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Ethanol (EtOH) exerts deleterious actions on reproductive function at all three levels: the hypothalamus, pituitary, and gonad (HPG). Nitric oxide (NO), a newly identified messenger molecular in a variety of biological systems, has been suggested as playing a role in HPG hormone regulation. NO stimulates luteinizing hormone releasing hormone secretion from the hypothalamus and has variable effects on luteinizing hormone release from the pituitary. NO is inhibitory to testosterone production, and it may also directly inhibit some steroidogenic enzymes. Related studies in the accompanying paper have demonstrated that inhibiting NO synthase (NOS) using various NOS inhibitors can prevent the EtOH-induced suppression of testosterone on the male HPG axis, and this action is mainly, although not entirely, due to a direct gonadal effect. To further investigate the role of NO in the HPG axis, we assessed the HPG NO-NOS system by determining NOS mRNA levels, protein levels, and enzyme activity in the presence and absence of EtOH. At the testicular level, EtOH's action did not appear to be mediated by increasing NO content. However, EtOH was able to potentiate NO's suppressive effect on the testicular synthesis system. One locus where EtOH and NO interacted was at the steroidogenic enzyme level. N(G)-nitro-L-arginine methyl ester, a NOS inhibitor, was found to antagonize the EtOH-induced fall on P-450 17alpha-hydroxylase/C17-20 lyase mRNA levels when administered along with EtOH. EtOH had no apparent effect on the pituitary NO-NOS system and its effects on the hypothalamic NO-NOS system do not explain its ability to reduce luteinizing hormone releasing hormone secretion.

Ethanol (EtOH) suppression of the hypothalamic-pituitary-gonadal (HPG) axis results in broad reproductive malfunction. In the HPG axis, the suppressive effects of EtOH are manifested by decreased serum testosterone, reduced testicular luteinizing hormone (LH) receptor numbers, lowered serum LH and pituitary beta-LH mRNA levels (in castrated animals), and impaired luteinizing hormone releasing hormone (LHRH) release from the hypothalamus. Increasing evidence has suggested that nitric oxide (NO) plays a role in regulation of the HPG axis. NO was shown to stimulate LHRH secretion from the hypothalamus and to have variable effects on LH release from the pituitary. At the gonadal level, NO is inhibitory to testosterone production. NO may directly inhibit some testicular steroidogenic enzymes. To investigate the effect of EtOH, NO, and their interaction on the male HPG axis, three NO synthase (NOS) inhibitors, N(G)-nitro-L-arginine methyl ester, N(G)-nitro-L-arginine, and 7-nitro indazole were used to study overall HPG function in the presence and absence of EtOH. Animals were given intraperitoneal injections of saline, EtOH, various NOS inhibitors, or EtOH, along with NOS inhibitors 2 hr before sacrifice. Serum testosterone and LH concentrations, pituitary beta-LH mRNA levels, hypothalamic LHRH mRNA levels, and LHRH content were determined. It was found that blocking NOS by these NOS inhibitors prevented EtOH-induced suppression of testosterone and, in some cases, serum LH. However, this was not accompanied by concurrent changes with NOS blockade on LHRH mRNA, hypothalamic pro-LHRH or LHRH content or pituitary LH beta mRNA levels. It appears that the protective effect of NOS blockade was largely, although not completely, due to a direct effect at the gonadal level.

Teenage drinking is a major problem in the United States, as well as abroad. Besides psychosocial implications, ethanol (EtOH) has detrimental effects on the reproductive system. Clinical problems associated with reduced reproductive hormones include osteoporosis, decreased muscle function, anemia, altered immune function, prostate involution, and decreased reproductive abilities. Education coupled with strategies aimed at preventing these deleterious consequences even in the face of continued EtOH intake is extremely important. We have tested the possibility that naltrexone, a drug currently used in patients to decrease alcohol craving, might also prevent the fall in the male hormone, testosterone, caused by EtOH exposure. Rats aged 35 days old (prepubertal), 45 days old (midpubertal), and 55 days old (late pubertal) were injected (intraperitoneally) with either saline, EtOH, naltrexone, or EtOH plus naltrexone. In the two older age groups, EtOH significantly suppressed testosterone, which was prevented by administration of naltrexone. In the youngest animals, there was no treatment effect presumably due to low basal levels of testosterone. EtOH similarly reduced luteinizing hormone (LH), but this suppression was not prevented by naltrexone. There was no consistent effect of any treatment on hypothalamic concentration of pro-LH releasing hormone (RH) (LHRH), LHRH, or on steady-state levels of LHRH mRNA. We conclude that, as animals progress through puberty, EtOH suppresses LH and testosterone. The testosterone decline can be prevented by opiate blockade with naltrexone, an effect primarily seen at gonadal level. Thus, naltrexone, a drug already used clinically to reduce EtOH intake, also has protective physiological effects on the endocrine system.

