Insulin mRNA content in pancreatic beta cells of coxsackievirus B4-induced diabetic mice

Molecular and Cellular Endocrinology,

55 (1988) 193-202

Insulin mRNA content in pancreatic beta cells of coxsackievirus B4-induced diabetic mice Nando Wadsworth Center for Laboratories

IS. Chatterjee

(Received

Key wor&:

Hyperglycemia;

Hybridization;

and Catherine

and Research, New York State Department 19 June 1987: accepted

Polyadenylated

RNA;

24 September

Diabetogenic

Nejman ofHealth,

Albany, NY 12,‘01, U.S.A

1987)

virus

Summary Molecular hybridization was used to measure poly(A)-containing mRNA and insulin mRNA, and to evaluate viral persistence, in pancreatic beta cells of coxsackievirus B4-induced diabetic mice. Cellular RNA was hybridized with [‘H]poly(U) to measure poly(A)-containing total mRNA, ‘2P-labeled preproinsulin I and II probes to measure insulin mRNA, and a j2P-labeled virus-specific probe to evaluate persistence. The infected mice (SO-90%) showed subnormal blood glucose at 72 h postinfection and were hyperglycemic at 6 and 8 weeks. Poly(A)-containing total mRNA decreased by about 26% at 72 h and 6 weeks and by 49% at 8 weeks, while preproinsulin I mRNA by 30% and preproinsulin II by 46% at 8 weeks postinfection compared to control. Viral sequences were abundant at 72 h and in fair amounts later. It appears that persistent viral infection produces a pathological state, which impairs beta cell function to reduce insulin mRNA and consequently insulin synthesis apparently leading to hyperglycemia.

Introduction Type I diabetes mellitus (juvenile-onset diabetes) is caused by insulin deficiency in susceptible individuals (Cahill, 1975; Felig, 1980; Gepts, 1981). Extensive epidemiologic observations and many case reports (Gamble and Taylor, 1969; Gamble et al., 1973; Barrett-Connor, 1985; Niklasson et al., 1985) and studies of animals (Craighead, 1975; Notkins, 1977), have associated several viruses including group B coxsackieviruses, in the etiology of the disease. Infections caused by the diabetogenic strains of the virus have been reported to produce abnormalities in Address for correspondence: Nando K. Chattejee, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201, U.S.A. 0303-7207/88/$03.50

0 1988 Elsevier Scientific

Publishers

Ireland.

sugar metabolism and diabetes in humans (Coleman et al., 1974; Gladisch et al., 1976; Yoon et al., 1979; Champsaur et al., 1980; Ahamad and Abraham, 1982), and diabetes-like disease in mice (Yoon et al., 1978; Webb et al., 1979). In diabetes-prone mice (inbred strains which readily develop diabetes, see Yoon et al., 1978) coxsackievirus B4 (CB4) infection results in decreased immunoreactive insulin in the pancreas and serum, increased blood glucose concentrations, and impaired glucose tolerance. However, the mechanism by which this virus infection reduces insulin concentration has not been established. Histopathologic investigations indicate that pancreatic beta cell destruction may be partly responsible for the decrease in pancreatic insulin; neurologically regulated hormones may also contribute to the abnormalities in glucose homeostasis Ltd.

