FGF21 is not required for glucose homeostasis, ketosis or tumour suppression associated with ketogenic diets in mice.

AIMS/HYPOTHESIS: Ketogenic diets (KDs) have increasingly gained attention as effective means for weight loss and potential adjunctive treatment of cancer. The metabolic benefits of KDs are regularly ascribed to enhanced hepatic secretion of fibroblast growth factor 21 (FGF21) and its systemic effects on fatty-acid oxidation, energy expenditure (EE) and body weight. Ambiguous data from Fgf21-knockout animal strains and low FGF21 concentrations reported in humans with ketosis have nevertheless cast doubt regarding the endogenous function of FGF21. We here aimed to elucidate the causal role of FGF21 in mediating the therapeutic benefits of KDs on metabolism and cancer. METHODS: We established a dietary model of increased vs decreased FGF21 by feeding C57BL/6J mice with KDs, either depleted of protein or enriched with protein. We furthermore used wild-type and Fgf21-knockout mice that were subjected to the respective diets, and monitored energy and glucose homeostasis as well as tumour growth after transplantation of Lewis lung carcinoma cells. RESULTS: Hepatic and circulating, but not adipose tissue, FGF21 levels were profoundly increased by protein starvation, independent of the state of ketosis. We demonstrate that endogenous FGF21 is not essential for the maintenance of normoglycaemia upon protein and carbohydrate starvation and is therefore not needed for the effects of KDs on EE. Furthermore, the tumour-suppressing effects of KDs were independent of FGF21 and, rather, driven by concomitant protein and carbohydrate starvation. CONCLUSIONS/INTERPRETATION: Our data indicate that the multiple systemic effects of KD exposure in mice, previously ascribed to increased FGF21 secretion, are rather a consequence of protein malnutrition.

Classification of images acquired with colposcopy using artificial neural networks.

OBJECTIVE: To explore the advantages of using artificial neural networks (ANNs) to recognize patterns in colposcopy to classify images in colposcopy. PURPOSE: Transversal, descriptive, and analytical study of a quantitative approach with an emphasis on diagnosis. The training test e validation set was composed of images collected from patients who underwent colposcopy. These images were provided by a gynecology clinic located in the city of Criciúma (Brazil). The image database (n = 170) was divided; 48 images were used for the training process, 58 images were used for the tests, and 64 images were used for the validation. A hybrid neural network based on Kohonen self-organizing maps and multilayer perceptron (MLP) networks was used. RESULTS: After 126 cycles, the validation was performed. The best results reached an accuracy of 72.15%, a sensibility of 69.78%, and a specificity of 68%. CONCLUSION: Although the preliminary results still exhibit an average efficiency, the present approach is an innovative and promising technique that should be deeply explored in the context of the present study.

Augmented renal clearance in septic patients and implications for vancomycin optimisation.

The aim of this study was to evaluate the effect of augmented renal clearance (ARC) on vancomycin serum concentrations in critically ill patients. This prospective, single-centre, observational, cohort study included 93 consecutive, critically ill septic patients who started treatment that included vancomycin by continuous infusion, admitted over a 2-year period (March 2006 to February 2008). ARC was defined as 24-h creatinine clearance (CL(Cr))>130 mL/min/1.73 m(2). Two groups were analysed: Group A, 56 patients with a CL(Cr)≤130 mL/min/1.73 m(2); and Group B, 37 patients with a CL(Cr)>130 mL/min/1.73 m(2). Vancomycin therapeutic levels were assessed on the first 3 days of treatment (D(1), D(2) and D(3)). Serum vancomycin levels on D(1), D(2) and D(3), respectively, were 13.1, 16.6 and 18.6 µmol/L for Group A and 9.7, 11.7 and 13.8 µmol/L for Group B (P<0.05 per day). The correlation between CL(Cr) and serum vancomycin on D(1) was -0.57 (P<0.001). ARC was strongly associated with subtherapeutic vancomycin serum concentrations on the first 3 days of treatment.

Bypassing the duodenum does not improve insulin resistance associated with diet-induced obesity in rodents.