Mammalian reproductive function is under control of the integrated hypothalamic-pituitary-gonadal (HPG) axis. Castration in male rats has been utilized as an effective tool to investigate hormonal interactions in the mammalian HPG axis. Recently, nitric oxide (NO) has been suggested to play a role in HPG hormonal regulation. In order to gain further insight into the function of the NO-NOS system in reproductive neuroendocrine control, particularly in the gonadal feedback regulation of the hypothalamic-pituitary unit, we examined steady state levels of nNOS mRNA, nNOS protein, and the important physiological index, NOS enzyme activity, of the intrinsic NOergic system in both hypothalamus and pituitary in castrated male rats and their sham-operated counterparts one week after surgery. In the pituitary, we found a significant four-fold increase in nNOS mRNA, p < 0.0003 compared to sham. Castration also resulted in a four-fold rise in pituitary nNOS protein, p < 0.02 compared to sham. Pituitary NOS enzyme activity was stimulated 2 fold, p < 0.003 after castration. In the hypothalamus, conversely, we observed no significant castration-modulated difference in either nNOS mRNA, nNOS protein or NOS enzyme activity. Thus, it appears that the hypothalamic NO-NOS system is either not required for hypothalamic adaptations to castration, although important in the release of LHRH under normal physiological conditions, or alternatively, the hypothalamus may become more sensitive to the effects of NO in the castrated state. In the pituitary, NO may attenuate the gonadotropin response to castration as a local balancing mediator.

It is the purpose of this study to investigate the effects of acute ethanol (EtOH) on the female rat hypothalamic-pituitary-gonadal (HPG) axis. The molecular and cellular mechanistic details of such effects have been studied intensively in the male rat. However, there has been relatively little in-depth study of EtOH's effects on the adult, postpubertal female rat. Adult female rats with confirmed 4- or 5-day estrous cycles were given a single injection of EtOH or saline between noon and 1:00 PM on proestrous and were killed at 4:00 PM. EtOH caused a sharp 97% reduction in luteinizing hormone (LH) serum levels (p < 0.001), compared with controls with no concomitant change in LH mRNA. EtOH also significantly reduced hypothalamic LH releasing hormone (LHRH) by 49% (p < 0.01), with no change in content of the precursor pro-LHRH compared with saline-injected controls. The ratio of LHRH to pro-LHRH was also significantly reduced by EtOH (p < 0.05), compared with control. There was no EtOH-induced change in LHRH mRNA. Compared with saline, EtOH reduced both serum estradiol by 37% (p < 0.02) and progesterone by 47% (p < 0.001). These results show that EtOH has profound disruptive effects on the female HPG axis. Our data suggests that EtOH decreases the releasable LHRH pool either by decreasing conversion of pro-LHRH to LHRH and/or by increasing local LHRH degradation. This acutely restricts the release of LH and subsequent estradiol and progesterone secretion.

