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Summary Aims This study was designed to assess further the possible links between enterovirus infection and Type 1 diabetes mellitus (DM). Methods Sera from 110 children in the age range 0–15 years was obtained shortly after the diagnosis of Type 1 DM, in paediatric centres throughout the UK. They were tested for the presence of enteroviral sequences by the polymerase chain reaction (PCR) of the 5′ nontranslated region (5′ NTR). One hundred and eighty-two controls tested were matched for age, geographical location and time of year. Results A significantly greater number of diabetic children (27% vs. 4.9%, P

The empire comes south After conquering the Yangtze River basin and proclaiming the Qin Empire in 221 bce , Qin Shi Huang, “The First Emperor of China,” sent thousands of his soldiers over the mountains into the valleys and coastlands of what is now South China. He also sent convicts and women to establish a population of northerners there. After years of hard fighting against local people, Qin commanders built a city on the site of modern Guangzhou (Canton), the main seaport for trade into the southern seas. When the Qin Empire collapsed after Qin Shi Huang’s death in the year 210 bce , this coastal outpost became the center of a regional kingdom ruled by the senior commanding officer, Zhao To. As armies fought for control of the empire in the north, Zhao To proclaimed himself King of Nan Yue (Southern Yue). Zhao To is among the first historical figures with a role in Vietnamese history. Sometime during the first quarter of the second century bce , he extended his authority over the people living in the Red River plain of northern Vietnam. Yue had been the name of a state on the south-central coast of China (the modern province of Zhejiang) during the sixth to fourth centuries bce . It was appropriated by Zhao To and eventually applied to the Red River plain by ancient Chinese dynasties; in Vietnamese, it is pronounced Viet.

This report describes classical Type 1 insulin deficient diabetes mellitus (DM) arising in twins aged 14 months, both of whom had evidence of enterovirus infection. The diagnosis of Type 1 DM was made in the second twin within 12 days of the first. Enterovirus infection was detected in each twin at diagnosis by polymerase chain reaction (PCR). Both twins were negative for enterovirus by PCR 5 months following diagnosis, although both were then positive for islet cell antibodies. Sequencing of the amplicons produced by PCR suggested that the viruses from each twin were not the same but that they were both variants related to echovirus 6. © 1998 John Wiley & Sons, Ltd.

Immunoglobulin (Ig) fractions from the plasma of a group of newly diagnosed insulin-dependent diabetes mellitus (type 1) patients and set of control subjects were assessed for their effects on isolated mouse islet function. It was found that Igs from type 1 patients caused a significant inhibitory effect on insulin secretion when incubated with mouse islets as compared with controls (25.6 +/- 2.9 pg islet-1 h-1 vs 44.7 +/- 7.7 pg islet-1 h-1, P0.05). The plasma samples from which the Igs were obtained were then tested for the presence of antibodies to the mouse islet cell surface (ICSA). Four of the nine patients were positive for ICSA, and plasma samples from eight control subjects were all negative. ICSA-positive samples appeared to have the greatest inhibitory effect on insulin secretion when compared with their respective controls (53.3 +/- 7.0 pg insulin islet -1 min-1 vs 30.9 +/- 3.7 pg insulin islet -1 min-1, (P0.05). In contrast, it was also found that ICSA-positive Ig fractions had no significant effect on glucose oxidation when co-incubated with mouse islets as compared with the controls (11.3 +/- 2.3 pmol islet-1 h-1 vs 11.2 +/- 2.9 pmol islet-1 h-1). These studies suggest that Igs from newly diagnosed type 1 patients containing ICSA may impair insulin secretion from isolated mouse islets by mechanisms which do not involve the inhibition of B-cell glucose metabolism.

Many different viruses belonging to several genera have the potential to damage beta cells. The mechanisms they employ are varied, and infection may result in either a direct destruction of islets and rapid insulin deficiency, or in a more gradual loss of functioning islets with the onset of diabetes many years later. Several case histories involving extensive cytolysis of beta cells can be directly linked to viral infection, whilst an example of diabetes occurring many years after viral infection is found in individuals who had a congenital infection with rubella virus. Here, the virus induces an autoimmune reaction against beta cells. Autoimmune phenomena have also been observed in islets following infections with viruses other than rubella, and thus activation of autoimmune mechanisms leading to beta-cell destruction may be a relatively frequent occurrence. Recent evidence shows that picornaviruses are not exclusively lytic, and can induce more subtle, long-term changes in beta cells, which may be important in the aetiology of diabetes. The exact mechanisms involved are not known, but it is clear that several viruses can directly inhibit insulin synthesis and induce the expression of other proteins such as interferons, and the HLA antigens. Strain differences in viruses are important since not all variants are tropic for the beta cells. Several laboratories are in the process of identifying the genetic determinants of tropism and diabetogenicity, especially amongst the Coxsackie B (CB) virus group. The sequence of one such diabetogenic CB4 strain virus has been determined. It is clear therefore that there are many viruses with the potential to induce diabetes, and a viral involvement in the pathogenesis of diabetes has been established in some instances. Further research work at both a fundamental and epidemiological level is now urgently needed to define the nature of the interaction of such viruses with the beta cell.