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(Yoon et al., 1978, 1984; Webb et al., 1979). Recently, we have shown that alterations in the functional capacity of pancreatic beta cells appear to contribute to CB4-induced, long-term hyperglycemia in mice (Chatterjee et al., 1985). Changes in total protein synthesis and insulin synthesis in intact beta cells during early and late infection were correlated with abnormalities in sugar metabolism. Additionally, changes in cellular proteins of beta cells were monitored to assess cell damage and repair. The results showed that although the beta cells from diabetic mice sustained severe damage, they continued to synthesize insulin at a reduced rate. To ascertain how insulin synthesis is reduced, in the present study, we have quantitated poly(A)-containing total mRNA and insulin mRNA levels in these beta cells, using molecular hybridization of cellular RNA with radioactive poly(U) or radioactive probes for rat preproinsulin I and II genes. To examine if these cells are persistently infected with the virus, cellular RNA was hybridized with a radioactive, 1X cDNA probe of the viral RNA. Radioactive probes for the rat insulin genes have been used by other workers (Brunstedt and Chan, 1982; Giddings et al., 1982, 1985; Orland and Permutt, 1987) to measure mouse and rat insulin mRNAs under various experimental conditions. The results of our study demonstrate significant reduction of both poly(A)-containing mRNA and insulin mRNAs during late infection. Presence of viral RNA indicates persistent infection in beta cells, which could produce a pathological state that impairs cell function. Materials and methods Materials Unlabeled deoxynucleoside triphosphates and restriction enzymes - PstI, BarnHI, and EcoRI were purchased from Pharmacia P.L. Biochemicals. E. coli DNA polymerase I, yeast tRNA, and calf thymus DNA were from Boehringer Mannheim. Avian myeloblastosis virus (AMV) reverse transcriptase was from Life Sciences Incorporated. [a-32P]dTTP (3000 Ci/mmol) was from New England, Nuclear, [ 3H]poly(U) (20 Ci/mmol) and [a- 32P]dCTP (800 Ci/mmol) were from Amersham

Corporation, Restriction endonuclease HindIIIdigested X-DNA markers were from Bethesda Research Laboratories. Nitrocellulose membrane or disks (0.45 pm) was from Schleicher and Schuell or Sartorius Membranefilter. Nytran transfer membrane (0.45 pm) and DEAE membrane were from Schleicher and Schuell. Collagenase fraction IV was from Cappel Worthington Biomedical. Other chemicals were of reagent grade. Rat preproinsulin I cDNA, pRI-7 (Chan et al., 1979) and preproinsulin II cDNA clone, RRl/ pcr(II)IN (Lomedico et al., 1979) were generous gifts of Dr. D.F. Steiner, University of Chicago, and Dr. H.M. Goodman, Harvard Medical School, respectively. Preproinsulin I cDNA was amplified by subcloning in MC 1060 E. coli cells using published procedures (Maniatis et al., 1982) in our laboratory. Animals and virus CD1 male mice, virus screened, 5-6 weeks old, were obtained from Charles River Laboratory, Wilmington, MA. A diabetogenic strain, E2, of CB4 was used in this study. The source of the virus, its origin and complete passage history, virus growth in Buffalo green monkey kidney cells, purification, and plaque assay have been described previously (Chatterjee et al., 1985). Briefly, the virus was grown at 37” C for 24 h in monolayers of the cells in Eagle’s minimum essential medium containing 15% newborn bovine serum and 0.01% gentamicin. The infected cells were frozen and thawed 3 times and the supernatant was clarified by centrifugation at 2000

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aqueous glucose solution (20%) per g of body weight intraperitoneally and was then bled from the tail 60 min later under general anesthesia with ether. Blood glucose in whole blood was measured by the oxidase method using a kit from Sigma Chemical (Technical Bulletin 510). Mice with blood glucose concentrations > 3 standard errors (SE) above the mean of the uninfected mice were considered abnormal (hyperglycemic) (Yoon et al., 1978). In some experiments, nonfasting glucose level was determined for each mouse in addition to the 60 min GTT. In these experiments, any mouse with a nonfasting glucose concentration > 3 SE of the uninfected control mean was considered abnormal. Prepor~tion of enriched beta cells The procedure for isolation and purification of beta cells from the pancreas by digestions with several enzymes (collagenase fraction IV, 1ychymotrypsin, DNase I) and centrifugation through a Ficoll gradient has been described (Chatterjee et al., 1984). Most of these cells (70-80%) appeared to be insulin-positive by specific staining. This method of beta cell purification allowed us to process these cells from a large number of mice (up to 80), and it required less time than beta cell purification from isolated islets of so many mice. Preparation of RNA from beta cells or whole pancreas RNA was extracted by the guanidine-HCI method (Chirgwin et al., 1979), which was developed to obtain biologically active, essentially protein- and DNA-free, RNA in fairly large amounts from sources enriched in ribonuclease, e.g., the pancreas. All glassware was rendered nuclease free by baking for 2 h at 180°C or by autoclaving. Solutions were autoclaved or rendered nuclease free by treatment for 20 min with 0.2% diethyl pyrocarbonate. Cells from 20-80 pancreata or chopped tissue from 5-7 pancreata were used to extract RNA from each group of mice. Several extractions were needed to obtain RNA for this study. Briefly, the cells or the tissue were homogenized in a buffer (7.6 M guanidine-HCl and 0.1 M potassium acetate, pH 5.0; about lo6

