Effect of tumor necrosis factor-alpha on insulin signal transduction in rat adipocytes: relation to PKCbeta and zeta translocation.

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.

Differential effect of the antidiabetic thiazolidinediones troglitazone and pioglitazone on human platelet aggregation mechanism.

Troglitazone and pioglitazone, antidiabetic thiazolidinediones, are known to improve insulin resistance. However, the effect of these drugs on platelet aggregation remains unclear. The chemical structure of troglitazone contains vitamin E. Accordingly, we studied the effect of troglitazone, pioglitazone, and vitamin E on thrombin-induced platelet aggregation, metabolism of phosphoinositide, protein phosphorylation, protein kinase C (PKC)-alpha and -beta, and phosphatidylinositol (PI) 3-kinase activation in vitro in human platelets. Maximum platelet aggregation by ADP, collagen, and thrombin decreased in the presence of 0.1-1 micromol/l troglitazone and 500 nmol/l vitamin E for 60 min compared with controls. However, pioglitazone did not inhibit ADP-, collagen-, or thrombin-induced platelet aggregation. Pretreatment with troglitazone and vitamin E, but not with pioglitazone, resulted in decreases in thrombin-induced phosphatidic acid production, hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, and 47-kDa protein phosphorylation. Thrombin-induced PKC-alpha and -beta activation in membrane fraction was suppressed by pretreatment with troglitazone and vitamin E, but not with pioglitazone. Separately, troglitazone and pioglitazone stimulated PI 3-kinase activity, but thrombin-induced PI 3-kinase activation was suppressed by pretreatment with troglitazone and pioglitazone for 60 min. These results suggest that troglitazone and vitamin E, but not pioglitazone, have a potent inhibitory effect on platelet aggregation via suppression of the thrombin-induced activation of phosphoinositide signaling in human platelets. Finally, the chemical structure of vitamin E may contribute to the inhibitory effect of troglitazone on platelet aggregation in human platelets.

Alterations in insulin-induced postreceptor signaling in adipocytes of the Otsuka Long-Evans Tokushima fatty rat strain.

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is a new spontaneous non-insulin-dependent diabetes mellitus (NIDDM) model rat strain developed in Tokushima, Japan. After 18 weeks of age, decreases of 45% and 40% respectively in insulin- and phorbol ester-stimulated [3H]2-deoxyglucose (DOG) uptake were observed, compared with those in Long-Evans Tokushima (LETO) rats (control). Insulin-specific binding and 95 kDa autophosphorylation of insulin receptor in OLETF rats were not different from those in LETO rats. Insulin-induced diacylglycerol (DG) production and Mono Q column-purified protein kinase C (PKC) translocation in adipocytes of OLETF rats were decreased compared with those of LETO rats. Insulin-induced PKC beta translocation from cytosol to membrane was also decreased in adipocytes of OLETF rats. Increases of the PKC beta I, beta II, epsilon and zeta isoforms in membranes of OLETF rats were markedly smaller than those of LETO rats. Analysis of mRNA levels of PKC isoforms in adipocytes of OLETF rats showed decreases of basal level and insulin-induced delayed responses of PKC beta I, beta II, epsilon and zeta mRNA in OLETF rats. On the other hand, insulin- or phorbol ester-induced phosphatidylinositol 3-kinase (PI 3-kinase) activation was decreased in adipocytes of OLETF rats compared with those of LETO rats. These results suggest that insulin resistance in OLETF rats, a spontaneous NIDDM model rat, may be associated with deterioration of insulin-induced DG-PKC signaling and subsequent decrease in PI 3-kinase activation.

Effect of glucocorticoid receptor antagonist RU 38486 on acute glucocorticoid-induced insulin resistance in rat adipocytes.

