Physiology and Endocrinology Symposium: FGF21: Insights into mechanism of action from preclinical studies.

Fibroblast growth factor 21 (FGF21) is a multifaceted metabolic regulator which has several potential applications in the treatment of metabolic disease. When administered in vivo, FGF21 exhibits a plethora of actions, modulating metabolic homeostasis in a diverse manner. However, the mechanism and site of action underlying these effects were, until recently, entirely uncertain. Using mouse models lacking either FGF receptor isoform 1 (FGFR1) or ßKlotho (KLB), a transmembrane co-factor critical for FGF21 action, our group and others sought to determine the tissue on which FGF21 acts and the receptor complex responsible for mediating its in vivo efficacy. Importantly, when KLB was ablated from all tissues mice were completely refractory to FGF21 action. Therefore, to determine the precise tissue of action we utilized mice with tissue specific deletion of FGFR1 in either adipose tissue or neurons, respectively. Surprisingly, in animals with neuronal FGFR1 loss there was no change in the metabolic activity of FGF21, suggesting a lack of central FGF21 action in the pharmacologic setting. In contrast, we found dramatic attenuation of metabolic efficacy in mice with adipose-specific FGFR1 ablation following either acute or chronic dosing with recombinant FGF21. Furthermore, several recent studies have suggested that the metabolic effects of FGF21 may occur via modulation of adipokines such as adiponectin and leptin. Importantly, the action of FGF21 via adipose tissue results in alterations in both secretion as well as systemic sensitivity to these factors. Therefore, while FGF21 itself does not seem to directly act on the CNS, leptin and other endocrine mediators may serve as intermediary facilitators of FGF21's secondary central effects downstream of an initial and direct engagement of FGF21 receptor complex in adipose tissue. Further studies are required to delineate the precise mechanistic basis underlying the interplay between peripheral and central FGF21 modes of action in both the physiological and pharmacological settings.

Increased responses to the actions of fibroblast growth factor 21 on energy balance and body weight in a seasonal model of adiposity.

The present study aimed to investigate the actions of fibroblast growth factor 21 (FGF21) on energy balance in a natural model of relative fatness, the Siberian hamster. Hamsters were studied under long days (LD) to promote weight gain, or short days to induce weight loss, and treated with rhFGF21 (3 mg/kg/day) via s.c. minipumps for 14 days. On days 7-9, detailed assessments of ingestive behaviour, metabolic gas exchange and locomotor activity were made. FGF21 caused substantial (P < 0.0001) weight loss in the fat LD state but not in the lean SD state: at the end of the study, FGF21-treated hamsters in LD lost 18% of body weight compared to vehicle controls, which is comparable to the natural body weight loss observed in SD. Epididymal fat pads, a correlate of total carcass fat content, were reduced by 19% in FGF21 treated hamsters in LD, whereas no difference was found in SD. Body weight loss in LD was associated with a reduction in food intake (P < 0.001) and a decreased respiratory exchange ratio (P < 0.001), indicating increased fat oxidation. Treatment with FGF21 maintained the normal nocturnal increase in oxygen consumption and carbon dioxide production into the early light phase in hamsters in LD, indicating increased energy expenditure, although locomotor activity was unaffected. These data suggest a greater efficacy of FGF21 in hamsters in LD compared to those in SD, which is consistent with both the peripheral and possibly central actions of FGF21 with respect to promoting a lean phenotype. The observed differences in FGF21 sensitivity may relate to day length-induced changes in adipose tissue mass.

FGF21: The center of a transcriptional nexus in metabolic regulation.

Fibroblast growth factor 21 (FGF21) is emerging as a key regulator of energy homeostasis and presents a novel target for the development of therapies for the treatment of diabetes, cardiovascular disease and obesity. Recent publications have demonstrated that FGF21 resides downstream of a complex network of transcriptional regulators which modulate its expression in response to a wide array of physiological stimuli or pharmacologic agents. The manner in which these mechanisms are integrated to regulate FGF21 transcription, production and subsequent secretion is poorly understood. While FGF21 is detected in many metabolically active tissues and is regulated by several of the known transcription factors involved in metabolic control little is known about how these pathways are integrated. In this review, we discuss the data presented to date on regulation of FGF21 by a wide array of transcription factors and explore how it relates to metabolic regulation in vivo.

