Henry K. Driscoll

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 cell lines. Retinoic acid stimulates insulin secretion from INS-1 cells. Transglutaminase is a candidate for mediating retinoid-induced changes in insulin secretion.

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.

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.

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 RARβ were transfected with RARβ 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/106 cells) and at 11.0 mM (A-def, 6.9 ± 0.24; ATRA, 1,000 nM, 13.6 ± 1.86 ng/106 cells). The cellular insulin content was increased about threefold (A-def, 39.2 ± 2.95; ATRA, 1,000 nM, 118 ± 8.54 ng/106 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). RARβ-transfected cells showed overexpression of mRNA to RARβ and dose-dependent inhibition of growth, with almost-complete inhibition at ATRA concentrations as low as 100 nM. Overexpression of RARβ 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 RARβ facilitated these effects on growth and secretion. These findings may reflect the known effect of ATRA on differentiation of cells and mediation through RARβ.

Effects of vitamin A deficiency and repletion on rat glucagon secretion

To determine whether vitamin A is involved in pancreatic a 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 a cell line, In-Rl-G9. Arginine 19 mM plus glucose 2.8 M-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 In-RIG9 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 a cell level.

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.

Severe hypoglycemia and coronary artery calcification during the diabetes control and complications trial/epidemiology of diabetes interventions and complications (DCCT/EDIC) study.

AIM Recently, major attention has been paid to the role of hypoglycemia as a cardiovascular risk factor. While EURODIAB-investigators concluded that severe hypoglycemia is not a cardiovascular risk factor in type 1 diabetes, other investigators found the opposite. The primary purpose of this study was to investigate the role of severe hypoglycemia in atherosclerosis during the DCCT- and EDIC-years with special attention to overall glycemic levels. RESEARCH DESIGN AND METHODS The effect of severe hypoglycemic rates on coronary artery calcification (CAC) was evaluated for the entire cohort (n = 1205) and glycemic stratified cohorts (HbA1C < 7.5% [58 mmol/mol], HbA1C ≥ 7.5%). RESULTS The association between CAC and mean DCCT-hypoglycemia rate was stronger than the association between CAC and mean EDIC-hypoglycemia rate. Although the DCCT-severe hypoglycemia rate without HbA1C-stratification was not significantly associated with a CAC-score ≥ 100 Agatston units (p = 0.093), the interaction between above glycemic ranges and DCCT-hypoglycemic rate was significant (p < 0.05). A sub-analysis of patients belonging to the lower glycemic range (HbA1C < 7.5%), adjusted for baseline age, gender, baseline diabetes duration, baseline neuropathy, baseline albumin excretion rate, systolic blood pressure, LDL-cholesterol, smoking status, body mass index and DCCT-A1C, indicated significant (p = 0.02) associations between DCCT-severe hypoglycemia rate and CAC-score ≥ 100. One unit increase in the natural logarithm transformed DCCT-severe hypoglycemia rate increased the risk of having a CAC ≥ 100 by 30%. CONCLUSIONS Our results suggest a cumulative effect of hypoglycemic events on cardiovascular risk. They provide a possible link between above mentioned contradictory reports. Our findings support the relevance of personalizing glycemic goals in diabetes management beyond HbA1C.

Acetaminophen attenuates obesity-related renal injury through ER-mediated stress mechanisms.

Background/Aims: Obesity is an independent risk factor for the development of kidney disease. The purpose of this study was to determine how obesity may contribute to renal damage and whether acetaminophen ingestion can diminish obesity-associated renal cell injury in the obese Zucker rat model. Methods: Male obese Zucker rats (4 weeks old, n=6) were treated with acetaminophen (30 mg / kg body weight / day) for 26 weeks. Age matched obese control (OC), obese vehicle (OV, 0.073 mL DMSO/kg/d), and lean Zucker rats (LC) were used to determine the effects of treatment and obesity. Results: Compared to lean control rats, renal lipid deposition, expression of the endoplasmic reticulum (ER) stress protein GRP78 and activation of the ER stress-related eIF2α-ATF4-CHOP, caspase 12, and JNK-MAPK signaling pathways were increased in the obese control and obese vehicle rats. These alterations were associated with the elevated renal cell apoptosis and urinary albumin excretion. Acetaminophen treatment decreased renal lipid deposition, ER-stress related signaling, apoptosis and albuminuria. Conclusion: These data suggest that the protective effects of low dose acetaminophen on renal injury are mediated, at least in part, through attenuation of ER stress.

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.

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.