The effects of a diabetogenic strain of encephalomyocarditis (EMC) virus on total protein and insulin biosynthesis in mouse islets of Langerhans have been studied in tissue culture. In dispersed mouse islets, the rates of protein biosynthesis were assessed by measuring the incorporation of [3H]leucine into proteins. In infected dispersed islets incubated in 20 mM-glucose, both insulin and total protein biosynthesis were decreased at 6 h; only insulin biosynthesis was significantly decreased at 3 h. In whole islets, EMC virus brought about a decrease in glucose-stimulated protein and insulin biosynthesis as early as 2 h after infection without concomitant effects on insulin release. This inhibition of protein biosynthesis was still apparent at 20 h post-infection, at which time insulin release was found to be markedly elevated, and the islet insulin content was moderately decreased. At 44 h post-infection, glucose-induced insulin biosynthesis was preferentially inhibited. Infected islets at this later time point also displayed elevated levels of insulin release, and a marked loss of islet insulin content. When insulin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were assessed by dot-blot hybridization using appropriate cDNA probes, levels of insulin mRNA were shown to decrease steadily during the first 20 h of infection, in contrast with the levels of GAPDH mRNA. At 44 h post-infection, both types of mRNA were markedly decreased. It is suggested that there is an initial early ‘shut-off’ of protein synthesis without other detectable changes in islet function. This is followed by a phase where both insulin mRNA levels and insulin synthesis are dramatically decreased.

Infections with Coxsackie viruses (especially Coxsackie B4) are thought to be involved in the pathogenesis of diabetes. Many interdependent variables determine the outcome of an infection with a Coxsackie virus, one of them being the tropism of the virus for a specific tissue. The extent to which Beta cell tropic variants of Coxsackie B4 virus occur naturally was assessed. Human isolates of this virus were tested in an in vitro system in which elevated insulin release from infected islets incubated at a non-stimulatory (2 mmol/l) glucose concentration appears to be related to viral attack. Using this technique, 8/24 isolates tested, impaired secretory function in mouse islets. Some strains of Coxsackie B4 virus, therefore, will directly infect mouse islets in vitro leading to changes in islet cell function. In conclusion, these findings confirm that variants of Coxsackie B4 virus with the potential to damage Beta cells occur quite frequently in the natural population. In certain circumstances the damage they inflict on Beta cells may cause destruction of these cells, or precipitate overt diabetes.

Skin lipids and pH are two factors classically considered of importance in homeostatic characteristics of skin. Skin physiology in cats has received little attention. The aim of this study was to evaluate the long-term influence of sex, sexual status, season, and dietary fat and energy on these parameters. Twenty-four European short-haired laboratory cats, 14 months of age, were followed over a 1-year period. They were divided into 8 groups of three, according to: sex (12 males and 12 females), sexual status (intact or neutered) and diets [(high energy 4300 kcal/kg as fed, 21% fat) vs. (moderate energy 3500 kcal/kg as fed, 10% fat)]. Both diets were fed to all cats for 6 months following a cross-over design. Parameters regularly evaluated were skin pH and hair total lipid content (extraction from samples of 0.6–1.2 g of clipped hairs). The pH of the skin varied from 6.6–6.8 initially to 7.2–7.4 at the end of the study. This increase was significant only in intact animals (male and female). The dietary changes did not affect skin pH. Hair total lipid content was not affected by sex or the diets but slightly increased in all groups over the study period from 1.5–2.4 to 2.4–3.3%. In conclusion, skin pH appeared to be potentially modified by sexual status, but not by sex or dietary lipids and energy. Hair lipids do not appear to be affected by sex or increases in dietary lipids and energy. The slight increase in pH and hair lipids during the study was attributed to the change from young adult to adult stage of all cats. Funding: Royal Canin.