Chronic central administration of apelin-13 over 10 days increases food intake, body weight, locomotor activity and body temperature in C57BL/6 mice.

The peptide apelin has been located in a wide range of tissues, including the gastrointestinal tract, stomach and adipose tissue. Apelin and its receptor has also been detected in the arcuate and paraventricular nuclei of the hypothalamus, which are involved in the control of feeding behaviour and energy expenditure. This distribution suggests apelin may play a role in energy homeostasis, but previous attempts to discern the effects of apelin by acute injection into the brain have yielded conflicting results. We examined the effect of a chronic 10-day intracerebroventricular (i.c.v.) infusion of apelin-13 into the third ventricle on food intake, body temperature and locomotor activity in C57BL/6 mice. Apelin-13 (1 microg/day) increased food intake significantly on days 3-7 of infusion; thereafter, food intake of treated and control individuals converged. This convergence was potentially because of progressive conversion of apelin-13 to [Pyr(1)]apelin-13 which has a four-fold lower receptor binding affinity at the orphan G protein-coupled receptor, APJ. Locomotor activity was also higher in the apelin-treated mice, especially during the nocturnal peak, when most feeding occurs, and the first hours of the light phase. Body temperature was also elevated during this increased period of activity, but was otherwise unaffected. Apelin-13-infused animals gained more weight than the saline-infused controls, suggesting the elevated locomotor activity did not offset the increased food intake. Elevated locomotion and the consequent increases in body temperature were probably secondary effects to the increased food intake. These results suggest that apelin-13 may play a central role in the control of feeding behaviour and is one of only two peripheral ligands known to stimulate rather than inhibit intake. As apelin production is elevated during obesity, this may provide an important feed-forward mechanism exacerbating the problem. Antagonists of the apelin receptor may therefore be useful pharmaceuticals in the treatment of obesity.

Feeding behaviour in galanin knockout mice supports a role of galanin in fat intake and preference.

It has been widely suggested that saturated fat consumption has fuelled the current obesity epidemic. Macronutrient choices appear to be important not only as potential factors influencing obesity, but also independently as risk factors for diabetes, cardiovascular disease and cancer. The neuropeptide galanin has previously been implicated in the regulation of fat intake, although its precise role has been contested. The present study investigated mice with targeted knockout of the galanin gene (GKO). We demonstrate that, when only a high fat diet (HFD) was available, wild-type (WT) animals consumed significantly more energy than the GKO mice (89.85 +/- 4.57 kJ/day versus 76.84 +/- 3.55 kJ/day, P < 0.001, n = 17 versus 15). Consistent with this, WT animals gained more body weight when fed the HFD than GKO animals (3.48 +/- 0.44 g versus 2.02 +/- 0.62 g, P < 0.001, n = 17 versus 15). In a macronutrient choice scenario, WT mice ate almost three-fold more fat than GKO animals (0.63 +/- 0.02 g versus 0.23 +/- 0.01 g, P < 0.001, n = 18 versus 24). Chronic administration of galanin by mini-osmotic pumps into the lateral ventricle of GKO animals partially reversed the fat avoidance phenotype. Fat intake was significantly lower in the phosphate-buffered saline-treated GKO group compared to galanin-treated GKO animals (0.32 +/- 0.01 g versus 0.38 +/- 0.01 g, P < 0.005, n = 17 versus 17). These data are compatible with the hypothesis that galanin specifically regulates fat intake, and implies that an antagonist to one or more of the galanin receptor subtype(s) may be of use in the treatment of some forms of obesity.

Spitzer spectral observations of the deep impact ejecta.