cells/O.1 ml or tissue from 1.5-2 pancreata/ml) by passing the viscous mixture several times through a series of plastic syringes (Yale), equipped with 21-gauge and 25-gauge needles. The resultant nonviscous solution was then layered over a 1.5 ml CsCl cushion - containing 5.7 M CsCl, 0.1 M EDTA, and 0.2% diethyl pyrocarbonate - in a Spinco SW 65 Ti rotor, and centrifuged at 35000 rpm for 16 h at 25 *C. The small pellet containing RNA - separated from proteins and DNA, was carefully suspended using a 1.5 ml centrifuge tube in 0.4 ml of the gua~dine-HCI buffer; RNA was precipitated by adding 0.6 volume of 95% ethanol and keeping the mixture for 18 h at - 20°C. The precipitated RNA was pelleted by centrifugation for 15 min at 4’ C in an Eppendorf centrifuge, suspended in guanidine-HCI, and reprecipitated with ethanol once more. The resulting RNA pellet was then suspended in a small volume of 0.15 M NaCl and precipitated with 2.5 volumes of alcohol as before. The final RNA pellet was dissolved in 100 ~1 15% formaldehyde and used in hybridization as quickly as possible. The RNA in formaldehyde could be stored at - 71°C for a very short time. However, the efficiency of hybridization was often reduced, possibly due to this storage. The ratio of A,&A,, of the RNA was usually 2.0, indicating very little protein conta~nation. Hybridization of RNA with [3H]pofy(U) Poly(A)-containing total mRNA was determined by hybridization of total cellular RNA with radioactive poly(U) as described (Gillespie et al., 1972). Briefly, 60-~1 hyb~dization mixture contained 450 mM NaCl, 45 mM sodium citrate, 10 mM Tris-HCl (pH 7.2), 50% formamide, various amounts of unlabeled beta cell RNA, HeLa cell rRNA, or poly(A), and 30 ng (0.05 PCi) of [3H]poly(LJ). Mixtures were incubated at 37 OC for 24 h and then treated with 5 pg/ml ribonuclease A, and 20 pg/ml DNase I at 30” C for 2 h. The nuclease-resistant hybrids were collected on 25-mm nitrocellulose disks and counted for radioactivity. Hybridization with poly(A) and rRNA acted as the positive and negative controls in these experiments. Most of the radioactive poly(U) formed nuclease-resistant hybrids with poly(A), while essentially none with rRNA.

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Isolation and purification of preproinsulin I and II inserts Details of the procedures employed in this section have been described in a cloning manual (Maniatis et al., 1982). Briefly, plasmid DNA containing the cDNA inserts were purified by centrifugation of bacterial DNA in preparative CsCl gradients. To purify preproinsulin I insert, 30-40 pg of pRI-7 plasmid DNA was digested with a combination of two restriction enzymes - BamHI and EcoRI (Chan et al., 1979) while for preproinsulin II insert RRl/pcr(II)IN plasmid DNA was digested with PstI enzyme by incubation for 16 h at 37°C (Lomedico et al., 1979). The sizes and purity of the inserts were checked by analytical agarose gel electrophoresis prior to use in nick translation. Nick translation The procedure of Rigby et al. (1977) was used for the translation. The specific activity of the probe was > 1 X lOa cpm/pg.

