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Heat stroke leads only rarely to permanent neurological deficits and the convalescence is almost complete. There are, however, some sporadic descriptions of disturbances that lasted for up to 4 months. Little has been mentioned in the literature on residual changes in personality and late neurological side effects. The present study was conducted to follow systematically late personality and behavioral abnormalities in a population of heat stroke victims. This study analyzed 21 young subjects (age: 21 +/- 2 years), who were inflicted by heat stroke. They were invited for a physiological and psychological follow-up examination at least 6 months post-hospitalization. The psychological assessment was comprised of the self-report symptom checklist-90R (SCL-90R), which inquires about symptoms during the 2 weeks preceding the interview. The results indicated that the subjects are psychologically healthy because their scores fell within the normal range. Comparison with a carefully matched control group strengthened this finding. The conclusion was that prominent neurological or behavioral sequelae in heat stroke victims are rare. The psychological assessments clearly indicate that heat stroke did not leave long-term adverse residues. However, one should be aware of the possible complications and follow the patient for several months after the event.

Antibiotic treatment tends sometimes to result in sensations of fatigue and decreased physical performance. The effects of antibiotics were therefore studied in 50 healthy, male trainees, aged 18-25 years, assigned in a random, double-blind fashion to one of the following treatments: tetracycline, ampicillin, trimethoprim/sulphamethoxazole, placebo I and placebo II. Duration of treatment was five times the half-life of each agent and the placebo was matched accordingly. Muscle enzyme activity (serum glutamine oxaloacetate transaminase, lactate dehydrogenase, creatine phosphokinase), maximal aerobic capacity (VO2max), muscle strength (MS), and rating of subjective sensation of fatigue were assessed prior to and upon conclusion of treatment. Compared to pretreatment values, plasma enzymes activity was elevated in all five groups (P < 0.005). No differences in VO2max or in MS were found among the subjects treated with either one of the antibiotics or those given a placebo. A significant difference in VO2max was found between the groups treated for 1 day (antibiotic and placebo) and the groups treated for 3 days (antibiotic and placebo) (P < 0.0001). The rating of subjective sensation was not affected by any of the agents. We concluded that in healthy individuals, a short-term antibiotic treatment had no deleterious effect on aerobic capacity or on muscle strength and was not associated with subjective side effects. The time interval between the two maximal tests could, however, have affected the aerobic capacity. Physiological disturbances associated with a sensation of fatigue following a longer period of antibiotics cannot be excluded.

The production of insulin-like growth factor-I (IGF-I) in extrahepatic tissues supports both autocrine and paracrine functions and is regulated differently from that in liver, which supports endocrine function. In rat liver, transcription initiation primarily occurs at four distinct, widely separated sites in exon 1 of the IGF-I gene, whereas in exon 2, transcription initiation occurs at a cluster of sites. To understand the molecular basis for tissue-specific regulation of IGF-I gene expression, we have mapped transcription start site usage in the following extrahepatic tissues: testes, lung, kidney, heart, brain, muscle, and stomach, with liver serving as a control. In adult rats, kidney and brain exhibited a pattern of exon 1 transcription similar to that seen in liver, i.e. roughly equivalent use of start sites 2 and 3. In contrast, testes and lung preferentially used start site 3, while stomach, heart, and muscle predominantly used start site 3. Start sites 1 and 4 were used in all tissues at extremely low levels. In those tissues studied in which exon 2 transcripts are expressed (testes, lung, stomach, and kidney), the pattern of exon 2 transcription initiation was identical to that in adult rat liver. During postnatal development, the use of all transcription start sites in exons 1 and 2 was coordinate in lung and stomach. Selection of transcription start sites in the kidney, on the other hand, was subject to regulation during postnatal development. Specifically, within exon 1, start site 3 was expressed constitutively throughout peri- and postnatal development. In contrast, the usage of start site 2 was not detected at late fetal or early postnatal stages, but appeared and rapidly increased only at the stage of weaning. Exon 2 transcripts in kidney also did not appear until the postnatal period. These data suggest tissue-specific and developmentally regulated transcription factors regulating IGF-I promoter activity or, alternatively, tissue-specific and developmental stage-dependent differences in the stability of IGF-I mRNAs resulting from the use of different transcription start sites. These different mRNAs may be of significance in the differential regulation of IGF-I production for autocrine or paracrine function.

