Prevention of diabetes in the BB rat by essential fatty acid deficiency. Relationship between physiological and biochemical changes.

Essential fatty acid (EFA) deficiency exerts a striking protective effect in several animal models of autoimmune disease. We now report that EFA deprivation prevents diabetes in the BB rat, an animal model of human insulin-dependent diabetes mellitus. In diabetes-prone (DP)-BB rats, the incidences of spontaneous diabetes and insulitis (the pathological substrate of autoimmune diabetes) were greatly reduced by EFA deficiency. This beneficial effect of the deficiency state was also seen in diabetes-resistant (DR)-BB rats that, after treatment with antibody to eliminate RT6+ T cells, would otherwise have become diabetic. The susceptibility of EFA-deprived DP-BB rats to spontaneous diabetes was restored when they were given dietary supplements of linoleate at 70 d of age (during the usual period of susceptibility), but not when they were repleted beginning at 120 d (after the peak incidence of diabetes). EFA deficiency did lead to growth retardation, but calorically restricted control rats demonstrated that the protective effect of the deficiency state was not a function of decreased weight. To examine the relationship between the biochemical changes of EFA deficiency and its physiological effects in this system, we compared the fatty acid changes that occurred in EFA-deficient animals that did and did not develop diabetes. Nondiabetic animals had significantly lower levels of (n-6) fatty acids (i.e., linoleate and arachidonate) and higher levels of oleate, an (n-9) fatty acid, than did diabetic animals. Levels of 20:3(n-9), the fatty acid that uniquely characterizes EFA deficiency, were similar in both groups, however. Among diabetic EFA-deficient rats, the age at onset of diabetes was found to correlate inversely with the level of (n-6) fatty acids, the least depleted animals becoming diabetic earliest, whereas there was no correlation with levels of 20:3(n-9). Among animals repleted with linoleate beginning at 70 d, restoration of susceptibility to diabetes correlated with normalization of the level of arachidonate. In summary, EFA deprivation reduced the frequency of diabetes in both DP and RT6-depleted DR-BB rats. This protective effect was strongly associated with depletion of (n-6) fatty acids, particularly arachidonate, but not with accumulation of the abnormal 20:3(n-9). Conjecturally, arachidonate and/or a metabolite may play a key role in mediating inflammatory injury in this animal model of autoimmune diabetes.

Diagnostic whole body scan (pre-therapy scan) in differentiated thyroid cancer: A single center community hospital experience.

OBJECTIVE: Diagnostic whole body scan (pre-therapy scan) with either I-123 or I-131 (radioactive isotopes of iodine) is performed to assess the extent of thyroid cancer especially distant metastasis prior to administering the therapeutic dose of I-131. Our aim of the following study was to determine the utility of the diagnostic pre-therapy scan in the management of differentiated thyroid cancer. MATERIALS AND METHODS: It was a case-control study carried out by retrospective chart review, of a randomly selected 100 patients with differentiated thyroid cancer who had followed in our community hospital over the course of 1 year. We collected data on multiple variables in the subjects - including age, gender, pre-operative size of the nodules, diagnosis, stage of the malignancy, size of the tumor, multifocality, lymphovascular invasion, dose of radioiodine used for remnant ablation, recurrence rates and persistence rates. Continuous variables were compared using the independent sample Mann-Whitney U-test whereas the Chi-square test was used for nominal variables. RESULTS: The mean dose of radioactive iodine administered was 97.56 (±27.98) in the pre-therapy scan group and it was 97.23 (±32.40) in the control group. There was no difference between the two groups (P - 0.45). There was also no difference in the recurrence rates between the groups (P = 1.0). There was a trend toward a higher degree of persistent cancer in the group that had the pre-therapy scans (P - 0.086). CONCLUSION: Pre-therapy scan may not affect the dose of radio-iodine I-131 used for remnant ablation of differentiated thyroid cancer and does not influence the recurrence rates. This was especially true with respect to I-131 remnant ablation for low risk tumors.