1 X cDNA probe E2 viral RNA was reverse transcribed into 1X cDNA by the procedure described in the cloning manual (Maniatis et al., 1982). Briefly, an incubation of 50 ~1 containing 100 mM Tris-HCl (pH 8.3) 10 mM MgCl,, 140 mM KCl, 20 mM pmercaptoethanol, 25 units of RNasin, unlabeled nucleoside triphosphates (dATP, dGTP, dTTP, 0.5 mM each; 20 PM dCTP), 25 PCi of [a- “‘P]dCTP, 125 pg of denatured and fragmented calf-thymus DNA primer, 1 pg of E2 viral RNA, and 70 units of AMV reverse transcriptase. The mixture was incubated at 40°C for 120 min. RNA from the hybrids was removed by incubation with 100 mM NaOH for 45 min at 65°C. The 1X cDNA was neutralized prior to hybridization.

Hybridization All glassware, plastic containers, and tubes were siliconized and baked or autoclaved. Solutions were autoclaved or treated with 0.2% diethyl pyrocarbonate as before. A microanalytical method for the detection of specific RNA sequences by dot-blot hybridization (Cheley and Anderson, 1984) was modified. RNA

in 15% formaldehyde was taken in 10 x SSC (1 x SSC, standard saline citrate, contained 150 mM NaCl and 15 mM sodium citrate), incubated at 65 ’ C for 10 min, and immediately chilled on ice. For dot-blot, aliquots of RNA were applied to nitrocellulose or Nytran sheets using a Schleicher and Schuell ‘Minifold’ filtration apparatus. For quantitation, RNA was applied onto 25mm disks. The sheets and the disks were dried for 2 h at 80” C in a vacuum oven prior to hybridization. The efficiency of hybridization was better using Nytran disks over nitrocellulose disks due to higher nucleic acid binding capacity of the former. Dot-blot or disk hybridization was carried out in a 50% formamide buffer (Cordell et al., 1979) containing about 0.7 X 10h cpm probe/ml for 24 h at 42OC. For dot-blot 20-25 ml hybridization buffer was adequate. Disk hybridization was carried out in glass scintillation vials with 2 ml of the buffer. Nitrocellulose sheets or disks with RNA were prehybridized for 2 h prior to addition of radioactive probe, The sheets or disks were washed at room temperature 3 times with 2 x SSC, 0.1% sodium dodecyl sulfate (SDS) 10 min each, and 2 times at 50°C with 0.1 x SSC, 0.1% SDS for 30 min each. The sheets were dried and autoradiographed, while the dried disks were counted for radioactivity in toluene and Omnifluor. The specificity of hybridization was checked by analyzing the size distribution of the RNA that hybridized to labeled preproinsulin I probe by Northern technique. Although not shown, the hybridized RNA was about 600 nucleotides in length, which agrees with the previous size estimate of rat insulin mRNA (Chan et al., 1979). The procedure for hybridization in liquid medium has been described (Berk and Sharp, 1977). Briefly, 50-100 pg RNA and 1 X cDNA probe (about 60000 cpm) in 100 ~1 Pipes buffer containing 80% formamide, 0.4 M NaCl, 40 mM Pipes (piperazine-N-N’-bis(2-ethanesulfonic acid)) (pH 6.4), and 1 mM EDTA - were incubated for 10 min at 66’ C and then at 51 o C. At indicated intervals 10 ~1 aliquots were removed, treated with 50 units of Sl nuclease for 30 min at 45 OC to solubilize nonhybridized radioactive cDNA. The Sl-resistant cDNA-RNA hybrids were applied to 25-mm DE-81 disks. The disks were washed, dried, and then autoradiographed.