We examined the mechanism of acute glucocorticoid-induced insulin resistance in rat adipocytes using the glucocorticoid receptor antagonist RU 38486. Pretreatment with dexamethasone (DEX) and prednisolone for 60 minutes resulted in 50% inhibition of insulin-induced [3H]2-deoxyglucose (DOG) uptake at 10(-8) and 10(-7) mol/L, respectively, in rat adipocytes and 20% and 25% inhibition of insulin-induced [3H]2-DOG uptake, respectively, in soleus muscles. Our previous experiments indicated that DEX and prednisolone alone stimulate protein kinase C (PKC) in rat adipocytes. Accordingly, we examined [3H]DEX binding to PKC from MonoQ column-purified rat brain cytosol. Specific [3H]DEX binding to MonoQ column-purified PKC was observed (kd, 56.8 nmol/L; Bmax, 725 fmol/mg protein). Thus, insulin-induced PKC translocation from the cytosol to the membrane was suppressed by pretreatment with 10(-7) mol/L DEX and 10(-6) mol/L prednisolone for 80 minutes. During treatment with RU 38486 for 60 minutes, there was no change in the glucocorticoid-induced inhibitory effect on insulin-induced [3H]2-DOG uptake and PKC translocation from the cytosol to the membrane. Moreover, pretreatment with RU 38486 for 120 minutes slightly prevented the DEX-mediated inhibition of insulin-induced glucose uptake. These results suggest that acute glucocorticoid-induced insulin resistance may be mainly mediated through the other non-glucocorticoid receptor pathway.

Effects of excitation and inactivation in area 17 on paired cells in area 18.

This investigation examines how neighboring neurons of area 18 react when area 17 inputs are excited or depressed. In anesthetized cats, area 18 responses to a sine-wave grating in the receptive field were analyzed, while a second grating was positioned in its periphery and responses were recorded in area 17. This latter site was also inactivated with GABA. A waveform template process sorted out at least two individual, neighboring cells with similar orientation preferences in area 18. These cells frequently displayed opposite reactions to stimulation and inactivation in area 17. Experiments suggest that nearby neurons belonging to the same functional domain in the visual cortex may simultaneously carry disparate information.

Nitric oxide and its influence on oscillations of collicular responses in developing rats.

We investigated the influence of modulating NO synthesis on oscillatory components of ON and OFF evoked field potentials in developing rat superior colliculus. Nitric oxide (NO) is involved in neuronal transmission by adjusting neurotransmitter release in adults and in stabilizing synaptic connections in developing brains. NO synthesis was decreased by inhibiting nitric oxide synthase (NOS) with an acute microinjection of N-nitro-L-arginine methyl ester (L-NAME); whereas NO synthesis was augmented by an acute microinjection of L-arginine (L-ARG). The study is focused on rhythmic activity by analyzing fast Fourier transform (FFT). Collicular responses were recorded in anesthetized rats, at post-natal days (PND) 13-19 and adults. This time window was chosen because it is centered on eye opening. NO down- and upregulation resulted in a dual effect depending on age and response-type. NO synthesis inhibition decreased the magnitude of oscillations in ON responses in the youngest animals (PND13-PND14), whereas oscillations of frequencies higher than 20 Hz in OFF responses were increased in all age groups of developing rats. In adults NO downregulation increased oscillations in ON responses and decreased oscillations in OFF responses. L-arginine application produced effects opposite to those seen with L-NAME. Our data together with results reported in the literature suggest that the temporal patterns of the evoked activity are NO-dependent. This sculpting action of the evoked firing may play a role in the synchronization of action potentials in afferent axons which in turn contributes to synaptic stabilization.

Anterograde transsynaptic transport of WGA-HRP in rat olfactory pathways.

The transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was studied in rat olfactory pathways. After applications of tracer to the vomeronasal organ, the olfactory epithelium or injections into the olfactory bulb, WGA-HRP reaction product was observed in second-order neuron terminal areas of each pathway, e.g. within posteromedial cortical amygdaloid nucleus, primary olfactory cortex and contralateral primary olfactory cortex, respectively. The results indicate that anterograde transsynaptic transport of WGA-HRP occurs in olfactory pathways, as has been shown in visual, somatosensory and limbic systems, and thus, anterograde transsynaptic transport may be a mechanism for neurons to exchange materials and/or messages.

Retinal efferents from the pretectal area in the rat.

Horseradish peroxidase (HRP) labeled neuronal somas were consistently found in the medial pretectal area after intraocular injection of HRP. The labeled cells represent the source of centrifugal fibers to the rat retina.