Using a gas mask (GM) may involve considerable inconvenience, impairment of respiration and communication, and serious psychological reactions. The medical literature is primarily focused on the occupational aspects of using the GM by young and healthy workers. In contrast, there is hardly any information concerning the use of GMs by large, unselected populations, including children, the elderly, and the sick. Issuing GMs to all residents of Israel prior to Operation Desert Storm created an urgent need to define the populations whose health might be jeopardized by using the standard GM. Adding an active air supply system (AASS) to a standard GM may ease the burden on this high-risk group. We evaluated the physiological aspects of breathing with a GM, with and without AASS, in respect to pathophysiology of diseases, and reached a set of criteria for identifying those who may be endangered by a GM and are expected to benefit from the AASS. The method used to sort and identify those entitled to the AASS is described.

The insulin-like growth factors (IGF-I and IGF-II) play important roles in the regulation of growth and metabolism. While the liver is the main source of circulating IGFs, their production by numerous extrahepatic tissues suggests the existence of autocrine and paracrine modes of action in addition to typical endocrine mechanisms. The actions of the IGFs are mediated through their activation of specific cell surface receptors, primarily the IGF-I receptor, although some effects may be mediated through the IGF-II receptor and the insulin receptor. The stability of the IGFs and their interaction with their receptors are mediated by specific IGF binding proteins (IGF-BPs) which are found in the circulation and in extracellular fluids. Thus, the overall biological actions of the IGFs can be regulated by control of ligand biosynthesis, modulation of receptor levels and postreceptor signalling pathways, and changes in the levels and activity of IGF-BPs.

The effect of glucose polymer solutions on physical performance has been extensively investigated, mainly under controlled laboratory conditions. The influence of such beverages on fluid balance and on glycemic state in the field, during prolonged exercise of moderate intensity (a 134 km march) in the heat (32-41 degrees C, 60-14% relative humidity) was therefore studied. 48 endurance-trained men were randomly assigned to drink either a 7.2% glucose polymer (GP) electrolyte beverage or tap water (TW); there were 24 in each group. Each group was then divided into subgroups that either consumed fluid ad libitum, or were instructed to consume 1000 ml/hr. Mean fluid consumption of all subgroups was similar. There was a greater change in plasma volume for the TW than for the GP group (+7.9% vs. +4.8%, respectively; p less than 0.05). However, in neither the GP nor the TP group did dehydration exceed 2% of body weight. Blood glucose concentration increased significantly in subjects ingesting GP (p less than 0.01) while it decreased on each day of march in those drinking TW. It is concluded that the fluid intake recommended at present by the IDF is adequate to maintain hydration within the normal range during physical effort in the heat. The differences between the GP and the TW groups in this study do not justify the substitution of glucose-polymer solutions for water during prolonged, moderate exercise.

The renin-aldosterone system and plasma insulin were studied in 19 patients with familial Mediterranean fever (FMF). Their relationships to serum potassium level at rest and before and after oral glucose loading are described. An interesting finding is the occurrence of hyperkalemia in the absence of oliguria, in the advanced stages of renal failure. No differences were found in the activity of the renin-angiotensin-aldosterone system to explain these variations in serum potassium found in some of the patients. The response of the renin-aldosterone system to glucose loading showed no abnormality, and the regular relationship between serum potassium, plasma renin activity (PRA), aldosterone, insulin, and plasma pH is maintained. Levels of insulin, potassium, and bicarbonate in serum or plasma pH were found similar in FMF patients with normal renal function with and without proteinuria. Further decrease in renal function due to the progression of the underlying disease is manifested by an increase in FENa+ and FEK+ and a hyperchloremic metabolic acidosis, as is the case in other patients with chronic renal failure.

Traditionally, the two major systems of intercellular communication (i.e. the nervous and endocrine systems) were considered separate functional and anatomical entities. Recent studies have provided evidence that the biochemical elements of these systems have common early phylogenetic origins and have suggested that, with the exception of their anatomical diversity, all the systems of intercellular communication are biochemically similar. On the basis of these findings, we suggest that the overlaps between the nervous and endocrine systems, the widespread tissue production of hormones, and other phenomena are now more easily understood.

The threat of using chemical warfare (CW) by countries ruled by dictators and totalitarian governments still exists despite the Geneva Convention of 1925 that prohibited the use of CW. This situation forces nations and their armed forces to be in a state of preparedness in the event of a CW attack. A CW attack on an unprotected civilian population in a dense urban area can cause numerous casualities and become a mass disaster. However, this danger may be significantly reduced by: a) providing collective and individual protective measures, b) training the population in the use of protective measures, and c) early warning to provide sufficient lead time to use the various components of protection. Coping with a nonconventional warfare threat requires an innovative approach in the organization of the health care delivery system so as to maximize the number of survivors. The fact that the population is protected may deter the enemy from using CW since the potential destructive impact of CW is neutralized or at least reduced.