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Extraction of viral RNA Purification of E2 virus in CsCl gradients, extraction of RNA from the purified virus, and analysis of the RNA in 15-30% sucrose gradients have been described (Chatterjee et al., 1983). This RNA was used for the 1X cDNA probe synthesis. Statistical analysis Student’s two-tailed t-test was applied to compare experimental group means * SE for significance. Results Virus-induced abnormalities in glucose metabolism The effect of E2-virus infection on glucose metabolism is shown in Table 1. The infected mice (90%) showed subnormal blood glucose concentrations (hypoglycemia); the values were well below the control mean at 72 h p.i., possibly due to a rapid release of insulin into the blood from infected pancreatic beta cells. Blood glucose then began to increase with time of infection; and by 8 weeks p.i., the concentrations were much higher in the infected mice than in the controls. Blood glucose values higher than the mean + 3 SE of the uninfected animals (167 mg/dl) were considered abnormal (Yoon et al., 1978). By this criterion, we detected hyperglycemia in nearly 80% of the mice at 6 and 8 weeks p.i. When the nonfasting blood

TABLE

glucose and blood glucose after a 60 min GTT of individual animals were compared with the uninfected control means (123.9 + 13.5 nonfasting; 137.7 + 18.8 GTT; n = lo), 80% of the animals were hyperglycemic (abnormal GTT, blood glucose > 194.1) while 60% showed abnormal nonfasting glucose (> 164.4) at 8 weeks p.i. PO/y(A)-containing mRNA content We reported earlier that insulin synthesis was significantly inhibited in beta cells isolated from the virus-infected mice (Chatterjee et al., 1985). Furthermore, a reduction in total protein synthesis was also evident in these cells. Therefore, it was of interest to examine whether the differences in glucose metabolism and protein and insulin synthesis were related to the actual amounts of mRNA including insulin present in these cells. Fig. 1 shows the kinetics of poly(U) hybridization with total RNA from beta cells of the various groups of mice. The extent of ribonuclease-resistant hybrids formed increased with an increase in input RNA for up to 10 pg. Further increase in input RNA did not increase hybridization. Furthermore, hybrid formation was maximum with uninfected RNA compared to RNA from virus-infected mice. This indicates that the amount of

1

group were selected at random for GTT. Values are means f SE. Blood glucose values at 6 and 8 weeks p.i. were significantly different (P < 0.001) when compared to uninfected control. The uninfected group consisted of 30 mice; GTI was performed in 15 mice at 72 h and in the rest at 8 weeks p.i.

Group mice

of

Uninfected (control) Infected Experiment 1 Experiment 2 Experiment 3

Time (p.i.) 72 h

Fig. 1. Kinetics of binding of total RNA from beta ceils with Increasing amounts of RNA from beta cells of various groups of mice were hybridized with the poly(U) to determine the extent of ribonuclease-resistant hybrid formation in each case. RNA from uninfected mice (0); virus-infected mice at 72 h p.i. (0); 6 weeks p.i. (A); and 8 weeks p.i. (A). Each point represents the mean *SE from two experiments with replications. Background cpm (150) with no RNA addition has been subtracted at each point.

[ 3H]poly(U). 6 weeks

8 weeks

187.7+10.0 169.7_+15.0 174.4+ 5.4

185.0+_14.0 180.8+ 16.0 237.6 + 23.0

128.2 +_13 119.6~13.8 109.8klO.O 92.8 f 13.5

RNA (ml

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poly(A)-containing total mRNA is lower in the infected mice. Based on the data from three separate experiments with 10 pg of input RNA for each hybridization reaction, we detected a significant reduction in poly(A)-containing mRNA content: about 26% (P < 0.05) at 72 h and 6 weeks p.i. and 49% (P < 0.01) at 8 weeks p.i. Some of this mRNA from the infected beta cells are virusspecific (see Fig. 5) which should not interfere with the hybridization reaction, however. Insulin mRNA content To determine whether the virus-infected mice were also deficient in insulin mRNA, a series of molecular hybridization experiments were performed with total RNA isolated from whole pancreas or beta cells, and 32P-labeled, nick-translated probes of rat preproinsulin I and II genes. Fig. 2A shows the nature of highly purified restriction fragments (cDNA inserts), containing the genes for rat preproinsulin I and II, in agarose gels, which were used to prepare radioactive probes by nick translation for this study. Fig. 2B shows the results of a control dot-blot hybridization ex-