The deleterious effects of ethanol on the hypothalamic pituitary growth hormone axis in adult male humans and animals have been well documented. It is also well established that ethanol has toxic effects on testicular function in adult humans and animals. Much less is known, however, about the effects of ethanol on the growth hormone (GH) axis and testicular function in adolescence. Recent studies have established that adolescent problem drinking is a widespread and growing threat to the health of young people in the United States. In the present study, therefore, we investigated if acute ethanol exposure in peripubertal male Sprague–Dawley rats altered normal pituitary and testicular function. Serum levels of GH and testosterone were measured at 1·5, 3, 6, and 24 h after a single i.p. injection of either saline or 3 g/kg body weight ethanol. Histologic analysis as well as serum testosterone levels allowed us to assign animals to either early puberty (35-day-old animals), mid-puberty (41-day-old animals), or young adult (51- and 66-day-old animals) status. Ethanol produced significant decrements in serum testosterone in the 51-and 66-day-old animals, with a trend toward suppression in the 41-day-old group. Furthermore acute ethanol administration significantly decreased serum GH (P< 0·0001 by 3 way ANOVA) demonstrating a significant effect of ethanol on serum GH in all age groups and at all time points studied when compared with saline injected controls (P We conclude that, as in adult animals, acute exposure to ethanol causes a prolonged and severe decrement in serum GH which is possibly mediated at the level of secretion. In addition, there is attenuation in testosterone secretion. These data are all the more important since GH and testosterone play critical roles in organ maturation during this stage of development. Journal of Endocrinology (1997) 152, 477–487

Disorders of mineral metabolism may cause neurologic manifestations of the central and peripheral nervous systems. This is because plasma calcium stabilizes excitable membranes in the nerve and muscle tissue, magnesium is predominantly intracellular and is required for activation of many intracellular enzymes, and extracellular magnesium affects synaptic transmission. This chapter reviews abnormalities in electrolytes and minerals which can be associated with several neuromuscular symptoms including neuromuscular irritability, mental status changes, cardiac and smooth muscle changes, etc.

Many studies have consistently shown that castration induces a prompt increase in serum levels and pituitary content of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as a concomitant rise in steady state levels of the messenger RNAs directing their synthesis. The reports of effects of castration on the overall physiology of hypothalamic luteinizing hormone-releasing hormone (LHRH) — steady state levels of LHRH mRNA, post-translational processing and secretion — have, however, not been consistent. The goal of the studies reported here was to provide the first analysis of the effect of castration, at multiple post-operative time points, on steady state levels of LHRH mRNA and on the levels of hypothalamic proLHRH. All these data are correlated with hypothalamic levels of the mature LHRH decapeptide and with serum and pituitary levels of immunoreactive LH and FSH. Adult male rats were either castrated or sham-castrated (controls) and then sacrificed at 1, 3, 5, 7, 14, 21 or 28 days postoperatively. As expected, there was a prompt and sustained rise in serum immunoreactive LH and FSH in castrates compared with sham-operated animals. Intrapituitary LH levels rose above levels in the sham-operated animals by 14 days post castration. Intra-pituitary FSH showed a biphasic response, first falling significantly below control levels, then rising above control levels at 21 days. Steady state levels of LHRH mRNA in castrates, measured by reverse transcription/polymerase chain reaction, were increased about 2-fold above control levels by 1 day postoperatively, but were virtually identical to control levels at each of the other time points despite marked changes in the gonadotropins. ProLHRH content in castrates was 1·8-times that seen in controls at 1 day post castration (PPP We conclude that: (1) changes in steady state levels of LHRH mRNA after castration are small and transient and (2) increased proLHRH coupled with unchanged LHRH levels at 1 day post castration, and castrate animal pro-LHRH at control levels coupled with falling LHRH at later post-castration time points indicate that the effect of gonadectomy on post-translational processing of pro-LHRH to LHRH is, likewise, small and transient. In aggregate our data suggest that most of the increase in serum LH and FSH seen in male rats after castration is not mediated at the hypothalamic level. Journal of Endocrinology (1996) 148, 509–515