Skin physiology in cats has received little attention. The aim of this study was to evaluate the long-term influence of sex, time and the level of dietary fat and energy on the dynamics and qualities of the hair coat. Twenty-four European short-haired laboratory cats were followed over a 1-year period. They were divided into eight groups of three, according to: sex (12 males and 12 females), sexual status (intact or neutered) and diets [(high energy 4300 kcal/kg as fed, 21% fat) vs. (moderate energy 3500 kcal/kg as fed, 10% fat)]. Both diets were fed for 6 months to all cats following a cross-over design. The following parameters were evaluated throughout the study: thickness of hair coat and hair lengths (neck, rump, lateral, flank), hair regrowth (after periodic clippings of 25 cm2 areas), and telogen/anagen ratio. The thickness of the hair coat initially varied from 1.2–1.7 cm on the neck, 1–1.4 cm on the rump, 1.8–2.5 cm on the flank, and hair shaft lengths were 1.7–2.5, 3.7–3.9 and 2.5–3.2 cm, respectively. Comparison of values revealed few statistical differences: increase of the thickness of hair coat in neutered cats (male and female) during the study, and increase of the length of lateral hairs in all groups during the study. Over all periods and in all groups, the curve of growth was similar (rapid then slower). Some transient variations were attributed to temporary changes in ambient conditions. In conclusion, neither sex, nutrition or season (in housed cats) influenced the general quality of hair coat, in particular hair regrowth. Funding: Royal Canin.

Ketone bodies promote insulin secretion from isolated rat pancreatic islets in the presence of 5 mM-glucose, but are ineffective in its absence. At concentrations of 10 mM or less, the relative abilities of the ketone bodies to potentiate release are in the order D-3-hydroxybutyrate greater than DL-3-hydroxybutyrate greater than acetoacetate. The response curve relating insulin release to D-3-hydroxybutyrate concentration displays a threshold at 1 mM and a maximum at 10 mM. D-3-Hydroxybutyrate (5 mM, but not 10 mM) promotes insulin secretion in the presence of 5 mM concentrations of both L-arginine and DL-glyceraldehyde, but not with L-leucine, L-alanine, L-glutamate or 4-methyl-2-oxopentanoate. The oxidation rates of the exogenous ketone bodies do not correlate well with their capacities to promote insulin release. Moreover, the oxidation of 5 mM-D-3-hydroxybutyrate can be inhibited by 25% with methylmalonate (10 mM) without any diminution of release. The potentiation with D-3-hydroxybutyrate occurs without an observable increase in total islet cyclic AMP. However, a small net efflux matches the relative abilities of the ketone bodies to promote insulin release. With islets from 48 h-starved animals the insulin response is both diminished and less sensitive than in fed animals, since insulin secretion is not significantly raised until a threshold of 5 mM-D-3-hydroxybutyrate is reached. These results suggest that, in the rat at least, there should be a reappraisal of the physiological role of ketone bodies in the promotion of insulin release.

1. Rabbit islets of Langerhans were disrupted by ultrasonic methods and the sonicated preparations were used to study proinsulin biosynthesis. 2. When [3h]leucine is incubated in such preparations, incorporation takes place into proinsulin, as evidenced by characterization on polyacrylamide gels, and by the conversion of this labelled material into insulin, by using trypsin. 3. The labelled proinsulin may also be purified by antiinsulin antibody bound to Sepharose. 4. With the broken-cell preparation it was shown that incorporation of leucine is accelerated by increasing the glucose content of the medium from 2mM to 16mM. However, 16mM-galactose or -sucrose did not stimulate incorporation significantly from basal values. This effect of glucose was abolished by cycloheximide. 5. The significance of these findings in relation to the mechanism of glucose stimulation of proinsulin biosynthesis is discussed.

Infection of DBA2 mice with the M strain of EMC virus was used to study the effects of virusinduced diabetes on the A and B cells of the islets of Langerhans. A transient hypoglycaemia was seen in 48% of mice 2–3 days after infection and probably resulted from increased serum insulin concentrations together with inhibition of glucagon secretion at that time. Islets from hypoglycaemic mice showed no significant alterations from control level in basal or fluoride-stimulated adenylate cyclase activity. Overall, 70% of infected mice became hyperglycaemic with a maximum incidence 6 days after infection. Hyperglycaemia was accompanied by a dramatic reduction in the total pancreatic insulin content and in insulin secretory responses to glucose and theophylline, while A-cell structure and function appeared relatively unaffected in diabetic animals. Basal adenylate cyclase activity was increased in hyperglycaemic mice at 7 days after infection, while fluoride-stimulated adenylate cyclase activity was normal throughout the course of infection. Ultrastructural alterations were observed in a small proportion of B cells from two days after infection and included abnormalities of mitochondrial structure and increased electron opacity of the cytoplasm of affected cells, which subsequently led to complete necrosis. The results suggest that EMC virus specifically affects the B cells of the islets and that disturbances of A cell function may be secondary to B cell damage.