Roux-en-y gastric bypass (RYGB) surgery rapidly improves glucose tolerance and reverses insulin resistance in obese patients. It has been hypothesized that this effect is mediated by the diversion of nutrients from the proximal small intestine. We utilized duodenal-jejunal bypass (DJB) as a modification of gastric bypass to determine the effect of nutrient diversion from the foregut without gastric restriction on insulin resistance in obese rats. The effects of DJB or Sham surgery on glucose homeostasis were determined in both high-fat-fed Long-Evans and Wistar rats. Body weight and food intake were measured weekly postoperatively, and body composition was monitored before and after surgery. Glucose tolerance was tested before and as early as 1 month postoperation; additionally, in Wistar rats, insulin sensitivity was determined by a hyperinsulinemic-euglycemic clamp (HIEC). DJB did not affect body weight, body composition, glucose tolerance, or insulin concentrations over the period of the study. The average glucose infusion rate (GIR) during the HIEC was 6.2 ± 1.16 mg/kg/min for Sham rats compared to 7.2 ± 1.71 mg/kg/min for DJB rats (P = 0.62), and neither endogenous glucose production (EGP; P = 0.81) nor glucose utilization (glucose disappearance (R(d)), P = 0.59) differed between DJB and Sham rats. DJB does not affect insulin resistance induced by a high-fat diet in Long-Evans and Wistar rats. These data suggest that duodenal bypass alone is an insufficient mechanism to alter insulin sensitivity independent of weight loss in obese, nondiabetic rodents.

Central nervous system delivery of the antipsychotic olanzapine induces hepatic insulin resistance.

OBJECTIVE: Olanzapine (OLZ) is an atypical antipsychotic whose clinical efficacy is hampered by side effects including weight gain and diabetes. Recent evidence shows that OLZ alters insulin sensitivity independent of changes in body weight and composition. The present study addresses whether OLZ-induced insulin resistance is driven by its central actions. RESEARCH DESIGN AND METHODS: Sprague-Dawley rats received an intravenous (OLZ-IV group) or intracerebroventricular (OLZ-ICV group) infusion of OLZ or vehicle. Glucose kinetics were assessed before (basal period) and during euglycemic-hyperinsulinemic clamp studies. RESULTS: OLZ-IV caused a transient increase in glycemia and a higher rate of glucose appearance (R(a)) in the basal period. During the hyperinsulinemic clamp, the glucose infusion rate (GIR) required to maintain euglycemia and the rate of glucose utilization (R(d)) were decreased in OLZ-IV, whereas endogenous glucose production (EGP) rate was increased compared with vehicle-IV. Consistent with an elevation in EGP, the OLZ-IV group had higher hepatic mRNA levels for the enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Phosphorylation of hypothalamic AMP-activated protein kinase (AMPK) was increased in OLZ-IV rats compared with controls. Similarly, an intracerebroventricular infusion of OLZ resulted in a transient increase in glycemia as well as a higher R(a) in the basal period. During the hyperinsulinemic period, OLZ-ICV caused a decreased GIR, an increased EGP, but no change in R(d). Furthermore, OLZ-ICV rats had increased hepatic gluconeogenic enzymes and elevated hypothalamic neuropeptide-Y and agouti-related protein mRNA levels. CONCLUSIONS: Acute central nervous system exposure to OLZ induces hypothalamic AMPK and hepatic insulin resistance, pointing to a hypothalamic site of action for the metabolic dysregulation of atypical antipsychotics.

Neonatal arthritis disturbs sleep and behaviour of adult rat offspring and their dams.

This study aims to evaluate the impact of neonatal arthritis on adult pain threshold, sleep and general behaviours in rats and their lactating dams. Male pups were injected in the hind paw with complete Freund's adjuvant or saline on postnatal day (PN) 1. After weaning, dams were tested for anxiety, sleep recording or hormone profiling (ACTH, corticosterone and prolactin) and brain sampling (pineal melatonin and hippocampus serotonin). At adulthood (PN90), distinct subgroups of neonatal arthritic (AR) and control rats (CR) were also assessed for anxiety and pain thresholds, sleep recording, and blood/brain sampling. Compared to their respective controls at PN12, dams of arthritic rats (DAR) showed a longer latency in expressing pup retrieval and dam-pup interaction. DAR and AR showed a lower pain threshold, anxiety-like behaviour, and sleep fragmentation. Compared to controls, DAR displayed longer sleep latency, reduced paradoxical sleep latency and sleep efficiency, a decrease in prolactin and serotonin levels and increased melatonin levels. This model of unilateral hindpaw inflammation has a wide range of long-term effects in both lactating dams and their adult offspring.

Long term treatment with ACE inhibitor enalapril decreases body weight gain and increases life span in rats.