OBJECTIVE It is not clear whether intensive pharmacological therapy can be effectively sustained in non-insulin-dependent diabetes mellitus (NIDDM). The relative risks and benefits of intensive insulin therapy in NIDDM are not well defined. Accordingly, we designed a feasibility study that compared standard therapy and intensive therapy in a group of NIDDM men who required insulin due to sustained hyperglycemia. RESEARCH DESIGN AND METHODS A prospective trial was conducted in five medical centers in 153 men of 60 ± 6 years of age who had a known diagnosis of diabetes for 7.8 ± 4 years. They were randomly assigned to a standard insulin treatment group (one morning injection per day) or to an intensive therapy group designed to attain near-normal glycemia and a clinically significant separation of glycohemoglobin from the standard arm. A four-step plan was used in the intensive therapy group along with daily self-monitoring of glucose: 1) an evening insulin injection, 2) the same injection adding daytime glipizide, 3) two injections of insulin alone, and 4) multiple daily injections. Patient accrual and adherence, glycohemoglobin (HbA1c), side effects, and measurements of endpoints for a prospective long-term trial were assessed. RESULTS Accrual goals were met, mean follow-up time was 27 months (range 18–35 months), and patients kept 98.6% of scheduled visits. After 6 months, the mean HbA1c in the intensive therapy group was at or below 7.3% and remained 2% lower than the standard group for the duration of the trial. Most of the decrease in the mean HbA1c in the intensive group was obtained by a single injection of evening intermediate insulin, alone or with daytime glipizide. By the end of the trial, 64% of the patients had advanced to two or more injections of insulin a day, aiming for normal HbA1c. However, only a small additional fall in HbA1c was attained. Severe hypoglycemia was rare (two events per 100 patients per year) and not significantly different between the groups, nor were changes in weight, blood pressure, or plasma lipids. There were 61 new cardiovascular events in 40 patients and 10 deaths (6 due to cardiovascular causes). CONCLUSIONS Intense stepped insulin therapy in NIDDM patients who have failed glycemic control on pharmacological therapy is effective in maintaining near-normal glycemic control for > 2 years without excessive severe hypoglycemia, weight gain, hypertension, or dyslipidemia. Cardiovascular event rates are high at this stage of NIDDM. A long-term prospective trial is needed to assess the risk-benefit ratio of intensified treatment of hyperglycemia in NIDDM patients requiring insulin.

We have previously reported a decrease in Luteinizing Hormone (LH) levels in serum afterin vivo acute ethanol exposure in male rats. Accompanying these changes, a rapid and marked decrease of β-LH mRNA was observed. A similar decrease was not detected in the common α-subunit or β-FSH mRNA. The studies presented here examined the possible mechanisms of decreasing β-LH mRNA by using S1 nuclease protection assay to evaluate the effect of acute ethanol exposure on the levels of β-LH heteronuclear RNA (hnRNA). There was no significant difference detected in the level of β-LH hnRNA after ethanol exposure. Polysome distribution analysis was used to evaluate the association and disassociation of β-LH mRNA with polyribosomes since non-polyribosome associated mRNA may be more vulnerable to degradation by RNAases. The results indicated a decrease in the association of the β-LH mRNA with polysomes following acute ethanol exposure. This decrease in polyribosome association would increase the exposure of the β-LH transcript making it more susceptible to RNases. We conclude that the decrease in steady-state β-LH mRNA levels after ethanol exposure occurs because of increasing degradation of the transcript rendered vulnerable by displacement from polysomes and not through a decreased transcriptional rate.