periment with preproinsulin II probe and increasing amounts of RNA (spots a through d) from uninfected mouse pancreas (lane l), pancreatic beta cells (lane 2) HeLa cells (lane 3) or without any RNA (lane 4). The probe is specific as it hybridized only with RNA from the pancreas or beta cells which contains insulin mRNA. The hybridization assay is sensitive and reproducible since the spots were clearly visible and were increasingly darker with an increase in input RNA. Finally, insulin mRNA can be readily detected using total RNA from the pancreas or purified beta cells. Although not shown, similar results were also obtained with preproinsulin I probe. Fig. 3 is a composite of the results of several dot-blot hybridization experiments with preproinsulin I and II probes using total RNA from whole pancreas (3A) or beta cells (3B) from uninfected or virus-infected mice at different times p.i. Fig. 3C shows the densitometric tracings of the spots of 3B. As before, positive hybridization was obtained with RNA from the pancreas or beta cells; however, the spots were more prominent with the latter RNA. With increasing amounts of beta cell

Fig. 2. Characteristics of preproinsulin I and II cDNA inserts. A: Photograph of the highly electrophoresis with HindHI-digested h-DNA markers (M) and staining with ethidium bromide. hybridization of 32P-labeled, nick-translated preproinsulin II probe with different RNAs. Spots a RNA from uninfected mice pancreas (10, 20, 40, 60 pg), lane 1; pancreatic beta cells (5, 10, 20, 30 30 pg), lane 3; and no RNA addition, lane 4.

purified inserts after agarose gel B: Autoradiograms after dot-blot through d, increasing amounts of pg), lane 2; HeLa cells (5, 10, 20.

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PREPROINSULIN

I

PREPROINSULIN

II

d C

b 0 1234

3 d c

d

b 0

b a

c

1234

1234

from uninfected mice, at similar levels of input RNA. Thus, the extent of hybridization appears to be lower with RNA from the infected mice, indicating the presence of less insulin mRNA in these mice. A different method was used next to measure the reduction of insulin sequences. In this method, input RNA was spread on 25mm disks, and hybridization was quantitated by counting the radioactivity on the disks. Data from three separate hybridization experiments at each point of RNA addition were pooled to present in Fig. 4. The extent of hybridization increased linearly with an increase in input RNA for up to 40 pg; however, it was significantly lower with RNA from the infected mice, especially at 6 and 8 weeks p.i. In some cases (e.g., with RNA from mice at 72 h p.i.), addition of higher amounts of RNA de-

2,000

PAEPROINSULIN

I T

T

E

4

PREPROINSULIN

II

Fig. 3. Autoradiograms after dot-blot hybridization of 32Plabeled, preproinsulin I and II probes with total RNA from whole pancreas (A) or beta cells (B). Hybridization with increasing amounts of RNA (O-150 pg in spots a through d in A; and 5, 10, 20, 30 pg in spots a through d in B) from mice: uninfected, lane 1; virus-infected at 72 h p.i., lane 2; 6 weeks p.i., lane 3; and 8 weeks pi., lane 4. The densitometric tracings of the spots in B, using a BioRad Model 620 Video Densitometer. are shown in C.