Plasma from BB/Wor rats increases insulin secretion by perfused rat pancreas.

Diabetes-prone (DP) BB rats develop spontaneous autoimmune diabetes mellitus in the context of multiple abnormalities of humoral and cellular immunity. Diabetes-resistant (DR) BB rats have phenotypically normal immune systems and rarely become spontaneously hyperglycemic, but can be rendered diabetic by in vivo immune elimination of T cells that express the RT6 surface alloantigen. To determine if humoral factors in these animals influence beta-cell function, we studied the effect of BB rat plasma on glucose-induced insulin secretion from the isolated perfused rat pancreas. We found that plasma dialyzed to remove molecules less than 14 kD from nondiabetic DR and DP BB rats significantly enhanced total insulin secretion [4806 +/- 711 ng (+/- SEM; n = 6) and 4968 +/- 1235 ng (n = 7), respectively] from perfused pancreata when compared with the effects of either plasma from Wistar-Furth rats (2585 +/- 336 ng; n = 9) or medium containing no plasma (1862 +/- 92 ng; n = 38). The presence of chemically induced diabetes was also associated with enhanced insulin secretion [3276 +/- 414 ng (n = 8) using alloxan and 3956 +/- 470 ng (n = 7) using streptozocin], but the greatest degree of enhancement was observed with plasma from spontaneously diabetic BB rats (6521 +/- 751 ng; n = 17). The enhancement of insulin secretion by BB rat plasma, both diabetic and nondiabetic (DR and DP), was characterized by preservation of first and second phase hormone release. Heat inactivation of acutely diabetic BB rat plasma did not affect its ability to stimulate insulin secretion. We conclude that the plasma of BB rats, both before and after the onset of autoimmune diabetes mellitus, contains a factor other than complement of greater than or equal to 14 kD that enhances insulin secretion in vitro from the isolated perfused pancreas.

Retinoic acid receptor transcripts and effects of retinol and retinoic acid on glucagon secretion from rat islets and glucagon-secreting cell lines.

Using intact rat islets, hamster In-R1-G9 cells, and mouse alphaTC-1 clone 9 transgenic tumoral glucagon-secreting cells, we determined the effects of retinol (ROH) and retinoic acid (RA) on glucagon secretion. Since vitamin A effects may be mediated through nuclear RA receptors (RARs) and cytoplasmic ROH- and RA-binding proteins (CRBP and CRABP), cells were also assayed for RARs, CRBP, and CRABP mRNA by Northern blot analyses. Islets and cells were cultured in 2.8 mmol/L glucose and vitamin A-deficient (A-def) medium or in different concentrations of ROH and RA. Using intact islets, RA 10 and 100 nmol/L inhibited glucagon secretion to approximately 60% of control levels. Using In-R1-G9 cells, ROH 0.175 to 5.0 micromol/L inhibited glucagon secretion to 60% to 83% of control levels, and RA 100 and 1,000 nmol/L inhibited glucagon secretion from 72% to 43% of control levels, respectively. Using alphaTC-1 cells, ROH 1.75 micromol/L inhibited glucagon secretion to 80% of control levels, and RA 1 to 100 nmol/L inhibited secretion from 83% to 68% of control levels. Inhibition of secretion was dose-dependent. RARalpha RNA transcripts were detected in alpha TC-1 and In-R1-G9 total RNA extracts; RAR gamma transcripts were detected in alphaTC-1 cells. We conclude the following: (1) ROH and RA inhibit glucagon secretion in cultured rat islets and glucagon-secreting cell lines, and in cell lines the effect of RA is dose-dependent; (2) on a molar basis, RA is on the order of 10- to 100-fold more potent than ROH, a finding consistent with RA being the active metabolite of ROH at the alpha-cell level; and (3) this inhibition may be mediated through classic pathways of retinoid action involving nuclear RARs and gene expression of specific proteins.

Vitamin A status affects the development of diabetes and insulitis in BB rats.