The Israeli civilian-military health services contingency program for mass and wartime casualties has more than four decades of experience. The contingency program includes key civilian and military organizations that are involved in the planning, policy making and delivery of health care and support services to the wounded. During the Persian Gulf war the unified civilian and military command--the supreme hospitalization authority--implemented a national hospital and emergency medical services preparedness system designed to treat the victims of chemical warfare attacks.

During the Persian Gulf war, the entire Israeli population was under the threat of chemical missiles. One of the main effects of chemical agents (e.g., organophosphorus) is respiratory distress, which requires treatment with mechanical ventilation and oxygen enrichment. In the event of a chemical missile attack, the civilian hospitals may enter a state of insufficiency for treating such victims due to the limited amount of equipment, staff and oxygen/air sources. A possible technological solution is a multiple respirator system (MRS) with a multiple oxygen enrichment system designed for use in the battlefield. The advantages of these technologies in the civilian hospital setting during a chemical mass casualty situation are: (a) rapid deployment, (b) high transportability, (c) capability of operation in any location, (d) modularity, and (e) less medical staff for operation. Two types of MRS are described and issues concerning their selection are discussed. The authorities responsible for national health policy may wish to adopt and incorporate these technologies into their hospital and emergency services preparedness system.

The physiological effect of the "passive children's hood" (PCH) was studied in 24 children: 8 toddlers (3-4.5 years old), 8 pre-school pupils (4.5-6 years) and 8 first- and second-grade pupils (6-8 years). This device consists of a children's gas mask and a transparent PVC (polyvinyl chloride plastic) covering (hood). Inspiratory CO2 and O2 (FiCO2 and FiO2, respectively), temperature and humidity were monitored at 10-min intervals while the children were occupied with sedentary activities (playing and watching TV) in a sealed room. Ambient temperature and relative humidity were approximately 27 degrees C and 75% respectively. In the PCH space the temperature was 2 degrees C higher and humidity was near saturation at the end of exposure. FiCO2 in 12 children exceeded 2%, which is the upper acceptable limit according to industrial standards. In four of them FiCO2 was greater than 4% and FiO2 less than 16%. Twenty-two children tolerated the PCH for 92 +/- 35 min (range 24-133 min) with no physiological complications. A significant correlation was found between childrens' age and tolerance time (r = 0.47, P less than 0.025). We conclude that children whose masks are not well adjusted may be exposed to rebreathing CO2-enriched air.

Adequate staffing of hospitals during a prolonged, potentially unconventional war is a key component in the disaster plan of institutions. In an attempt to determine policy regarding hospital staffing, a state-wide survey was conducted in Israel among hospital personnel during the recent Persian Gulf war. This survey aimed to explore the willingness of staff to report to their duties (WTR) following an unconventional missile attack described in a hypothetical scenario. Of the 2,650 questionnaires distributed among all categories of staff in 10 hospitals (42%) across the country, 51% were returned. Overall, 42% of the responding staff were WTR under the presented scenario. However, WTR would increase to 86% if safety measures were provided. Males, personnel with headquarter duty of hospital site managers, and parents of children older than 14 years of age, were the most WTR. The finding of extensive interhospital variation in WTR indicates the need for evaluating WTR on an institutional basis when establishing both the hospital and the regional disaster plan. Data are presented on the extent of WTR, the factors inhibiting WTR, and possible measures to improve WTR.

Pyridostigmine is known as a pre-treatment drug against intoxication with organophosphorus nerve agents. During the Persian Gulf war, we encountered a cluster of nine cases of pyridostigmine self-poisoning, of which three presented with mixed drug poisoning. The clinical and laboratory features of pyridostigmine toxicity are presented. Doses ranged between 390 and 900 mg. Pyridostigmine ingestion resulted in mild to moderate cholinergic symptoms such as abdominal cramps, diarrhea, emesis, nausea, hypersalivation, urinary incontinence, fasciculations, muscle weakness and blurred vision. No central nervous system manifestations were evident. The symptoms developed within several minutes and lasted up to 24 h. All patients underwent gastric emptying followed by administration of activated charcoal. Atropine (1-8 mg) was required in only three patients. Measurement of serum cholinesterase inhibition was found to be a reliable and sensitive diagnostic tool in pyridostigmine poisoning. No clear correlation was found between the extent of cholinesterase inhibition and the incidence or severity of the cholinergic signs. The clinical recovery was faster than the spontaneous recovery of the enzyme. Pyridostigmine intoxication is self-limited and well tolerated by young healthy adults.