Diabetes was induced in 20–30% of adult CD1 mice 15–20 days after infection by a tissue culture propagated strain of Coxsackie B4 virus. Serum insulin and insulin release from isolated islets indicated a relative insulin deficiency in diabetic animals. In some animals diabetes appeared to be permanent whilst in many it was of a temporary nature. Histology showed only slight damage in both islet and acinar tissue. Infection with pancreas and heart adapted strains of Coxsackie B3 virus failed to produce diabetes in mice and, in contrast to the effects of Coxsackie B4, severe acinar cell damage was seen.

Blood insulin, blood glucose and the biosynthesis and release of insulin have been studied in mice made obese with a single injection of gold thioglucose. In such mice, blood glucose levels were normal, though serum insulin rose in parallel with the development of obesity. When compared with controls, insulin secretion and synthesis were increased in isolated islets of Langerhans from obese mice, over a wide range of glucose concentrations. However, in obese animals, insulin biosynthesis was augmented above control levels at 2 mmol/l glucose, whilst the increase in insulin secretion accompanying obesity only became evident at glucose concentrations greater than 5 mmol/l. After 2 min incubation, cyclic AMP rose more in islets from obese mice than in controls, though cyclic AMP levels did not significantly differ in either group after 10 min incubation with glucose. Glucose oxidation was also increased in islets of Langerhans from obese mice. It seems possible that changes in glucose oxidation, as well as in cyclic AMP levels, contribute to the alteration in the B cell response in this type of obesity.

The effect of various glycolytic intermediates on the biosynthesis of rabbit insulin, in vitro, has been studied. It was shown that pyruvate, d -fructose, d -glyceraldehyde and dihydroxyacetone were without effect in stimulating the incorporation of labelled leucine into rabbit insulin, even though such substances may be oxidised by islets. A small, though not statistically significant effect of β-hydroxybutyrate on incorporation was noted. Dibutyryl cyclic AMP (2 mM) also stimulated leucine incorporation into insulin. Neither azaserine (which lowers tissue NAD) nor 6-aminonicotinamide (which inhibits glucose-6-phosphate dehydrogenase) affected the stimulatory effect of 16 mM glucose on synthesis. Despite its lack of effect on insulin synthesis, d -glyceraldehyde was found to be a potent stimulant for insulin release. It is concluded from these experiments that the stimulatory effects of glucose on insulin synthesis are not exercised by glycolytic intermediates involving the later stages in glycolysis below the hexose phosphate level. In experiments using [ 14 C] glucose and [ 14 C] pyruvate it was found that little of the glucose or pyruvate carbon was incorporated into rabbit insulin although both these substrates were readily oxidised by islet tissue. It is concluded that the effects of glucose on insulin synthesis are not due to the provision of extra carbon for amino acid synthesis.

Mouse pancreatic islets cultured in vitro were infected with a tissue culture-adapted or a mouse pancreas-adapted strain of Coxsackie B4 (CB4) virus. The effects of the viruses on the islets were assessed by examination of their biochemical functions. It was found that the mouse pancreas-adapted strain of CB4 induced a ‘leakage’ of insulin from islets incubated at a basal (2 mmol l−1) glucose concentration, both at two and four days following infection. However, at a stimulatory concentration of glucose (20 mmol l−1) the rate of insulin secretion appeared to be normal in these islets. At two days the rate of total protein synthesis in islets infected with mouse pancreas-adapted CB4, incubated at high glucose concentration, was reduced; at four days the degree of inhibition was more severe, the rate at basal glucose concentration falling to half that of the control islets and at the stimulatory glucose concentration to a quarter of the control islets. (Pro)insulin biosynthesis was also inhibited, the rate being reduced to less than half the mean control value in islets infected with mouse pancreas-adapted CB4 virus at 20 mmol l−1 glucose at two days; at four days the rate was greatly reduced at both 2 and 20 mmol l−1 glucose. It is concluded from this study that (1) only certain strains of CB4 virus can infect mouse pancreatic islets in vitro and (2) that infection with strains of virus tropic for the islets leads to an impairment of metabolic functions of the B-cells, and is not necessarily lytic.