Spitzer Space Telescope imaging spectrometer observations of comet 9P/Tempel 1 during the Deep Impact encounter returned detailed, highly structured, 5- to 35-micrometer spectra of the ejecta. Emission signatures due to amorphous and crystalline silicates, amorphous carbon, carbonates, phyllosilicates, polycyclic aromatic hydrocarbons, water gas and ice, and sulfides were found. Good agreement is seen between the ejecta spectra and the material emitted from comet C/1995 O1 (Hale-Bopp) and the circumstellar material around the young stellar object HD100546. The atomic abundance of the observed material is consistent with solar and C1 chondritic abundances, and the dust-to-gas ratio was determined to be greater than or equal to 1.3. The presence of the observed mix of materials requires efficient methods of annealing amorphous silicates and mixing of high- and low-temperature phases over large distances in the early protosolar nebula.

Examination of the involvement of protein kinase A in D2 dopamine receptor antagonist-induced immediate early gene expression.

Immediate early genes (IEGs) are induced by different signaling pathways. It has been proposed that D2 dopamine receptor blockade induces IEG expression through activation of protein kinase A (PKA), although few studies have examined this issue in vivo. We infused the PKA inhibitor H-89 into the striatum of male rats, followed 30 min later by systemic administration of eticlopride. Eticlopride-induced c-fos and zif268 mRNA expression in striatum was not blocked by H-89. In addition, eticlopride did not produce measurable levels of PKA activity in striatum, whereas the cAMP activator Sp-8-Br-cAMPs increased levels of activated PKA. Neither the adenosine A2a receptor agonist CGS 21680 nor the phosphodiesterase-4 inhibitor rolipram, each of which should increase PKA activation, potentiated eticlopride-induced IEG expression. To test whether other signaling pathways are involved in eticlopride-mediated gene induction, we also infused inhibitors of the mitogen-activated and calcium/calmodulin-dependent protein kinases into animals and then treated them with eticlopride. The data suggest that eticlopride-induced IEG expression is not solely dependent on these kinases either. These data suggest that PKA activation may not be necessary for induction of IEGs by D2 dopamine receptor antagonists and that other intracellular signaling pathways may be involved.

Degree of immediate early gene induction in striatum by eticlopride determines sensitivity to N-methyl-D-aspartate receptor blockade.

Cortical afferents excite striatal efferent neurons through activation of N-methyl-D-aspartate (NMDA) receptors, which can be modulated by D2 dopamine receptors. It is suggested that activation of PKA by D2 receptor blockade leads to NMDA receptor phosphorylation in the dendrites or phosphorylation of transcription factors in the nucleus. Thus, the levels and cellular localization of activated PKA may determine if D2 antagonist-mediated gene expression is dependent on NMDA receptor activation. We have previously demonstrated that NMDA receptor antagonists block gene expression induced by a high dose of eticlopride in medial and central but not lateral striatum. Here, we examined the effects of NMDA receptor antagonists on striatal gene expression after administration of a low dose of eticlopride. The results showed that NMDA receptor antagonists blocked gene induction by eticlopride throughout striatum. Less PKA activation by the low dose of eticlopride might explain why the expression was more sensitive in the lateral striatum to NMDA receptor blockade than in our previous study. To increase levels of PKA activation to the extent that NMDA receptor blockade would have less effect on eticlopride-mediated gene induction in all regions of striatum, we administered the phosphodiesterase inhibitor IBMX to animals treated with eticlopride. The combined administration of IBMX and eticlopride induced gene expression that was only partially attenuated (c-fos) or unaffected (zif268) by NMDA receptor blockade. These data support the suggestion that the degree of second messenger activation by D2 receptor blockade determines whether D2 dopamine receptor antagonist-mediated gene expression is dependent on NMDA receptor activation.

Effects of conantokins on L-3,4-dihydroxyphenylalanine-induced behavior and immediate early gene expression.