During the period 18 January-28 February 1991, a total of 39 Iraqi modified Scud missiles landed in Israel, most of them in the densely populated Tel Aviv area. There were 23 missile attack alerts. These attacks caused 1,059 cases of injury; there were two deaths and 232 patients were admitted to emergency rooms for injuries directly related to the explosions, only one of which was severe. A survey among 91 of the injured showed that 46.6% of the wounds were caused by glass splinters, 31.1% were blunt contusions, and 22.2% were acute psychological reactions. No case of blast injury was reported. Inappropriate injection of atropine was reported in 230 cases. Acute anxiety was the reason for admission of 544 patients to emergency rooms. Another 40 patients sustained various traumas while rushing to the sealed room. The relatively low number of injured people is striking in view of the density of population in the areas hit. Various explanations are discussed.

Respiratory function was evaluated in 12 healthy and 13 asthmatic volunteers following a single oral dose of pyridostigmine in a double-blind, placebo-controlled cross-over study. Respiratory function tests were performed at rest and after submaximal exercise at the time corresponding to the expected peak cholinesterase inhibition by pyridostigmine. A single dose of 60 mg pyridostigmine given to nonasthmatic subjects led to a decrease of 28.4% in cholinesterase activity when compared to the baseline and a statistically (but not physiologically) significant decrease in FEV1 (forced expiratory volume in 1 sec) both at rest (P less than 0.015) and after exercise (P less than 0.05). This effect showed a strong correlation to the degree of cholinesterase inhibition (r = -0.936, P less than 0.0001). According to these findings, a smaller dose of pyridostigmine (30 mg) was given to subjects with mild bronchial asthma. At that dose, pyridostigmine resulted in a similar inhibition of cholinesterase activity to a mean of 76.7% of the baseline. A significant decrease in the pulse rate was also found (P less than 0.005). However, no changes in respiratory function were observed when compared with the effects of placebo. The effect of post-exertion atropine inhalation on respiratory function was also unchanged with pyridostigmine at that dose. We conclude that, in general, at this dose pyridostigmine is a safe drug for asthmatics; however, the distribution of individual results in this group cannot preclude the existence of a subpopulation of asthmatic patients who are more vulnerable to the effects of pyridostigmine.

The counterregulatory hormone responses to semisynthetic human insulin and purified porcine insulin were compared in 20 healthy volunteers (ten men and ten women) and 16 patients (8 men and 8 women) with type I diabetes mellitus (IDDM). In both groups blood glucose fell to similar levels following insulin administration; no difference in counterregulatory hormone response or hypoglycemic awareness was noted when comparing human to porcine insulin. However, when men were compared to women, significant differences were noted in basal glucagon, cortisol, and growth hormone levels, as well as in norepinephrine, prolactin, and cortisol responses to hypoglycemia. These differences could not be attributed to insulin species, different doses of insulin, or degree of hypoglycemia. These findings suggest that hormonal response to and awareness of hypoglycemia are similar in healthy subjects and patients with IDDM following administration of human and porcine insulin and that hormonal responses in men and women should be studied separately to avoid confusion in interpreting results arising from differences in sex.

Following the destruction of two trains in the Urals 2000 km east of Moscow, as a consequence of the conflagration caused by an explosion from a leaking natural gas pipeline, 3000 people were injured;* most of them (2200) died* immediately and the others (about 800) were badly burned. At the request of the Soviet Union Government a medical military delegation was sent to give assistance to the injured people. This report describes the treatment given by the delegation to 40 patients with burns of between 40 and 90 per cent TBSA during a period of 10 days. An insight into a Soviet Union Trauma Center is given and the good treatment given by the Soviet colleagues is emphasized.

Fifty-two patients with severe chloroquine resistant Plasmodium falciparum malaria were treated in a randomized double blind study with either quinine and a single dose of pyrimethamine-sulfadoxine (Fansidar) or quinine alone. Although no statistically significant differences were observed, the 25 patients who received both drugs responded faster and had a more favorable outcome (no deaths) when compared to the 27 who received quinine alone (2 deaths).