An immunomodulatory role for LHRH was suggested when we reported the presence of immunoactive and bioactive LHRH and its mRNA in rat splenic and thymic lymphocytes. In this paper we report that human peripheral T-cells as well as its subsets CD4+ and CD8+ contained immunoactive and bioactive LHRH. Furthermore, analysis of phytohemagglutinin (PHA)-activated T-cell lysates for LHRH by RIA demonstrated that the mean concentration of LHRH in PHA-activated T-cells increased from 45 +/- 4.5 to 64 +/- 7 pg/10(6) cells after 24 h of culture and from 47 +/- 3.6 to 117 +/- 11.8 pg/10(6) cells (P0.01) after 48 h. While the LHRH concentration in PHA-activated cells increased over the last 24 h of culture h from 64 +/- 7 to 117 +/- 11.8 pg/10(6) cells (P0.001), there was no change in mean concentration of LHRH in T-cells kept in medium alone. In a preliminary study we found that fresh T-cells contain 20 +/- 1.4 pg pro-LHRH/10(6) cells, and PHA stimulation increased the pro-LHRH content similar to the increase in LHRH. As with unfractionated T-cells, a significant PHA-induced time-dependent enhancement of intracellular LHRH was noted in CD4+ and CD8+ T-cells. RNA extracted from lymphocytes was subjected to reverse transcription-polymerase chain reaction analysis using LHRH and histone-3.3, primers, the latter as an internal control. The polymerase chain reaction-generated data demonstrated that the relative amount of LHRH mRNA in cultured, but non-PHA-stimulated (resting), cells diminished dramatically between 5-24 h, but recovered by 48 h of culture. The relative amount of LHRH mRNA in PHA-stimulated cells revealed a markedly different pattern. LHRH message expression in PHA-activated cells increased slightly at 5 h of culture and was maximally stimulated by 24 h, but declined by 48 h of culture. The PHA activation-induced time-dependent enhancement of intracellular accumulation of LHRH peptide at 5 and 24 h was accompanied by increased LHRH message. However, the increased concentration of LHRH peptide at 48 h coincided with decreased LHRH message expression. The data from total protein synthesis in PHA-activated cells showed a progressive increase in protein synthesis, a pattern entirely similar to the changes in the cell content of LHRH.(ABSTRACT TRUNCATED AT 400 WORDS)

The measurement of alterations in low abundance mRNAs such as the hypothalamic hormones luteinizing hormone-releasing hormone (LHRH) and growth hormone-releasing hormone (GHRH or GRF) from individual hypothalamic tissues in rats has previously been difficult and usually required either isolation of poly(A) mRNA or the pooling of numerous animals to obtain a reasonable signal on Northern blots. Although more sensitive detection methods exist, such as the use of RNA probes or solution hybridization (RNase protection), we have found the most reliable, sensitive, rapid, and accurate method is the reverse transcription-polymerase chain reaction (RT-PCR) using histone H3.3 as an internal control for both steps of this procedure. H3.3 is a cell-cycle independent and constitutively expressed gene in all tissues. We have developed an RT-PCR assay for LHRH and GRF mRNA quantitation and comparative analysis for hypothalamic and extrahypothalamic brain tissues and present the use of RT-PCR for LHRH quantitation in ethanol (EtOH) studies.

The recent availability of genetically altered rat lines differing in sensitivity to ethanol (EtOH) has allowed deeper investigation into the mechanisms of EtOH-induced cellular toxicity in several systems. Since the male central reproductive axis has been demonstrated to be exquisitely sensitive to EtOH, studies were undertaken to determine if the gonadotropin suppression reported earlier could be duplicated in one of these selected rat lines. Castrated high alcohol sensitivity (HAS), low alcohol sensitivity (LAS) and control alcohol sensitivity (CAS) rats were given EtOH or saline acutely. Castrated non-selectively bred Sprague Dawley rats were treated similarly and used as an additional control. At sacrifice, serum and pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were obtained and the mRNA levels for both gonadotropins assessed. In the selectivity bred animal there was essentially no change in serum or pituitary LH or FSH levels between EtOH and saline treated animals. The mRNA levels for both LH and FSH similarly were unaffected by EtOH, in striking contrast to the non-selectively bred Sprague Dawley rats where serum LH, FSH and beta-LH mRNA levels are markedly suppressed after EtOH exposure. The selectively bred lines of rats genetically manipulated for high or low EtOH sensitivity, as well as their non-selected controls, appeared to have a hypothalamic-pituitary reproductive unit that is resistant to EtOH. This is in contrast to Sprague-Dawley rats, where suppression of this axis previously has been consistently demonstrated.