RNA (a through d) from uninfected mice (lane 1) the spots became increasingly darker, which is also evident from their densitometric tracings. However, with RNA from the infected mice (lanes 2 through 4), the intensity of the spots and their densitometric tracings were quite variable. Nevertheless, the intensity of most of the spots were relatively lower compared to those with RNA

RNA (ug) Fig. 4. Kinetics of hybridization of total RNA from beta cells with preproinsulin I and II probes. Increasing amounts of RNA on 25-mm disks were hybridized with either probe to determine radioactivity present in RNA-cDNA hybrids. RNA from uninfected mice (0); virus-infected mice at 72 h p.i. (0); 6 weeks pi. (A); and 8 weeks p.i. (A). Each point represents the mean k SE from three experiments. Background cpm (60-80) with no RNA addition has been subtracted at each point.

200

creased the extent of hybridization, which could be due to aggregation of RNA. Based on the data of 40 pg RNA addition, the extent of hybridization with RNA from mice at 8 weeks p.i. was about 30% lower for preproinsulin I and 46% for preproinsulin II (P < 0.001) compared to that with RNA from uninfected mice, indicating that insulin mRNA is significantly lower in the diabetic mice. Vital RNA in beta cells Previously we reported the presence of infectious virus in the pancreas and beta cells at 72 h p.i.; no infectious virus could be detected at 6 or 8 weeks p.i. - when the mice were hyperglycemic (Chatterjee et al., 1985). This observation suggested that persistent infection in beta cells by the virus could produce the disease. It was of interest therefore to examine if these cells contained E2

Fig. 5. Autoradiograms following hybridization of beta cell RNA with 32P-labeled, 1X cDNA probe of E2 viral RNA in a liquid medium. Each spot contains the hybrids formed between the probe and 10 pg RNA from mice: uninfected, row a; virus-infected at 72 h p.i., b; 6 weeks p.i_, c; or 8 weeks p.i., d. Hybridization was for 22 h, 45 h, and 72 h in lanes 1 through 3, respectively. Sl nuclease-resistant hybrids following each hybridization were applied to DE-81 disks and autoradiographed.

viral RNA, especially at late time points of infection. Fig. 5 shows the results of such an experiment following hybridization of beta cell RNA with a 32P-labeled, 1X cDNA probe of the viral RNA. RNA from uninfected cells (row a), a control, did not hybridize following hybridization for 22 h, 45 h, or 70 h (1 through 3). RNA from the infected mice (rows b through d), on the contrary, hybridized quite well. Hybridization was maximum with RNA from cells at 72 h p.i. (row b) a time point when infectious virus was detected, indicating maximum virus replication. A fair amount of hybridization with RNA from cells at 8 weeks p.i. (row d) indicates the presence of viral RNA, although detectable infectious virus had disappeared at this time point. This observation provides evidence to our suggestion that persistent infection by E2 virus produces diabetes in mice. Discussion The experiments reported here were designed primarily to assess, using molecular hybridization, the specific effects of diabetogenic CB4 virus infection on mRNA content in general and insulin mRNA content in particular in pancreatic beta cells of diabetes-prone mice, during progressive stages of diabetes. We believe that the findings of this study will augment our understanding of the regulation of insulin gene translation in diabetes mellitus, since coxsackievirus etiology in diabetes is well documented (Yoon et al., 1979; BarrettConnor, 1985; Niklasson et al., 1985). The results demonstrate that E2 virus infection produces hyperglycemia in a large number of mice, which is evident at 6 and 8 weeks pi. Furthermore, the infection significantly reduces total mRNA content as well as insulin mRNAs in beta cells, apparently leading to the development of diabetes. These findings supplement our previous observation demonstrating significant reduction of total protein and insulin synthesis in beta cells from these mice, and provide further evidence to our suggestion that virus-induced alterations in the functional capacity of the beta cells contribute to the long-term hyperglycemic state (Chatterjee et al., 1985). Subclinical impairment of cells of the islets of Langerhans in virus-induced diabetes was also implied recently in the study of Oldstone et al. (1984).