1. The effects of azaserine and nicotinamide, agents which inhibit and stimulate hepatic NAD synthesis respectively, on the content of NAD+ and NADH in isolated rat islets of Langerhans incubated in vitro were studied. The effects of these compounds on the rates of insulin release, from isolated islets incubated in vitro, in response to various secretagogues were also measured. 2. Preincubation of islets in the presence of azaserine (0.3mm) caused a marked depletion of the normal islet-cell content of both NAD+ and NADH and prevented the secretion of insulin in response to stimulatory concentrations of d-glucose, xylitol, d-xylulose, l-arginine hydrochloride and l-leucine. 3. Preincubation of islets in the presence of nicotinamide (2mm) increased the islet content of NAD+ and enhanced the rate of release of insulin in response to d-glucose. Also when nicotinamide was present the inhibitory effect of azaserine on insulin release and the azaserine-induced depletion of the islet content of NAD+ and NADH was prevented. 4. Preincubation with azaserine was without effect on the stimulation of insulin release caused by theophylline or dibutyryl cyclic AMP. 5. It is suggested that insulin release caused by sugars and amino acids is dependent on the maintenance of NAD concentrations, though this may not be the case for release due to theophylline and dibutyryl cyclic AMP.

1. 1. Effects of glucose, fatty acids and ketone bodies on glucagon release from isolated guinea-pig islets of Langerhans incubated vitro have been studied. It was foundt that glucagon release was markedly inhibited by low levels of fatty acids and ketone bodies. 2. 2. The release of glucagon from isolated islets was also measured in the presence of cyanide, 2,4-dinitrophenol, malonate and iodoacetate. Glucagon release was found to be increased when the oxidation of substrates such as glucose, fatty acids and ketone bodies was impaired. The possible physiological significance of these results is briefly discussed.

INTRODUCTION THE western regions of the United States are relatively deficient in poultry feedstuffs containing high-quality carbohydrate. As the result corn is shipped from the Midwest at high cost to producers and consumers of poultry products. Growers could produce much larger amounts of sugar beets than are allowed under the quota system of the Sugar Act. If sugar beets could be used in feeds, they could be produced on non-quota acres and possibly serve as a replacement crop on surplus land. Sucrose, as refined product and in various raw materials, has been fed to livestock for many years. Beet varieties such as Mangold are used widely for silage. Recently pig starter rations (Lewis et al., 1955) have included generous levels of sucrose. Feeding it to hogs and steers prior to slaughter (Greenwood et al., 1953) results in heavier carcasses and more palatable livers. The use of refined sucrose in poultry …

1. Insulin secretion was studied in isolated islets of Langerhans obtained by collagenase digestion of rat pancreas. In addition to responding to glucose and mannose as do whole pancreas and pancreas slices in vitro, isolated rat islets also secrete insulin in response to xylitol, ribitol and ribose, but not to sorbitol, mannitol, arabitol, xylose or arabinose. 2. Xylitol and ribitol readily reduce NAD+ when added to a preparation of ultrasonically treated islets. 3. Adrenaline (1μm) inhibits the effects of glucose and xylitol on insulin release. Mannoheptulose and 2-deoxy-glucose, however, inhibit the response to glucose but not that to xylitol. 4. The intracellular concentration of glucose 6-phosphate is increased when islets are incubated with glucose but not with xylitol, suggesting that xylitol does not promote insulin release by conversion into glucose 6-phosphate. 5. Theophylline (5mm) potentiates the effect of 20mm-glucose on insulin release from isolated rat islets of Langerhans, but has no effect on xylitol-mediated release. These results indicate that xylitol does not stimulate insulin release by alterations in the intracellular concentrations of cyclic AMP. 6. A possible role for the metabolism of hexoses via the pentose phosphate pathway in the stimulation of insulin release is discussed.