Heparin/platelet factor 4 (H:PF4) antibodies are the causative agent in heparin-induced thrombocytopenia (HIT). The antibodies are frequently formed after exposure to heparin, most commonly without any signs of clinical HIT. Heparin-induced thrombocytopenia antibodies have been detected by enzyme-linked immunosorbent assay (ELISA) in individuals who have not been exposed to heparin. It is possible that the antibodies could be elicited by PF4 associated with endogenous, heparin-like glycosaminoglycans (GAGs). This risk would be higher in individuals with endothelial dysfunction and chronic platelet activation. In the setting of an outpatient endocrinology clinic, both diabetic and nondiabetic patients were studied and compared with healthy volunteers. Heparin/platelet factor 4 antibody titers were measured by ELISA and analyzed to determine the frequency of clinically seropositive responses, and median and interquartile ranges of baseline antibody titers. The study found no increase in frequency of ELISA-positive patients among diabetic patients. Moreover, the diabetic population had lower overall level of H:PF4 antibody titer, especially the subgroups treated with thiazolidinedione drugs or angiotensin receptor blockers. Further studies are needed to determine whether subthreshold titers of HIT antibody may be reflective of the physiological state of platelet/endothelial balance.

OBJECTIVE To determine, after 1 yr of follow-up in type II diabetes patients, whether a statistically and clinically significant difference can be achieved in HbA1c between a standard therapy group and an intensively treated group, while maintaining HbA1c levels in both groups within ranges acceptable in regular community practice. Secondary objectives include assessment of patient adherence to protocol, side effects, and accuracy of data collection. RESEARCH DESIGN AND METHODS This is a prospective, randomized, controlled VA CS conducted with 151 patients at five VAMCs. Patients are males, age 40–69 yr, treated at entry with a maximum dose of sulfonylurea or with insulin, exhibiting an HbA1c level > 3 SDs above the normal mean (5.05 + 3 × 0.50 = > 6.55%). Standard control is achieved with insulin and intensive control with a step-up regimen including insulin alone or insulin/glipizide combinations. Education and management of cardiovascular risk factors are handled similarly in both groups. Primary macrovascular end points are nonfatal myocardial infarction, congestive heart failure, stroke, amputation, and cardiovascular death. Primary microvascular end points are appearance and progression of retinopathy, documented by centrally read seven-field-stereo fundus photographs. Other measured indicators include resting and ambulatory ECGs, ventricular function (MUGA scan), serum lipid and apolipoprotein levels, plasma fibrinogen, nonsymptomatic peripheral vasculopathy, neuroautonomic status by heart-beat variation on Valsalva maneuver, and microalbuminuria. CONCLUSIONS This study may be the basis for a long-term trial, involving 1400 patients, to assess the long-term effects of metabolic control on macro- and microvascular end points.

The interaction of ethanol (EtOH), prolactin (Prl) and luteinizing hormone (LH) was examined in two studies. In the first study, adult male C57 B1/6J mice were given a single intraperitoneal injection of either vehicle or Prl at 5, 10 and 20 mg/kg and a significant dose-related suppression of ethanol consumption was found. This injection did not cause any differences in food intake or body weight. Additionally, a 5 mg/kg dose of Prl was also given to adult male Long Evans Hooded rats and, similarly, there was a significant suppression of ethanol consumption. In a second study, when rats were given a free choice between water and 5% EtOH, three subgroups were found regarding the amount of EtOH consumption: low, medium and high. After 2 weeks of free choice, hypothalamic, but not serum Prl and LH levels, were significantly increased in EtOH-imbibing groups compared to controls. These findings suggest important interactions between EtOH consumption and ambient levels of Prl and LH.

The impact of ethanol (EtOH) on male rodent reproduction has been well characterized for luteinizing hormone (LH) with suppression of LH release from the pituitary being reported. We have previously reported that acute ethanol (EtOH) exposure in vivo results in rapid and marked suppression of β-LH gene expression and protein release from the pituitary. This suppression of β-LH gene expression was unaccompanied by a change in the common α-subunit mRNA. To further explore the impact of ethanol on male rodent reproduction, we have expanded our studies to follicle stimulating hormone (FSH) and hypothalamic luteinizing hormone releasing hormone (LHRH) as well as of pituitary protein kinase C (PKC). Previously castrated male rats were acutely exposed to EtOH and a dramatic reduction in both serum FSH and LH levels was noted at 1.5 and 3 hr after treatment. These levels returned to saline injected control values at 6 and 24 hr. Despite the fall in serum FSH, there was no change in intrapituitary FSH content at any time point; this lack of pituitary FSH depletion in the face of a fall in serum levels is suggestive of impaired FSH release. In contrast to the fall in β-LH steady-state mRNA levels seen previously and confirmed in the present studies, there was no change in β-FSH steady-state mRNA at any time point suggesting that EtOH has dichotomous effects on the expression of these two gonadotropins. Pituitary PKC levels were also assessed and found to be unaffected by EtOH at any time point. This enzyme is important in transmembrane signaling for the gonadotropins, and while ETOH is known to affect PKC in other cells, it appears that the effect of EtOH on the gonadotrops is not mediated at this level. Finally, hypothalamic luteinizing hormone releasing hormone (LHRH) content was assessed by radioimmunoassay, and no change was found at any time point in this hypothalamic peptide. We conclude that the effect of EtOH on pituitary gonadotropin gene expression is not uniform, with β-LH suppression occurring selectively while β-FSH is unaltered. The release of both gonadotropins appears to be impaired by EtOH, while pituitary PKC and hypothalamic LHRH content are unchanged.