BB/Wor rats develop autoimmune diabetes mellitus with many features in common with human insulin-dependent diabetes mellitus. Since retinoids are known to have effects on insulin secretion and immune function, these studies were designed to investigate the effects of retinoid deficiency on diabetes in BB/Wor rats and to identify a role for retinoid status in the pathogenesis of autoimmune diabetes mellitus. Litters of diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats were divided at weaning and fed a diet either (1) devoid of retinoids and leading to clinical deficiency at approximately 60 days of age (A-def diet)-following 10 days of clinical deficiency, rats on the A-def diet were changed to a diet containing 2 microg/g retinoic (A-def/RA diet); (2) containing 2 microg/g retinoic acid but deficient in retinol (RA diet); or (3) replete in retinol with 4 microg/g retinyl palmitate (RP diet). Rats receiving RP or RA diets were pair-fed to rats on the A-def/RA diet. Diabetes by 120 days of age was greatly reduced (P < .01) in DP rats that received the A-def/RA diet (four of 27) or RA diet (four of 29) versus the RP diet (13 of 31). Insulitis progressed with age in nondiabetic DP rats receiving the RP diet (P < .02) or RA diet (P < .05), but not the A-def/RA diet (P > .22). Insulin secretion was measured in perfused pancreas of nondiabetic rats after age 120 days and correlated negatively with insulitis (P < .05). DP rats receiving the RP diet had reduced insulin secretion as compared with other DP and DR rats (P < .05). In DR rats, retinoid status had no effects on insulitis through 120 days of age or on insulin secretion after 120 days of age. In conclusion, retinol deficiency reduces diabetes and insulitis in DP BB/Wor rats, and retinoic acid can at least partly substitute for retinol in the development of insulitis.

Retinoid-X receptors and the effects of 9-cis-retinoic acid on insulin secretion from RINm5F cells.

Retinoid-X receptors (RXRs) are 9-cis-retinoic acid (9CRA)-dependent gene transcription factors, which modulate the action of all-trans-retinoic acid (ATRA), fatty acids, thyroid hormone (TH), and vitamin D (VD) by forming dimers with themselves or ATRA, TH, peroxisome proliferator activator receptors (PPARs), or VD receptors (VDRs). To determine if 9CRA and RXRs have a role in secretion, RINm5F cells were assayed for RXR transcripts and effects of 9CRA and ATRA on secretion. A single RXR alpha transcript and two RXR beta transcripts, but not RXR gamma, were evident by Northern blot. Cells were cultured for 48 hours without and with 9CRA 1 to 1,000 nmol/L and then stimulated with glucose 0, 0.5, 2.8, 7, and 11 mmol/L 9CRA increased secretion at each glucose concentration, 9CRA increased secretion by 50% to 100% (ANOVA, P < .001) with consistent concentration-dependent responses (eg. at glucose 2.8 mmol/L 9CRA: 0 nmol/L, 5.02 +/- .20 ng/(10(6) cells.h); 1 nmol/L, 6.97 +/- .30; 10 nmol/L, 8.36 +/- .18; 100 nmol/L, 9.15 +/- .28; 1,000 nmol/L, 10.24 +/- .24; n = 6). Although RINm5F cells respond slightly if at all to glucose, 9CRA facilitated glucose-induced insulin release (eg, at 9CRA 100 nmol/L, glucose: 0.5 mmol/L, 7.47 +/- .22 ng/(10(6) cells.h); 2.8 mmol/L, 9.15 +/- .27; 7 mmol/L, 9.81 +/- .19; 11 mmol/L, 11.16 +/- .23; n = 6). ATRA increased secretion by 28% to 57% (ANOVA, P < .001: at glucose 2.8 mmol/L, ATRA: 0 nmol/L, 6.17 +/- .32 ng/(10(6) cells.h); 1 nmol/L, 7.91 +/- .29; 10 nmol/L, 9.75 +/- .14; 100 nmol/L, 9.66 +/- .33; n = 6). 9CRA was more potent than ATRA (eg, at 2.8 mmol/L; baseline, 8.17 +/- .32 ng/(10(8) cells.h); ATRA 100 nmol/L, 9.66 +/- .33; 9CRA 100 nmol/L, 10.81 +/- .15; P < .05, n = 6). When 9CRA was combined with ATRA, the combination was not additive or synergistic (eg, at 2.8 mmol/L: ATRA 100 nmol/L, 9.66 +/- .33 ng/(10(6) cells.h); 9CRA 100 nmol/L, 10.81 +/- .15; ATRA 100 nmol/L + 9CRA 100 nmol/L, 10.79 +/- .28; P < .05, n = 6). These studies show that (1) 9CRA stimulates insulin secretion from RINm5F cells. This effect appears to be at least equal to if not greater than that observed with ATRA, but additive or synergistic effects with ATRA were not evident; (2) 9CRA may facilitate glucose-induced release; and (3) multiple RXR transcripts are present in insulin-secreting cells, implying specific functions. Our findings support the idea that the effects of 9CRA on insulin secretion are mediated through RXR homodimers or heterodimers with retinoic acid receptors (RARs) or possibly other nuclear receptors. Retinoid deficiency or alterations in retinoid receptor function could lead to abnormalities of cell growth or secretion.