Renin-angiotensin system is involved in homeostasis processes linked to renal and cardiovascular system and recently has been linked to metabolic syndrome. We analyzed the influence of long term angiotensin I converting enzyme (ACE) inhibitor enalapril treatment in normotensive adult Wistar rats fed with standard or palatable hyperlipidic diets. Our results show that long term enalapril treatment decreases absolute food intake, serum leptin concentration and body weight gain. Moreover, in adipose tissue, enalapril treatment led to decreased ACE activity, enhanced the expression of peroxisome proliferator activated receptor gamma, adiponectin, hormone-sensitive lipase, fatty acid synthase, catalase and superoxide dismutase resulting in prolonged life span. On the other hand, the ACE inhibitor was not able to improve the transport of leptin through the blood brain barrier or to alter the sensitivity of this hormone in the central nervous system. The effect of enalapril in decreasing body weight gain was also observed in older rats. In summary, these results extend our previous findings and corroborate data from the literature regarding the beneficial metabolic effects of enalapril and show for the first time that this ACE inhibitor prolongs life span in rats also fed with palatable hyperlipidic diet, an action probably correlated with adipose tissue metabolic modulation and body weight reduction.

Oxidative stress contributes to renovascular hypertension.

BACKGROUND: Oxidative stress is a state in which excess reactive oxygen species (ROS) overwhelm endogenous antioxidant systems. It is known that this state has been involved in the development of hypertension. On the basis of previous data, we hypothesized that overactivity of NAD(P)H oxidase-derived ROS and the lowered activity of CuZnSOD, an endogenous antioxidant within the rostral ventrolateral medulla (RVLM), could contribute to 2K-1C (two-kidney one-clip) hypertension. Moreover, to test the functional significance of whether oxidative stress was involved in the maintenance of sympathetic vasomotor tone and blood pressure in 2K-1C hypertension, we administered Ascorbic Acid (Vit C), an antioxidant, into the RVLM or systemically. METHODS: Experiments were performed in male Wistar rats (6 weeks after renal surgery--Goldblatt hypertension model--2K-1C). The mRNA expression of NAD(P)H oxidase subunits (p47phox and gp91phox) and CuZnSOD were analyzed in the RVLM using real-time PCR technique. The mean arterial blood pressure, heart rate, and renal sympathetic nerve activity were analyzed. Blood samples were collected and measured using thiobarbituric acid-reactive substances (TBARS). RESULTS: The mRNA expression of NAD(P)H oxidase subnits (p47phox and gp91pox) was greater in 2K-1C compared to the control group in the RVLM, and CuZnSOD expression was similar in both groups. In the RVLM, Vit C resulted in a fall in arterial pressure and in the sympathetic activity only in the 2K-1C rats. Thiobarbituric acid-reactive substances (TBARS) were significantly greater in 2K-1C rats and the acute infusion of Vit C significantly decreased arterial pressure and renal sympathetic activity in 2K-1C. CONCLUSIONS: The results support the idea that an increase in oxidative stress within the RVLM and systemically plays a major role in maintaining high arterial blood pressure and sympathetic drive in 2K-1C hypertension.

A reassessment of the hyperphagia/weight-loss paradox during sleep deprivation.

STUDY OBJECTIVES: Sleep deprivation is a well-known paradigm to investigate the deleterious effects of prolonged wakefulness. Previous studies have shown that, during sleep deprivation, rats are hyperphagic but, paradoxically, lose body weight. This phenomenon has been attributed to increased metabolism. However, most previous studies have failed to account for food spillage, which may be considerable during sleep deprivation. DESIGN: In the present study, we revisited the issue of feeding changes in sleep-deprived rats and introduced different procedures to allow accurate estimation of food spillage prior to, during, and after 120 hours of sleep deprivation by a single platform technique. SETTING: Animal Sleep Research Laboratory, Psychobiology Department, Universidade Federal de São Paulo, Brazil. MEASUREMENTS AND RESULTS: The main finding was that, once corrected for spillage, food intake was not significantly increased during sleep deprivation. Increases in food removed from feeders were accompanied by proportional increases in food spillage, resulting in no net changes in food intake. Further, weight loss did occur during the sleep-deprivation period, especially in the first 24 hours, and it was actually explained by a reduction in food intake. CONCLUSION: The hyperphagia/weight-loss paradox previously seen during prolonged sleep deprivation does not necessarily occur with shorter periods of deprivation. Although we found no evidence of hyperphagia for up to 5 days of sleep deprivation in chow-fed rats, our data suggest that an impairment in the ability to increase food intake in response to increased energy expenditure contributes to the energy deficit during sleep deprivation in rats.

Effects of paradoxical sleep deprivation and cocaine on genital reflexes in hyperlipidic-fed rats.