OBJECTIVE This study investigated the hypothesis that baseline calcified coronary atherosclerosis may determine cardiovascular disease events in response to intensive glycemic control within the Veterans Affairs Diabetes Trial (VADT). RESEARCH DESIGN AND METHODS At baseline, 301 type 2 diabetic participants in the VADT, a randomized trial comparing the effects of intensive versus standard glucose lowering on cardiovascular events, had baseline coronary atherosclerosis assessed by coronary artery calcium (CAC) measured by computed tomography. Participants were followed over the 7.5-year study for development of cardiovascular end points. RESULTS During a median follow-up duration of 5.2 years, 89 cardiovascular events occurred. Although intensive glucose-lowering therapy did not significantly reduce cardiovascular events in the substudy cohort as a whole, there was evidence that the response was modified by baseline CAC, as indicated by significant P values for treatment by log(CAC + 1) interaction terms in unadjusted and multivariable-adjusted models (0.01 and 0.03, respectively). Multivariable-adjusted hazard ratios (HRs) for the effect of treatment indicated a progressive diminution of benefit with increasing CAC. Subgroup analyses were also conducted for clinically relevant CAC categories: those above and below an Agatston score of 100. Among those randomized to intensive treatment, for the subgroup with CAC >100, 11 of 62 individuals had events, while only 1 of 52 individuals with CAC ≤100 had an event. The multivariable HR for intensive treatment for those with CAC >100 was 0.74 (95% CI 0.46–1.20; P = 0.21), while for the subgroup with CAC ≤100, the corresponding HR was 0.08 (0.008–0.77; P = 0.03), with event rates of 39 and 4 per 1,000 person-years, respectively. CONCLUSIONS These data indicate that intensive glucose lowering reduces cardiovascular events in those with less extensive calcified coronary atherosclerosis.

To determine risk factors in clinically significant macular edema (CSME) and if increased CSME in minorities is due to ethnicity or other factors in the Veterans Affairs Diabetes Trial (VADT).CSME prevalence based on 7-field stereo fundus photographs in 1268 patients with type 2 diabetes was related to ethnicity, demographics and biochemistries by univariate and multivariate analyses.Hispanics (H) made up 17.5% and African Americans (AA) 17.7% of the cohort. CSME prevalence was 10%. In univariate analysis, CSME was more prevalent in H, 18%, and AA, 15.6% than in non-Hispanic Whites (NHW), 6.3%, p0.01. Univariate regression of CSME associated with younger age, younger onset of diabetes; longer duration; retinopathy severity; and high HbA1c, BP, urine albumin/creatinine, and amputation, all p0.01. In multivariate regression, CSME was associated with ethnicity/race (Hispanic White vs. non-Hispanic White, OR, (95% CI), 2.30, (1.35-3.92), p0.01; African American vs. non-Hispanic White, 2.30, (1.33-4.00), p0.01), diastolic BP (1.13 per 5 mm Hg, (1.02-1.23), p=0.03), amputation (3.0, (1.11-8.13), p=0.04), and retinopathy severity ( approximately 30, ( approximately 17 to approximately 59), p0.01).The prevalence of CSME in the VADT is associated with ethnicity as well as diastolic BP, amputation, and retinopathy severity.