Transneuronal transport of WGA-HRP in immature rat visual pathways.

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to study transneuronal transport in the developing rat visual pathways. Intraocular injections of WGA-HRP were made in neonatal albino rat pups at different ages from the day of birth, postnatal day 0 (P0), to one month of age. Transneuronal labeling in geniculostriate fibers and in tectoparabigeminal terminals was observed as early as P1 and showed little change with eye-opening. However, at early ages, consistent transneuronal labeling was found to require injection of up to 4 times the amount of tracer (0.18 mg WGA-HRP) as adults (0.04 mg WGA-HRP), delivered in two injections. Control injections of HRP alone produced heavy anterograde labeling at all ages, without requiring increased injections. The results suggest that transneuronal transport precedes synaptic transmission, and may illustrate a mechanism for exchanging molecules between neurons. One explanation for the requirement of increased tracer is that axonal and/or transneuronal transport of WGA-HRP may be selectively limited to certain cells in the postnatal retina.

Centrifugal fibers to the rat retina from the medial pretectal area and the periaqueductal grey matter.

Intraocular injections of several different retrograde tracers were made in albino rats. Retrogradely filled neurons were observed in two locations: the medial pretectal area and the contralateral periaqueductal grey matter, indicating that the pretectum and the mesencephalon are sources of centrifugal innervation to the rat retina.

Retinal projections to the inferior and medial pulvinar nuclei in the Old-World monkey.

Horseradish peroxidase (HRP) and wheat germ agglutinin conjugated to HRP (WGA-HRP) were used as anterograde tracers to study the macaque monkey retinofugal pathways. Labeled axons were mapped beyond the lateral geniculate nucleus to the inferior (PI) and medial (PM) pulvinar nuclei, where they terminated. Retinofugal fibers project to the medial part of PI, adjacent to the brachium of the superior colliculus. A smaller projection to PM leaves the ventrolateral aspect of the lateral geniculate nucleus and travels parallel to the stria terminalis to reach the dorsal surface of the thalamus and PM. HRP reaction product was bilateral in PI and predominantly contralateral in P)M.

Retinal axons to the medial terminal nucleus of the accessory optic system in old world monkeys.

Horseradish peroxidase (HRP) and HRP conjugated to wheat germ agglutinin (WGA-HRP) were used as anterograde tracers in the monkey visual pathways. In addition to known optic pathways, we observed labeled fibers which left the lateral terminal nucleus of the accessory optic system, traveled around the cerebral peduncle, and could be followed as far as the medial terminal nucleus.

Retinal innervation of the inferior colliculus in rat and monkey.

Visual pathways were studied using horseradish peroxidase (HRP) as an anterograde tracer after intraocular injections in rats and monkeys. Retinal axons continue caudally from the stratum opticum of the contralateral superior colliculus at the medial and lateral sides of the superior colliculus. Fibers near the midline spread out as a flat sheet within the pericentral nucleus of the inferior colliculus. The optic axons travel through the dorsal pericentral nucleus and some reach the posterior pericentral nucleus, but their numbers diminish along the route, suggesting that the fibers terminate. The fact that a similar pathway was observed in rodent and primate suggests that the retino-pericentral nucleus pathway exists in other mammals.

WGA-HRP as a transneuronal marker in the visual pathways of monkey and rat.

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected intraocularly in monkeys and rats. All known primary visual pathways were labeled and, in addition, ocular dominance columns in visual cortex were labeled in monkey, and in rat, parabigeminal, oculomotor, thalamic reticular, and visual cortical areas were labeled as well as neuronal soma in superior colliculus. We conclude that WGA-HRP is a useful transneuronal marker for visual pathways.

Anterograde transsynaptic transport of WGA-HRP in the limbic system of rat and monkey.

The lectin tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), was injected into the entorhinal cortex in rat and monkey brains. Tracer labeling was followed in the entorhinal projection to dentate gyrus and hippocampus, i.e. along the perforant pathway. Besides labeling perforant pathway terminals in the outer two-thirds of stratum moleculare in the dentate gyrus, reaction product was also observed within stratum granulosum. We conclude that labeling of dentate granule cells was the result of anterograde transsynaptic transport of WGA-HRP. The evidence thus provides an example of anterograde transsynaptic transport: in the limbic system; and at an excitatory synapse.