1. The release of growth hormone from isolated fragments of rat anterior pituitary tissue incubated in vitro was studied by employing a double-antibody radioimmunoassay. 2. In the absence of added stimuli, two phases of hormone release could be distinguished, an early phase of 2h duration and a subsequent late phase. In the early phase, hormone release was rapid but could be significantly decreased by calcium depletion and by 2,4-dinitrophenol whereas the rate of release in the late phase was uninfluenced by these incubation conditions. These results have been interpreted as indicating the existence of a secretory component in the early phase of release. 3. In subsequent experiments, the effects of various agents on the rate of hormone output during the late phase of incubation were investigated. Hormone release was increased by theophylline and by dibutyryl cyclic AMP (N6-2′-O-dibutyryl-adenosine 3′:5′-cyclic monophosphate), the response to both of these agents being related to the concentration of the stimulant employed. 4. The stimulation of growth hormone output by theophylline was significantly decreased by calcium deprivation and by 2,4-dinitrophenol. The response to dibutyryl cyclic AMP was diminished by 2,4-dinitrophenol, iodoacetate and 2-deoxyglucose but not by malonate or colchicine. 5. Arginine, β-hydroxybutyrate, albumin-bound palmitate and variation in the glucose concentration of the incubation medium over a wide range were without any statistically significant effect on the rate of hormone release from either control pituitary fragments or those subject to secretory stimulation by dibutyryl cyclic AMP. 6. It is suggested that the regulation of growth hormone secretion is mediated by cyclic AMP (adenosine 3′:5′-cyclic monophosphate). The secretion observed in response to cyclic AMP requires the presence of ionized calcium and a source of metabolic energy but is independent of pituitary protein synthesis de novo. The integrity of the glycolytic pathway of glucose metabolism appears to be essential for cyclic AMP-stimulated growth hormone secretion to occur.

1. Concentrations of glucose 6-phosphate and 6-phosphogluconate were studied in islets of Langerhans isolated from rat pancreas and incubated in the presence of various agents that induce insulin release. 2. In response to rising concentrations of extracellular glucose (2–10mm) there is a linear increase in the intracellular concentration of glucose 6-phosphate, though this is not the case for 6-phosphogluconate, the intracellular concentration of which only increases when the external glucose concentration exceeds 5mm. 3. Tolbutamide, octanoate and citrate, all of which promote insulin secretion from isolated islets, increase the intracellular concentrations of glucose 6-phosphate and 6-phosphogluconate. The results obtained in the presence of octanoate and citrate are compatible with an inhibitory effect of citrate on islet-cell phosphofructokinase. 4. Theophylline and glucagon when incubated with islets in vitro promote insulin release and cause a rise in 6-phosphogluconate concentration and not in that of glucose 6-phosphate. 5. It is suggested that the further metabolism of glucose 6-phosphate through a pathway other than glycolysis is essential for insulin release. One such pathway involves its oxidation to 6-phosphogluconate, which seems to be a necessary accompaniment of insulin secretion due to glucose. The possibility that agents other than glucose promote insulin release by enhancing the oxidation of glucose 6-phosphate through this pathway is discussed.

1. 1. A method for the purification of insulin on a small scale from pancreas has been devised, in which the insulin is specifically precipitated with antibody and further purified by paper chromatography and paper electrophoresis. 2. 2. This technique has been used to purify insulin labelled by incubating tritium-labelled leucine with slices of ox pancreas in vitro. .

Sera from 123 patients with diabetes mellitus of recent onset, 155 patients with diabetes of more than two years' duration, and 250 normal persons were collected over a period of two and a half years. All sera were tested for neutralizing antibody to Coxsackie virus types B1-6, and a sample was tested for complement-fixing antibody to a number of viral, rickettsial, and mycoplasmal antigens.In diabetics of recent onset no evidence was found of any excess of antibodies to mumps virus or some common respiratory viruses. Insulin-dependent diabetes within three months of onset were found to have higher antibody titres to Coxsackie B virus, particularly of type B4, than either normal subjects or patients with diabetes of longer duration.

Inosine is a potent simulant of insulin release from rat but not from rabbit islets of Langerhans. Further investigation showed that nucleoside phosphorylase activity is exceptionally low in rabbit islets. The ability of inosine to promote insulin release seems to be related to islet nucleoside phosphorylase activity, which can display marked species differences.

The intraperitoneal inoculation of CD(1) mice with Coxsackie virus B(4) resulted in the raising of blood sugar levels to diabetic values 12 days after the administration of the virus. Serum insulin remained inappropriately low. Light microscopy changes in the islets of Langerhans showed mononuclear cell infiltration of the islets and marked degranulation of the beta cells. The acinar tissue appeared to be little changed. It is concluded that Coxsackie B(4) virus may cause a diabetic state compatible with islet cell damage.

Serum specimens from 162 patients with insulin-dependent diabetes of recent onset and 319 controls were tested for neutralizing antibodies to Coxsackie viruses types B1 to B5. Antibody to type B4 virus was more often found in diabetics than in controls, particularly in the 10-19 year age group. Though controls were not matched for geographical area it was thought that this was unlikely to explain the difference found. The month of onset of diabetes in the patients studied showed a pronounced seasonal incidence, which resembled that found in earlier studies.