Conantokins, peptides from Conus snails, are N-methyl-D-aspartate (NMDA) receptor antagonists. NMDA receptor antagonists potentiate L-3,4-dihydroxyphenylalanine (L-DOPA)-induced rotation in 6-hydroxydopamine-treated rodents, an index of anti-Parkinsonian potential. This study examined the effects of conantokin-G, conantokin-T(G), CGS 19755, and ifenprodil on L-DOPA-induced contralateral rotation and immediate early gene (IEG) expression in 6-hydroxydopamine-treated rats. Rats received unilateral infusions of 6-hydroxydopamine into the medial forebrain bundle. Three weeks later, rats were treated with an NMDA receptor antagonist, followed by an injection of L-DOPA. Contralateral rotations were recorded for 2 h. In addition, the expression of zif268 and c-fos were examined. Conantokin-G, conantokin-T(G), and CGS 19755 potentiated L-DOPA-induced rotation. Conantokin-G and ifenprodil had no effect on L-DOPA-induced IEG expression, whereas conantokin-T(G) and CGS 19755 attenuated expression. These data suggest that conantokins may be useful in treating Parkinson's disease. Furthermore, different NMDA receptor antagonists have distinct effects on striatal gene expression.

Buprenorphine potentiates L-DOPA-induced contralateral rotation in 6-hydroxydopamine-treated rats.

Animals are commonly given opioid analgetics such as buprenorphine for post-operative pain management. In this study, the effect of the analgetic buprenorphine, a partial mu receptor agonist and kappa receptor antagonist, on L-DOPA-induced contralateral rotation was measured in 6-hydroxydopamine (6-OHDA) treated rats. Male Sprague-Dawley rats received dopamine-depleting brain lesions by infusion of 6-OHDA into the medial forebrain bundle. After the procedure, buprenorphine was administered (430 microg/kg, s.c.) to 17 of 54 animals. Three weeks after 6-OHDA treatment, animals were given benserazide HCI (25 mg/kg, i.p.) and L-DOPA (4 mg/kg, i.p.). Contralateral rotations were monitored for 2 h. Animals receiving buprenorphine had significantly higher rates of rotation as compared with non-buprenorphine-treated animals (P = 0.023). The results suggest that buprenorphine sensitizes animals to the effects of L-DOPA.

Differential effects of N-methyl-D-aspartate receptor blockade on eticlopride-induced immediate early gene expression in the medial and lateral striatum.

The function of striatopallidal neurons is regulated by N-methyl-D-aspartate (NMDA) and dopamine D2 receptors. Previous studies show that immediate early gene induction by D2 receptor blockade is suppressed by NMDA receptor antagonists. Because the pharmacology of NMDA receptors depends on the incorporation of different NR2 subunits and NR2 subunits show regional and cellular differences in their expression in striatum, our study examined whether different NMDA receptor antagonists would have differential effects on eticlopride-induced immediate early gene expression in striatum. Male Sprague-Dawley rats were pretreated with vehicle, CGS 19755, MK-801 or ifenprodil. Rats then received injections of eticlopride and were killed 40 min later. In situ hybridization histochemistry was used to determine the expression of c-fos and zif268 in the striatum. Eticlopride increased immediate early gene expression in striatum, with the increase generally being greater in lateral than in medial striatum. Pretreatment with each of the NMDA receptor antagonists dose-dependently decreased the expression of the immediate early genes. This suppression of eticlopride-induced gene expression was significant only in the medial-central aspect of striatum. Although there was a trend toward suppression of the gene induction in lateral striatum, it did not reach statistical significance and was not typically dose dependent. The data suggest that different types of NMDA receptor antagonists do not exert differential effects on D2 dopamine receptor-mediated function in the striatum. In addition, the data indicate that eticlopride-induced gene expression in the striatum is not uniformly dependent on NMDA receptor activation.

The cyanobacterial repressor SmtB is predominantly a dimer and binds two Zn2+ ions per subunit.