Time course of hypothalamic growth hormone-releasing hormone and somatostatin content in streptozocin diabetic rats: evidence for early changes in hypothalamic regulation.

Growth hormone secretion is markedly suppressed early in streptozocin induced diabetes mellitus of the rat. Our studies were designed to delineate early changes in hypothalamic regulation by growth hormone-releasing hormone (GHRH) and somatostatin (SS) with the aim of determining the best time period for hypothalamic secretion studies. Although hypothalamic GHRH content (ng/hypothalamus) and SS concentration (ng/mg wet weight) were unchanged at 17 to 20 days in previous studies, we anticipated changes earlier in the time course from transient imbalances in release and synthesis. We examined hypothalamic GHRH content and SS concentration in control, diabetic, and insulin treated diabetic rats (n = 5-13; streptozocin 100 mg/kg i.p.) at 0, 2, 4, 7, 10 and 21 days. In diabetic rats GHRH content was greater at day 2 (142 +/- 9% of control-same day, P < 0.05) and day 4 (139 +/- 17%, P < 0.05), but was less at day 10 (67 +/- 4%, P < 0.01). GHRH content of insulin treated diabetic rats was elevated at day 2 (158 +/- 10%, P < 0.05), but subsequently was unchanged from control. In diabetic rats SS concentration was decreased at day 4 (78 +/- 5%, P < 0.01) and at day 21 (91 +/- 3%, P < 0.05). Our results show earliest changes compared to control in GHRH content at 2 days and in SS concentration at 4 days. These findings support early changes in hypothalamic secretion, define a time period of 1 to 10 days for further studies of release and gene expression, and suggest complex relationships of gene expression, peptide synthesis, and peptide release.

Effects of all-trans-retinoic acid (ATRA) and retinoic acid receptor (RAR) expression on secretion, growth, and apoptosis of insulin-secreting RINm5F cells.