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As mentioned under Introduction, RNA blot hybridization with nick-translated insulin probes was used in the past to measure preproinsulin mRNAs in the assessment of beta cell function and insulin synthesis in vivo. A strong correlation between serum insulin and preproinsulin mRNAs was apparent in the development of diabetes in genetically diabetic (&/db) mice (Orland and Permutt, 1987). The hybridization assay is highly sensitive (Giddings et al., 1985); it can detect about 5 pg of preproinsulin mRNA in 50 pg of cellular RNA (O.OOOOlW). Even total pancreatic RNA, about 20 pg, can be used to detect preproinsulin mRNA. In our studies, preproinsulin mRNAs were readily detected in less than 10 pg of beta cell RNA and in 30-40 pg of pancreatic RNA (Fig. 2B). The need for the use of a higher amount of pancreatic RNA in our study could be due to somewhat lower specific activity of the probes that we used. The data of Fig. 4 show essentially no reduction of insulin mRNA at 72 h p.i., an early time point in infection when the mice were hypoglycemic, indicating beta cell survival during the direct initial assault of virus infection. Since significant reduction in both preproinsulin I and II sequences was observed late in infection, virus-induced inhibition on the insulin mRNA content could be a delayed effect. Furthermore, since poly(A)-containing total mRNA also decreased late in infection (Fig. l), the inhibition appears to be directed toward other messengers as well. The reason for the decrease of total mRNA, but not insulin mRNA, at 72 h p.i. could be due to longer half-life of insulin messengers compared to other messengers. The inhibition of insulin synthesis that we previously observed in the infected beta cells (Chattejee et al., 1985), probably resulted from the short-age of mRNA, since such shortage will lower the rate and/or the amount of insulin synthesis in the cytoplasm. However, since a large number of in vitro studies have demonstrated that picornaviruses (e.g., CB4, poliovirus, etc.) inhibit host cell protein synthesis by causing a defect in one of the initiation factors needed for mRNA translation (Rose et al., 1978; Ehrenfeld, 1984), the possibility of initiation factor-directed defective translation of insulin mRNA in the beta cells cannot be ruled out.

Precisely how insulin mRNA content is reduced in the beta cells is currently unknown. However, two mechanisms seem feasible. (1) Altered mRNA stability. Virus infection could have accelerated its degradation. mRNA degradation by 2-SA-dependent ribonuclease (25A synthetase is interferon-induced) in several virus-infected cell systems is well known (reviewed by Lengyel, 1982). Interferon induction in mice by encephalomyacarditis virus, another picornavirus, has also been reported (Yoon et al., 1980). (2) Reduced transcription of insulin mRNA. Picornaviruses can reduce host cell mRNA level by inhibiting transcription (Apriletti and Penhoet, 1974; Schwartz et al., 1974; Crawford et al., 1981). In poliovirus-infected HeLa cells, the infection primarily blocks the transcription of mRNA by RNA polymerase II, presumably inactivating or modifying one of the specific factors required by the enzyme for transcription. Such a mechanism could also operate in E2 virus-infected beta cells. Further studies will be needed to determine the actual pathway. Presence of a fair amount of viral RNA in beta cells late in infection is interesting, which shows that the virus can persist in these cells without killing them. This is not unexpected, since group B coxsackieviruses readily establish persistent infection (Matteucci et al., 1985), the infected cells showing no changes in viability and proliferation. Persistent infection of islet cells of mice by another virus - lymphocytic choriomeningitis - has been reported (Oldstone et al., 1984), which led to diabetes. Our results support the hypothesis that CB4 can cause diabetes. The virus appears to replicate and persist in beta cells. It is currently unknown whether the virus also persists in other cells of the pancreas. The infection reduces insulin mRNA supply, which presumably results in inhibition of insulin synthesis and hyperglycemia. Thus, functional impairment in pancreatic beta cells appears to be a factor in virus-induced hyperglycemia. Acknowledgements We thank Drs. D.F. Steiner and S.J. Chan for a generous gift of their preproinsulin I cDNA, and Dr. H.M. Goodman for the gift of his preproinsulin II clone. This work was supported by Grant AM 33054 from the National Institute of Health.

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