The Synechococcus PCC7942 metallothionein repressor gene smtB has been cloned into a high expression vector and the protein purified to near homogeneity (>/=98%). Analytical ultracentrifugation studies demonstrate that the protein is predominantly dimeric in 0.1 M NaCl, pH 7.4, and 22 degrees C, exhibiting a monomer-dimer-tetramer equilibrium. The monomer-dimer (Ka(1,2)) and the dimer-tetramer (Ka(2,4)) association constants are 3.24 x 10(5) and 9.90 x 10(2) M-1, respectively. The repressor binds two Zn2+ ions per subunit with an overall Kd of 3.49 x 10(-6) M. In the presence of Zn2+, Ka(1, 2) increases by 2 orders of magnitude to 1.25 x 10(7) M-1 and the apparent weight-averaged sedimentation coefficient increases from 2. 00 to 2.22 S. The fact that the increase in sedimentation coefficient is greater than that predicted by increased dimerization is interpreted as caused by compaction of the structure in the presence of metal ions. At pH 6.0, 0.1 M NaCl, and 22 degrees C, the protein exhibits only a monomer-dimer equilibrium, with Ka(1,2) = 1.52 x 10(7) M-1 which is almost identical to that seen upon binding Zn2+ at pH 7.4. The compaction and conformational change in SmtB caused by Zn2+ is consistent with a role for this altered quaternary state in derepression of smtA in Synechococcus challenged with heavy metal ions.

Cyanobacteria carrying an smt-lux transcriptional fusion as biosensors for the detection of heavy metal cations.

The metal-responsive smt operator/promoter region of Synechococcus PCC7942 was fused to the luxCDABE genes of Vibrio fischeri. Plasmid DNA (pJLE23) carrying this fusion conferred metal ion-inducible luminescence to transformed cyanobacteria. Synechococcus PCC7942 (pJLE23) was sensitive to ZnCl2 concentrations within a range of 0.5-4 microM as demonstrated by induction of luminescence. Trace levels of CuSO24 and CdCl2 were also detected.

Dementia: diagnosis and evaluation.

OBJECTIVE: To describe an approach to the diagnosis of dementia based on effective assessment methods. DESIGN: We reviewed the literature and summarized the available diagnostic and prognostic studies of dementia that may be useful to the primary-care physician. RESULTS: Although controversy exists about certain aspects of the diagnostic workup, exclusion of potentially reversible causes of dementia is essential. Laboratory studies (for example, for detection of underlying metabolic abnormalities) and neuroimaging of the brain may be useful. The pattern of onset and the temporal course of the disease may suggest a cause and help direct the investigation. Functional losses can substantially impair the patient's ability to live independently. CONCLUSION: Dementia can be mistakenly considered as part of the normal aging process, and diagnosis necessitates a thorough, although not exhaustive, approach. Early identification of dementing illnesses improves the outcome for reversible disease and may also enhance the management of incurable dementias.

Immunization of mice against Naegleria fowleri infection.

Naegleria fowleri produces fatal meningoencephalitis in humans and in experimentally infected laboratory animals. The course of the disease in mice is dependent upon the infecting dose of amoebae, route of inoculation, and prior exposure to Naegleria antigens. DUB/ICR mice were immunized by various routes and antigen preparations, held for 21 days, and, together with noninfected control mice, challenged intravenously (i.v.) or intranasally (i.n.) with 10(7) or 10(6) N. fowleri per mouse, respectively. Mice immunized with liver or formalinized N. fowleri or live N. gruberi subcutaneously, intraperitoneally, i.v., or i.n. were significantly protected against a subsequent lethal challenge with N. fowleri i.v. or i.n. In general, i.v. inoculation afforded greated protection than other routes of immunization, intact cells immunized mice better than did cell fragments, and N. gruberi appeared to be a better immunogen than N. fowleri.

Modification of resistance of mice to Naegleria fowleri infections.

