHB, non-Hispanic Whites (NHW) and Mexican Americans (MA) were: 99, 140 and 144mg/dL, respectively. The mean percents of insulin-resistant NHB, NHW and MA with TG<130mg/dL were: 64, 31 and 36. The percents of insulin-resistant NHB, NHW and MA with TG<150mg/dL were: 75, 46 and 47. The percents of insulin-resistant NHB, NHW and MA with TG<176 mg/dL were: 81, 58 and 59. Significance was P<0.001 for each comparison to NHB. In conclusion, the prevalence of syndromes that use TG as a diagnostic criterion is lower in NHB than NHW or MA. NHB are more likely than NHW or MA to be insulin-resistant and have TG levels below threshold values. As syndromes are formulated to identify individuals at high risk for conditions such as cardiovascular disease and Type 2 diabetes, ethnic differences in TG levels should be considered.     

Insulin resistance in obesity is associated with elevated basal lactate levels and diminished lactate appearance following intravenous glucose and insulin.

Lactate metabolism is altered in obesity. Increasing obesity is associated with increased blood lactate levels after an overnight fast. In contrast, we have recently shown a marked decrease in the capacity for acute lactate generation in obese subjects following an oral glucose load, which we postulated might be linked to altered insulin sensitivity. In the present study, we systematically analyzed the relationship between insulin sensitivity (the Sensitivity Index [SI] derived using the minimal model), body mass index (BMI), and glucose, insulin, and lactate levels in the basal state and following intravenous (IV) glucose and insulin administration in lean and obese subjects. The results showed that SI and BMI were inversely related, as expected. Insulin sensitivity was more tightly associated with glucose, insulin, and lactate levels (both basal and integrated) than obesity per se. A significant inverse relationship was found between SI and basal lactate levels (r = -.56). Moreover, a significant and positive relationship was found between SI and incremental lactate area under the curve (reflecting acute lactate production) (r = .41). In a multiple regression analysis to separate the independent effects of obesity (BMI) and insulin sensitivity, after adjusting for age, sex, and race, SI accounted for 34% of the variance in basal lactate and 24% of the variance in incremental lactate area. Obesity independently accounted for 10% of the variance in basal lactate and 11% of the variance in incremental lactate area, neither of which were statistically significant. We conclude that elevations in basal lactate are associated with the development of insulin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)