Direct retinal input to the limbic system of the rat.

Retinal projections in rats were studied using anterograde horseradish peroxidase (HRP) tracing and electron microscopic (EM) degeneration. HRP-labeled axons were observed to leave the optic tract in the vicinity of the lateral geniculate nucleus and enter the stria terminalis where they coursed anteriorly to the bed nucleus of the stria terminalis (BNST), and the anterodorsal (TAD) and anteroventral (TAV) thalamic nuclei. After enucleation, selected areas studied with EM confirmed that retinal presynaptic terminals are located in the BNST and TAV. These results support the finding of Conrad and Stumpf that the retina has a direct link to the limbic system.

Intracortical termination of the retino-geniculo-striate pathway studied with transsynaptic tracer (wheat germ agglutinin-horseradish peroxidase) and cytochrome oxidase staining in the macaque monkey.

The tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), was used as a transneuronal marker in the macaque monkey to study retino-geniculo-striate pathway terminals in area 17. Concomitantly, we stained matching sections for metabolic capacity using the cytochrome oxidase staining technique. Terminal labeling by WGA-HRP and cytochrome oxidase activity staining revealed duplicate patterns in layers II and III, IVA, IVC alpha, IVC beta, and VI. The absence of WGA-HRP-labeled neuronal cell bodies in area 17 supports the conclusion that WGA-HRP is a transsynaptic marker in the macaque visual pathways.

Enucleation-induced transsynaptic labeling with WGA-HRP in the developing rat visual system.

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) is a neuroanatomical tracer which is transported transneuronally. In order to investigate whether transport of WGA-HRP is across synapses, labeling was studied in the developing retinotectal pathway where it is known that enucleation results in increased ipsilateral synaptic connections from the remaining eye. While little or no transneuronal labeling was evident in controls, after enucleation transneuronal labeling was consistently observed. Furthermore, the critical period for enucleation-induced transneuronal labeling coincides with the known critical period for enucleation-induced neuronal survival and synaptic formation. The results suggest that transneuronal exchange of WGA-HRP depends on the presence of synapses, and is therefore transsynaptic.

Functional development of the neonatal rat retinotectal pathway.

Electrophysiological activity in the neonatal rat superior colliculus was recorded to measure neuronal and synaptic activity, and, therefore, functional development. Neonatal rat pups were studied from five days to two weeks of age. The earliest activity in the superior colliculus were spontaneous discharges at a frequency of one unit per animal on postnatal day 6 (P6). Spontaneously discharging units were more numerous at P8, and the number peaked on P10. The first clear response to optic nerve stimulation was seen on P10, with relatively long and variable latencies. By P14, electrically evoked responses had much shorter latencies. The results are in line with the first response to light flash in the superior colliculus at P12/13. The evidence suggests that functional development of the rat retinotectal pathway begins at the end of the first week after birth, and that much of the functional maturation occurs mainly during the second week after birth.

Maturation of visual receptive field properties in the rat superior colliculus.

Visually responsive neurons were recorded in the superficial layers of rat superior colliculus from postnatal day 12 to 28. Receptive field properties such as size, type (ON, OFF, ON-OFF and motion sensitive) and direction selectivity were analyzed to disclose changes during maturation. Although some aspects of sensory properties are modified during development (latency, receptive field sizes, and proportions of receptive field types), a high level of sophistication is also present in young animals even before eyelid opening. For instance, direction selective and direction biased cells, which require complex synaptic relations, are already observed when the first light evoked responses emerge in the superior colliculus (P13), strongly suggesting that this property develops without visual experience. Furthermore, direction selectivity is present in the colliculus prior to the appearance of visually evoked activity in the cortex. This indicates that direction selectivity can not be attributable to incoming cortical afferents. This study provides the first direct evidence that, unlike the cat, the rat's cortico-tectal pathway is only weakly involved in the establishment of direction selectivity in collicular neurons.