To define the functions of retinoids and their receptors in insulin secretion, we tested the effects of all-trans-retinoic acid (ATRA) and retinoic acid receptor (RAR) expression on cell growth, differentiation, and secretion using insulin-secreting RINm5F cells. Wild-type cells with a low abundance of mRNA for RAR beta were transfected with RAR beta or chloramphenicol acetyltransferase (CAT control). Cells were cultured for 2-7 days in media without (A-def) or with ATRA, 1, 10, 100, and 1,000 nM. At day 2 of culture, ATRA stimulated insulin release in wild-type and transfected cells, and this effect was dose dependent. At 7 days, ATRA stimulated insulin secretion from wild-type cells twofold at glucose concentrations of 0.5 mM (A-def, 5.1 +/- 0.27; ATRA, 1,000 nM, 10.5 +/- 1.43 ng/10(6) cells) and at 11.0 mM (A-def, 6.9 +/- 0.24; ATRA, 1,000 nM, 13.6 +/- 1.86 ng/10(6) cells). The cellular insulin content was increased about threefold (A-def, 39.2 +/- 2.95; ATRA, 1,000 nM, 118 +/- 8.54 ng/10(6) cells). ATRA inhibited growth of wild-type cells as early as 3 days, and this effect was dose dependent. Whereas in the absence of ATRA, the cell number increased over fivefold between day 3 and day 5, ATRA, 1,000 nM, inhibited cell growth completely. ATRA, 1,000 nM, increased apoptotic RINm5F cells (day 3 A-def, 0.53 +/- 0.27% of total cells, and ATRA, 2.30 +/- 1.44; day 5 A-def, 0.38 +/- 0.23, and ATRA, 2.14 +/- 0.59; day 7 A-def, 0.90 +/- 0.29, and ATRA, 6.02 +/- 1.64). RAR beta-transfected cells showed overexpression of mRNA to RAR beta and dose-dependent inhibition of growth, with almost-complete inhibition at ATRA concentrations as low as 100 nM. Overexpression of RAR beta increased insulin secretion at ATRA, 100-1,000 nM. In summary, ATRA increased the insulin secretion and content of RINm5F cells, while inhibiting growth and increasing apoptosis. Increased expression of RAR beta facilitated these effects on growth and secretion. These findings may reflect the known effect of ATRA on differentiation of cells and mediation through RAR beta.

Effects of vitamin A deficiency and repletion on rat glucagon secretion.

To determine whether vitamin A is involved in pancreatic alpha cell function, we tested for (a) effects of vitamin A deficiency on glucagon release from perifused islets and perfused pancreases, and (b) the presence of cytosolic retinol-binding proteins (CRBP) and retinoic acid-binding proteins (CRABP), in the glucagon-secreting alpha cell line, ln-R1-G9. Arginine 19 mM plus glucose 2.8 mM-stimulated glucagon secretion was markedly impaired in islets and pancreases of vitamin A-deficient rats or rats that had at some time been cycled through vitamin A deficiency (ever A-def) despite repletion with retinoids for 2-4 weeks. Insulin secretion was impaired likewise. Repletion starting early in the development of vitamin A deficiency and for a longer period of time (18 or 60 days) did not restore glucagon secretion, but did normalize insulin secretion. CRBP and CRABP were present in ln-R1-G9 cells. We conclude that (a) vitamin A deficiency is associated with a defect in glucagon secretion; (b) The defect in secretion occurs early in the course of vitamin A deficiency; (c) The defect persists despite repletion; and (d) The requirement of vitamin A for secretion and the presence of CRBP and CRABP in glucagon-secreting cells support a physiologic role for vitamin A at the alpha cell level.

Vitamin A stimulation of insulin secretion: effects on transglutaminase mRNA and activity using rat islets and insulin-secreting cells.

Retinol or retinoic acid is required for insulin release. Retinoids increase transglutaminase activity, and transglutaminase has been implicated in islet insulin release. To examine whether transglutaminase could mediate effects of retinoids on insulin secretion, we measured (i) transglutaminase activity in islets from rats deficient in vitamin A or repleted with retinol or retinoic acid, (ii) transglutaminase activity in RINm5F and INS-1 insulin-secreting cells cultured in retinol or retinoic acid, (iii) mRNA for transglutaminase in RINm5F and INS-1 cells, and (iv) insulin secretion from INS-1 cells in response to retinoic acid. Islets from rats repleted with retinol or retinoic acid showed more than twice the transglutaminase activity of islets from vitamin A deficient rats. Retinoic acid increased RINm5F cells and INS-1 cell transglutaminase activity. Retinol did not increase transglutaminase activity. Transglutaminase mRNA was detected in INS-1 cells but not in RINm5F cells. Retinoic acid increased insulin secretion from INS-1 cells as observed previously in RINm5F cells. In conclusion, retinoic acid increases transglutaminase activity in both rat islets and two insulin-secreting from INS-1 cells. Transglutaminase is a candidate for mediating retinoid-induced changes in insulin secretion.