Previous studies have shown that insulin and IGF-I bind to their respective receptors and stimulate autophosphorylation of the receptor beta subunits in detergent extracts of neuronal and glial cells. In the present study, intact neuronal and glial cells in primary culture have been utilized to characterize insulin- and IGF-I-stimulated phosphorylation of their receptors. Following [32P]orthophosphate labelling and stimulation by insulin or IGF-I, the cells were solubilized and the phosphorylated receptors were partially purified on wheat germ agglutinin--agarose columns, and immunoprecipitated using anti-phosphotyrosine or anti-insulin receptor antibodies. Insulin stimulated the phosphorylation of its receptor beta subunit (95 kD phosphoprotein) in a dose-dependent manner, within at least 20 seconds in both neuronal and glial cells. Additionally, a 102-kD phosphoprotein was observed in insulin-stimulated neuronal cells. Maximal stimulation of receptor phosphorylation occurred at 1 minute for the glial cells, and 10 minutes for the neuronal cells. IGF-I stimulated the phosphorylation of two phosphoproteins in intact neuronal and glial cells; a 95-kD protein and a 102-kD protein, in a dose-dependent manner. These observations demonstrate that both insulin and IGF-I stimulate the phosphorylation of the beta subunits of their respective receptors in brain cells in a similar fashion to their effects on receptors from nonneural tissues.

The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [125I]insulin to cells obtained by mechanically dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t1/2 of association was 14 min and t1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [125I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the alpha-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver alpha-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [3H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [3H]NE uptake was sodium ion-dependent suggesting that 80-90% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (Glu, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the beta-subunit of the insulin receptors. These observations corroborate our contention that insulin exerts neuromodulatory effects mediated by the specific insulin receptors in the brain.

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.

The insulin receptors of frog brain and liver show features typical of other insulin receptors with regard to affinity and specificity of binding to insulins and proinsulin, solubility in Triton X-100, binding to and elution from wheat germ agglutinin, and insulin-sensitive tyrosine kinase activity. Likewise, the brain and liver receptors differ from one another in electrophoretic mobility and susceptibility to treatment with neuraminidase, analogous to brain and liver receptors of reptiles, birds, and mammals; while the functional implications of these differences are unknown, their evolutionary conservation for 400-500 million years suggests the possibility that they might have importance.

Lizard insulin receptors are evolutionarily highly conserved. Wheat germ agglutinin-purified brain membranes demonstrate the presence of an endogenous substrate (pp 105) for both the insulin and insulin-like growth factor-I receptors. Both insulin and I-insulin-like growth factor-I stimulate the phosphorylation of this endogenous substrate in a dose-dependent manner. Following insulin-stimulated autophosphorylation of the beta subunit, there is a lag period of about 5 min prior to observable phosphorylation of the endogenous substrate. Phosphoamino acid analysis of both the beta subunit as well as pp 105 reveal primarily phosphotyrosine in both the basal as well as the stimulated state.

Insulin receptors are present in membranes prepared from Alligator mississippiensis brain and liver. The apparent molecular weight (MW) of the alpha subunits are 132 kDa and 118 kDa in liver and brain respectively. Apparent MW of the beta subunit is 92 kDa in both brain and liver receptors. Despite the structural differences between brain and liver alpha subunits, brain insulin receptors demonstrate the normal coupling between alpha and beta subunits, i.e. following binding of insulin to the alpha subunit the beta subunit undergoes autophophorylation and stimulates tyrosine specific phosphorylation of exogenously added substrates. These findings suggest that functional insulin receptors are evolutionarily well conserved.

Studies from multiple laboratories with a range of methods raised the possibility that insulin production occurs naturally at extrapancreatic sites. Part A covers the presence of insulin-related materials in organisms that do not have an endocrine pancreas, including unicellular prokaryotes and eukaryotes as well as multicellular non-vertebrate animals (insects et al.) and plants. Part B covers possible production of insulin by extrapancreatic tissues of vertebrates that are remote from a source of pancreatic insulin e.g. early chick embryos and mammalian cells in culture. Part C covers possible extrapancreatic insulin production in mammals in vivo. Each section ends with an outline summary with evidence in favor of and against the hypothesis.

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of 125I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t1/2 of dissociation of 26 minutes. Cross-linking of 125I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the beta subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [3H]norepinephrine uptake. Insulin inhibition of [3H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in the Km, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.

Brain insulin receptors adsorb to and are recoverable from wheat germ agglutinin-agarose (WGA) columns. Similar results are obtained using dissuccinimidyl suberate (DSS)-crosslinked receptors or photo-affinity labeled receptors. WGA can be used for partial purification of brain insulin receptors provided the appropriate WGA preparation is chosen and the optimal ratio of receptor protein to lectin is achieved.