The effects of rat growth hormone (1 microgram/ml) on the synthesis and release of insulin by isolated rat islets of Langerhans were studied. There was no effect of growth hormone on the release of insulin from freshly isolated islets during 30 min incubation periods. By contrast, islets previously cultured for 16h with growth hormone exhibited at 40% increase in the release of insulin in response to glucose or to glucose and theophylline. These islets also showed specific increases in basal and glucose-stimulated insulin synthesis of 16% and 21% respectively, together with a 22% increase in the basal rate of total protein synthesis. The total insulin content of islets was not affected by culture with growth hormone. The adenylate cyclase activity of islet homogenates was unaffected by the presence of growth hormone during 30 min incubations. When homogenates from islets previously cultured with growth hormone were studied, basal adenylate cyclase activity was unchanged, while fluoride-stimulated adenylate cyclase activity was increased by 37%. It is concluded that growth hormone can directly affect the synthesis and release of insulin in islets of Langerhans, without relation to its metabolic activities in other target organs.

Isolated mouse pancreatic islets were infected in vitro with two strains of Coxsackie B4 virus – a tissue culture – adapted strain and a mouse pancreas-adapted strain. Within 48 h of infection changes had occurred in the biochemical activities of islets infected with the mouse pancreas-adapted strain of virus. Basal insulin release was increased two-fold in these islets, while glucose-induced insulin secretion remained unchanged. Insulin biosynthesis was greatly reduced at a sti, mulatory concentration of glucose (20 raM), thus leading to a reduced insulin content in these islets. These effects are of importance because they demonstrate that certain strains of Coxsackie B4 virus, like encephalo-myocarditis virus, may selectively alter B-cell function in vitro.

The direct effects of alpha- and beta-interferons on isolated mouse pancreatic islets were investigated in vitro and found to be similar. After 7 h incubation with interferon concentrations above 350 units/ml, glucose-stimulated (pro)insulin biosynthesis was significantly inhibited, with only a slight inhibition of total protein biosynthesis. Inhibition could be abolished in the additional presence of an anti-interferon antibody. Interferon did not affect insulin release, total insulin content, or glucose oxidation of the islets. The stimulation of (pro)insulin biosynthesis by adenosine, D-glyceraldehyde, mannose, N-acetylglucosamine and leucine was also inhibited by interferon, with no effect on insulin release. At concentrations of dsRNA (double-stranded RNA) said to induce interferon (1-100 micrograms/ml), glucose-stimulated (pro)insulin biosynthesis was inhibited without significantly affecting insulin release. The dsRNA may itself inhibit stimulated (pro)insulin biosynthesis or may function indirectly by the induction of interferon.

When isolated rabbit islets of Langerhans were incubated with 3 H-labelled leucine for 2 h in the presence of trypsin inhibitor, label was predominantly incorporated into material which behaved like porcine proinsulin on gel electrophoresis. Trypsin treatment produced material which was apparently rabbit insulin. In the absence of trypsin inhibitor, incorporation of labelled leucine is mainly into rabbit insulin.

A mild diabetes was induced in mice following the inoculation of a myocardial variant of encephalomyocarditis virus. The majority of infected mice developed a transient hyperglycaemia and only a few mice exhibited a chronic elevation of blood glucose. Infected mice were selected for study if their non-fasting blood glucose levels were above two standard deviations of values in control mice for at least 5 consecutive days. The pancreas from selected inoculated mice and control mice was removed 60 days after infection; random blood glucose levels at this time had returned to normal. Isolated islets of Langerhans from such previously ‘diabetic’ mice were used to determine the rate of insulin secretion, insulin biosynthesis and cyclic AMP accumulation in response to various glucose concentrations. At glucose (16 mmol/l), both insulin release and biosynthesis were depressed in previously infected animals. At glucose (2 and 10 mmol/l), while insulin biosynthesis was unchanged in infected animals, insulin release was increased. Cyclic AMP accumulation in response to glucose (20 mmol/l) was found to be significantly elevated in islets from infected animals and especially in response to glucose (20 mmol/l) in the presence of isobutyl methyl xanthine (1 mmol/l). Infected mice exhibited a reduction of total pancreatic insulin content in comparison with control mice. The insulin content of isolated islets of Langerhans from infected and control mice was found to be the same. Serum insulin levels, however, from infected mice were higher than in control sera. The results suggest that the myocardial variant of encephalomyocarditis virus in certain inbred strains of mice causes alteration in some of the biochemical functions of the B cells, 60 days after infection. It is not known, however, how permanent these alterations are.