Increasing incidence and characteristics of differentiated thyroid cancer in Huntington, West Virginia.

Since 1985, we have observed an increasing number of differentiated thyroid cancer cases in Huntington, West Virginia. We describe tumor incidence, patient and tumor characteristics, treatment modalities, and tumor recurrence and death. One hundred seventeen patients with differentiated thyroid cancer were identified between 1976 and 1999. Data were collected from patient records in our practice and the tumor registries at the three hospitals serving our community. The annual incidence of differentiated thyroid cancer increased significantly from fewer than 3 cases per 100,000 prior to 1996 to 9.4 cases per 100,000 in 1999. The median age at diagnosis was 49 years (range, 16-80). The median tumor size was 2.5 cm (range, 1.2-10). Forty-seven percent of the patients had bilateral disease, 28% had three or more tumors, 44% had thyroid capsular invasion, and 16% had gross extrathyroid invasion at surgery. Twenty-two percent had cervical lymph node involvement and 9% had distant metastases at diagnosis. During 1-month to 23-year follow-up, 11% had recurrence, and 5% died of thyroid cancer. In summary, differentiated thyroid cancer has increased dramatically in our community. The tumors appear to be aggressive at diagnosis as reflected by the high percentage of tumors with bilateral, multicentric, and locally invasive disease.

Calcified aldosterone-producing adrenocortical adenoma.

We report the first case of benign aldosteronoma of an ordinary size with calcifications. We review the clinical, clinical imaging, histopathological, and laboratory features of aldosterone-producing adrenal adenoma versus carcinoma. We conclude that no single feature is diagnostic, and the full range of data must be considered. Calcifications may not necessarily be a distinguishing point.

Two cases of malignant lymphoma complicating autoimmune (Hashimoto's) thyroiditis.

The occurrence of lymphoma of the thyroid in a setting of autoimmune thyroid disease is being recognized with increasing frequency. The usual presentation is a rapidly enlarging goiter. This article describes two cases of this condition and emphasizes how prompt diagnosis and treatment can yield satisfactory results.

Hepatic synthesis and urinary excretion of alpha2u-globulin by male rats: diurnal rhythm and response to fasting and refeeding.

The urine of sexually mature male rats contains a protein of hepatic origin, alpha2u-globulin, not found in the urine of immature or female rats; output of this protein is greatly reduced by fasting. We have examined the effects of feeding and of fasting for various lengths of time on urinary output and hepatic synthesis of alpha2u-globulin. Rats eating ad libitum showed diurnal rhythms of urinary alpha2u-globulin excretion reaching maxima between 2000 and 0800 hours, thus coinciding with the daily feeding period of the rat. Fasting for 12 hours extinguished this diurnal rhythm. When fasting was prolonged up to 36 hours, urinary excretion of alpha2u-globulin was reduced to very low levels. Hepatic synthesis of alpha2u-globulin under these nutritional conditions was investigated by incubating liver polyribosomes with [3H]leucine and a preparation of soluble enzymes for protein synthesis and separating the labeled alpha2u-globulin peptides by immunoprecipitation followed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. By this technique, it was shown that only membrane-bound ribosomes in the livers of mature male rats make this protein. Semi-quantitative measurement suggested that the proportion of liver polyribosomes synthesizing alpha2u-globulin was unchanged after 12 hours of fasting, but was reduced after 24 and 36 hours of fasting. It is proposed that the diurnal rhythm in alpha2u-globulin output in the urine represents translational control of its synthesis in the liver, whereas the more extensive reduction with prolonged fasting is partly due to a selective reduction in transcription of the messenger RNA for this protein.