Naegleria fowleri, which produces a fatal meningoencephalitis in humans, is also able to produce a progressive and fatal disease in mice. The course of the disease in DUB/ICR mice is dependent upon the infecting dose of organisms, whether administered intraperitoneally (i.p.) or intravenously (i.v.). All of the mice receiving 10(7) trophozoites/mouse i.v. or 4.85 X 10(7) trophozoites/mouse i.p. were killed within 10 days. Escherichia coli O26:B6 lipopolysaccharide, administered at a dose of 1 mg/kg 24 h prior to N. fowleri, afforded some protection for several days after challenge, but by day 8 there was no difference in survival of untreated and endotoxin-treated mice. No significant protection was afforded by a complex of lipid A with concanavalin A (ConA) or bovine serum albumin (BSA) or by dimethylmyristamide-BSA, dimethylmyristamide, BSA, beta-hydroxymyristic acid-ConA, beta-hydroxymyristic acid, ConA, myristic acid-BSA, or myristic acid. Mice surviving primary i.v. or i.p. challenge doses of N. fowleri, 5 X 10(6) and 10(7) trophozoites/mouse, respectively, were highly resistant to rechallenge with an i.v. dose of organisms (5 X 10(6) Naegleria/mouse) that produced uniformly fatal disease in untreated control mice.

Enhanced toxicity for mice of combinations of bacterial lipopolysaccharide and vincristine.

Sublethal doses of vincristine (VNC) and bacterial lipopolysaccharide (LPS) administered simultaneously to adult male mice resulted in markedly enhanced mortality. All of 10 strains of Pseudomonas aeruginosa tested, 4 of 7 strains of Bacteroides, and 6 of 10 strains of Listeria monocytogenes were able to substitute for purified LPS in enhancing mortality in VNC-treated mice. Inoculation of mice with each of 10 strains of Pseudomonas, each of 7 strains of Bacteroides, and about half of the 10 strains of Listeria tested elicited increased resistance to the lethal action of purified LPS. The patterns of responses of mice receiving a lethal combination of 2 mg of LPS/kg and 1 mg of VNC/kg resembled those of mice receiving a lethal dose of 10 mg of VNC/kg alone or 15 mg of LPS/kg alone with respect to (i) serum glutamic pyruvate transaminase activity, (ii) hematocrit values, and (iii) thrombocytopenia. The patterns of responses of mice receiving a lethal combination of LPS and VNC resembled those of mice receiving a lethal dose of LPS alone with respect to (i) hypothermia, (ii) retention of sulfobromophthalein, (iii) fibrinogen level, (iv) prothrombin activity, (v) blood urea nitrogen levels, and (vi) time of death. These data are consistent with the proposition that the combination of VNC and LPS produces a fatal renal failure. Histological studies confirmed that there was extensive renal damage in mice treated with lethal doses of LPS alone or a lethal combination of LPS and VNC.

Potentiation of the toxicity of mithramycin by bacterial lipopolysaccharide.

The lethality for BALB/c mice of 1,3-bis(2-chloroethyl)-1-nitrosourea, cytosine arabinoside, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, hydroxyurea, mithramycin, a polymyxin-like antibiotic (SQ 21,286), polyadenylic polyuridylic acid, procarbazine, 5-[3,3-bis(2-chloroethyl)-1-triazeno]-imidazole-4-carboxamide (TIC-mustard) or uracil arabinoside administered in combination with Escherichia coli lipopolysaccharide (LPS) was measured. Simultaneously administered mithramycin and LPS or TIC-mustard administered 24 h after LPS synergistically killed mice. Concanavalin A potentiated the lethality of TIC-mustard but not of eight other drugs tested. Pretreatment of mice with LPS or lipid A complexed to concanavalin A rendered mice resistant to the lethal action of LPS alone or combinations of LPS and mithramycin. Mithramycin-treated mice were killed by minute amounts of LPS. Mice sensitized to LPS by mithramycin were used to detect endotoxic activity in biological materials, such as commercially available enzymes, and in a complex of lipid A with concanavalin A.