1. An immunological method for the purification of small quantities of insulin has been devised. 2. This method has been used to isolate labelled insulin secreted from pancreas slices incubated in vitro. The insulin had previously been labelled by incubation of the slices with [(3)H]leucine in vitro. 3. There is some release of labelled insulin when such slices are further incubated in media of low glucose content. When the glucose content of the medium is raised, little additional radioactive insulin is released in the first hour after labelling. However, there is a marked increase in specific radioactivity of insulin released from slices in response to a high concentration of glucose in the second and third hours. Release of labelled insulin is again diminished in the final phase, 4hr. from the start of the experiment. 4. These results are discussed in relation to possible mechanisms of insulin release from the beta-cell.

1. A method was devised for the isolation of islets of Langerhans from rabbit pancreas by collagenase digestion in order to study the influx and efflux of K(+) in islets during insulin secretion. 2. Glucose-induced insulin release was accompanied by an increased rate of uptake of (42)K(+) by the islets of Langerhans, though this was not the case for secretion in response to tolbutamide. Ouabain significantly inhibited the uptake of (42)K(+) by islet tissue. 3. No significant increase in the rate of efflux of (42)K(+) was demonstrated during active insulin secretion. 4. Slices of rabbit pancreas were incubated in media of different K(+) content, and rates of insulin release were determined. Alteration of the K(+) concentration of the medium between 3 and 8mm had no effect on the rate of insulin release by pancreas slices. However, decrease of the K(+) concentration to 1mm resulted in inhibition of secretion in response to both glucose and to tolbutamide. Conversely, an increase in K(+) concentration increased rates of insulin release in response to both these stimuli. 5. It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes of K(+) in the islets do not appear to be important in the initiation of insulin secretion.

GLUCAGON is as essential as insulin in some species for the maintenance of blood glucose, although the factors influencing its release are less well understood. It has been thought, for example, that a low level of blood glucose might serve as a primary stimulus to secretion1–3, but the effects obtained with low glucose concentrations have been small, and may not have great physiological significance. High levels of amino-acids may also promote the secretion of glucagon3–5, but again the regulatory importance of the eifect is questionable. We here report a very marked inhibition of glucagon release by fatty acids and ketone bodies in vitro. It seems quite likely that the α cells of islets are more sensitive to variations in the levels of circulating fatty acids than to other metabolites.

A seasonal incidence in the onset of insulin-dependent diabetes in patients under 30 years of age has been suggested from the analysis of data derived from the clinic registers of two diabetic clinics. The increased incidence occurred in summer, autumn, and winter, with a peak in the autumn. A significant variation in annual totals of new cases of diabetes seen could not be demonstrated or excluded, but the pattern of the variation present showed significant positive correlation with the annual prevalence data for Coxsackie virus type B4, but not for other types of virus infection.

THE isolation of radioactive insulin, made by incubating mammalian pancreas with labelled amino-acids in vitro1,2, has presented considerable difficulties in small-scale experiments, because of contamination with the large excess of other pancreatic proteins. Immunological methods for purifying proteins are being used with increasing success in studies on protein biosynthesis3–5, and a simple means of applying such a technique to the isolation of insulin from crude pancreatic extracts is described here.

1. 1. An immunoassay for glucagon was devised to measure the release of gucagon from isolated guinea-pig islets of Langerhans. [ 125 I]Glucagon of specific activity 300–350 μC/μg prepared and used in the assay in which antibody-bound glucagon was separated from unbound glucagon by precipitation of the antibody-glucagon complex by 67% ethanol at 4°. 2. 2. Guinea-pig islets, isolated by collagenase digestion of guinea-pig pancreas, wee found to respond to changing concentrations of glucose and amino acids in a way similar to that reported for experiments with other species in vitro and in vivo .

EVIDENCE is accumulating that in some mammalian species there may be a direct effect of short chain fatty acids on the secretion of insulin from the pancreas. Thus it has been shown that octanoate causes a marked release of insulin from slices of rat pancreas1. Furthermore, butyrate was found to evoke a rapid and sustained elevation of serum insulin when infused into adult sheep2. These results contrast with the lack of effect on insulin secretion when longer chain fatty acids, such as palmitate, are incubated with pancreas slices3.