The present study was designed to investigate the effects of a hyperlipidic diet (HD) on penile erection (PE) and ejaculation (EJ) induced by cocaine in paradoxical sleep deprived (PSD) rats. Secondly, we aimed to verify the influence of HD cafeteria diet on steroid hormone levels. Twenty-one day-old male Wistar rats were randomly assigned into two groups: rats fed with commercial chow diet and rats fed with a palatable HD containing chow mixed with peanuts, milk chocolate and sweet cookies in the proportion of 3:2:2:1. After nine weeks of treatment, the animals were submitted to PSD or maintained as home cage control group for 96 h and challenged with cocaine (7 mg/kg, i.p.). Results showed that the HD led to a reduction in the frequency of erection in the PSD+cocaine group when compared to the PSD+cocaine fed with standard diet. Regardless of the diet, testosterone concentrations were significantly lower and progesterone was higher in the PSD rats than in the respective home-cage control rats. Although there were no hormonal alterations, the findings showed that a long-term HD might modify the stimulating effects of cocaine in potentiating genital reflexes in PSD rats.

Endocrinological and catecholaminergic alterations during sleep deprivation and recovery in male rats.

Since previous data of our group showed increased concentrations in HPA axis hormones in sleep deprived rats, we hypothesized that this augmentation could produce effects in other hormonal systems, particularly in the sexual system. Considering that little is known about how the hormonal system changes during the recovery period after sleep deprivation (SD), our objective was to examine from what point SD alters sexual and stress-related hormones along with plasma catecholamine concentrations during 4 days. We also sought to verify the time course of their recovery after an equivalent period of recovery sleep. Rats were deprived of sleep by the platform technique for 1-4 days and were allowed to recover for the same period. Plasma catecholamines [dopamine (DA) and noradrenaline (NOR)], testosterone, estrone, progesterone, prolactin, corticosterone and adrenocorticotropic hormone (ACTH) concentrations were measured. Comparisons between groups showed that the SD procedure used in the present study produced marked alterations in almost all studied hormones from 24 h of SD, except for estrone and prolactin (which required 96 h of SD to become altered). Testosterone and estrone decreased, whereas progesterone, prolactin, corticosterone, ACTH, DA and NOR increased. During recovery period, progesterone, prolactin and corticosterone concentrations returned to control levels, whereas testosterone, estrone, NOR and DA did not. In addition, after 48 h of recovery ACTH and NOR decreased below control concentrations, remaining low until 96 h of sleep recovery. Thus, SD showed long lasting, differential effects upon these neurochemicals suggesting that each has its own pattern of responses to SD as well as variable periods of recovery.

Increased hypocretin-1 (orexin-a) levels in cerebrospinal fluid of rats after short-term forced activity.

The hypocretins (orexins) are recently discovered neuropeptides initially associated with feeding behavior and sleep regulation. However, the normal function of these peptides is unclear and a number of studies have reported a role in energy homeostasis and locomotor activity. Exercise (or physical activity) is the most powerful way of challenging the internal homeostatic process. This study examines the circadian differences in response to forced activity and homeostatic challenges on hypocretin-1 (Hcrt-1) levels in the cerebrospinal fluid (CSF) of rats. Hcrt-1 levels were decreased after long-term immobilization at the end of active phase (zeigeber time-0, ZT-0) and increased after short-term forced swimming in the rest phase (ZT-8). Nevertheless, no effects were observed after short-term immobilization, total sleep deprivation or cold exposure. We concluded that despite the relation between hypocretins, stress and sleep regulation reported in the literature, short-term total sleep deprivation, immobilization and cold exposure did not induce increases in CSF Hcrt-1 levels at ZT-0 and ZT-8. On the other hand, the relationship between hypocretinergic system activation and motor activation is reinforced by decrease in Hcr-1 levels after long-term immobilization at ZT-0 and its increased levels after short-term forced swimming at ZT-8 in CSF of rats.

Increased hypocretin-1 levels in cerebrospinal fluid after REM sleep deprivation.

Rat cisternal (CSF) hypocretin-1 in cerebrospinal fluid was measured after 6 or 96 h of REM sleep deprivation and following 24 h of REM sleep rebound. REM deprivation was found to increase CSF hypocretin-1 collected at zeitgeber time (ZT) 8 but not ZT0. Decreased CSF hypocretin levels were also observed at ZT8 after 24 h of REM sleep rebound. These results suggest that REM sleep deprivation activates and REM sleep rebound inhibits the hypocretin system. Increased hypocretin tone during REM deprivation may be important in mediating some of the effects of REM sleep deprivation such as antidepressant effects, hyperphagia and increased sympathetic activity.