Specific insulin receptors are present in the liver and brain of the lizard Anolis carolinesis. In this study, the specific binding of 125I-insulin to the receptors showed time, temperature and pH dependency. Specific binding to crude membranes prepared from brain was 1-2% of the total radioactivity added compared to 4-5% in the crude membranes prepared from liver. Solubilization and wheat germ agglutinin purification of the membranes resulted in an increase in the specific binding (per mg of protein) between 6 and 32 times for liver membranes and 13-186 for brain membranes. Binding inhibition of tracer insulin by unlabeled porcine insulin was characteristic for insulin receptors with 50% inhibition for liver crude membranes at 60 ng/ml of porcine insulin and 0.7 ng/ml for purified brain insulin receptors. Chicken insulin was 2- to 3-fold more potent and proinsulin about 100 times less potent than porcine insulin. The alpha-subunits of liver and brain had apparent molecular weights on sodium dodecyl sulfate polyacrylamide gel electrophoresis of 135 kDa and 120 kDa respectively. Apparent molecular weights of beta subunits were 92 kDa for both tissues. Insulin stimulated phosphorylation of the beta subunit of both brain and liver receptors. Both tissues demonstrated tyrosine-specific phosphorylation, which was stimulated by insulin, of exogenously added artificial substrates. In addition, purified brain insulin receptor preparations contained an endogenous protein with apparent molecular weight of 105 kDa, whose phosphorylation was stimulated by insulin (10(-7) mol/l). This phosphoprotein was not immunoprecipitated by anti-insulin receptor antibodies. These studies suggest that the structural differences between brain and liver receptors previously demonstrated in the rat are also present in the lizard, which is about 300,000,000 years older than the mammalian species. Thus, there is strong evolutionary conservation of the brain insulin receptor.

Spontaneous hypertensive-corpulent rats (SHR/N-corpulent), homozygous for the corpulent gene (cp/cp), are obese, hyperinsulinemic and exhibit abnormal glucose tolerance and thus represent a model for type II diabetes and obesity. In view of their overall insulin resistance, we examined liver insulin receptor binding and tyrosine kinase activity from corpulent rats and lean littermates fed purified diets containing 54% sucrose or starch for about 12 wk. Specific 125I-insulin binding to crude liver membranes from female corpulent rats fed either starch or sucrose was reduced to approximately 50% of that seen in lean rats (14 vs. 7%). Affinity of insulin receptors was similar in all groups, suggesting that hyperinsulinemic corpulent rats possess fewer hepatic insulin receptors than do lean rats. Using similar numbers of wheat germ agglutinin-agarose (WGA)-purified insulin receptors with similar affinities for insulin, it was found that basal and insulin-stimulated phosphorylation of the synthetic tyrosine-specific kinase substrate poly(Glu, Tyr)4:1 was similar in lean and obese rats fed sucrose or starch. It is suggested that the contribution of the liver to the insulin resistance in obese SHR/N-cp rats probably lies distal to the insulin receptor tyrosine kinase.

Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [125I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC50 for [125I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the alpha subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B10) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the alpha subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the alpha subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosylation. Insulin induced autophosphorylation of the insulin receptor beta subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10(-9) to 10(-6) M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.

Adult obese Zucker rats (fa,fa) are hyperinsulinemic and insulin resistant. Specific insulin binding to crude membranes prepared from livers was 2.8% (per mg protein) in fatty animals compared with 7.9% in homozygous lean (Fa,Fa) and 9.0% in heterozygous lean (Fa,fa) animals. Insulin binding increased in liver membranes from fatty animals after a 72-h fast to 6.4%. The reduced insulin binding in livers from fatty rats was associated with elevated insulin-sensitive tyrosine kinase activity, which fell towards control values after the fast. The elevated tyrosine kinase activity was associated with an increased maximum velocity (Vmax) without a change in Michaelis-Menten constant (Km) for its substrates, ATP and poly(Glu,Tyr)4:1. These findings suggest that, in adult fatty rats, insulin-sensitive tyrosine kinase has increased intrinsic activity. Further, the effect of the prolonged fast on both insulin binding and kinase activity, suggest that in this model environmental factors, and not necessarily a genetic abnormality, may regulate liver insulin receptors and their kinase. Whether the inverse relationship of the kinase and insulin receptor number is the result of a compensatory mechanism remains to be elucidated.