Thermal injury is often associated with previous ethanol exposure, and close to 50% of patients admitted to a burn unit have a potentially high blood ethanol level. Cellular mechanisms by which ethanol and/or burn affect the hypothalamic-pituitary-gonadal (HPG) axis are not entirely understood. However, it is known that the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 influence negatively on the endocrine functions of the HPG. We report a time course study (6, 12, 24, and 48 hours) of the effects of ethanol, burn, or the combination of burn/ethanol on proinflammatory cytokines of the hypothalamus, pituitary and testes of male C57Bl/6 mice. We found that there were highly significant increases in each of these cytokines caused by ethanol, burn, and burn/ethanol compared with sham/vehicle (P < .001). This was true in hypothalamus, pituitary, and testes. Because these cytokines generally reduce reproductive function, it may be that proinflammatory cytokines of HPG axis mediate the deleterious effects of burn and/or ethanol on mammalian reproduction.

A variety of indirect data suggest that the luteinizing hormone (LH) lowering effects of ethanol (ETOH) are mediated at a hypothalamic level decreasing the synthesis and/or release of LH-releasing hormone (LHRH). Little direct data support this concept, however. The current study was, therefore, designed utilizing a perifusion system with frequent sampling for LHRH with and without ethanol added to determine if ethanol had a direct effect on basal or stimulated LHRH release. A variety of secretagogues, including dopamine, norepinephrine, naloxone, prostaglandin E2, and a high dose of potassium were utilized. Ethanol at a dose of 300 mg% did not alter either basal or secretagogue-stimulated LHRH release from the hypothalami of ethanol-naive male rats. Thus, ethanol did not appear to have a direct effect on LHRH in this system. Alterations in LHRH release by ethanol may occur at a suprahypothalamic level, involving neurotransmitter-LHRH interactions. Alternatively, the well-described lowering effect of ethanol on LH may be secondary to a direct pituitary locus of action, or involve a metabolic breakdown product of ethanol rather than ethanol itself.

Ethanol causes decreased function of the hypothalamic-pituitary-gonadal (HPG) axis. Ethanol resulted in inflammatory changes in HPG manifested by increased concentrations of pro-inflammatory cytokines. Since, such cytokines have deleterious effects on functions of HPG, it seemed possible that ethanol's suppressive action could be due, at least in part, to this inflammation. Since oxidative stress can cause inflammation, we have used the antioxidant vitamin E to test, whether reducing inflammation might protect reproductive functions from ethanol. Rats were fed an ethanol diet or pair fed identically without ethanol for a 3-week period. For the last 10 days, animals were given 30 IU/kg or 90 IU/kg or vehicle. Ethanol significantly increased hypothalamic, pituitary and testicular TNF-alpha and IL-6, all changes prevented by the higher dose of vitamin E. Also, ethanol induced changes in LHRH, LH, testosterone, and testicular germ cell apoptosis were similarly prevented by vitamin E. These data strikingly show that vitamin E protects the HPG from deleterious effects of ethanol and suggests that the mechanism of this protection might be both anti-inflammatory and antioxidant.

In recent, landmark clinical trials, insulin to maintain euglycemia in critically ill patients improved clinical outcomes, including decreased all-cause mortality. Novel antiinflammatory effects of insulin have recently been described. Thermal injury is an excellent model of critical illness. The addition of ethanol to the model is of great clinical relevance because nearly 50% of the patients admitted to hospitals for burn injuries have ethanol in their circulation. Utilizing a murine model of critical illness (ethanol and skin burn), we tested the hypothesis that insulin treatment in ethanol-exposed, burn-injured mice reduced hepatic inflammation, a potential mechanism for the benefit of insulin. Adult male C57BL/6 mice were given a single intraperitoneal injection of ethanol or saline, were given a 15% total body full-thickness skin burn, or were sham-burned and killed 24 hours later. In each group, half the animals were given subcutaneous injections of the long-lasting basal insulin glargine; the other half, the appropriate vehicle. Hepatic inflammatory markers, including polymorphonuclear infiltration, a chemokine, an important adhesion molecule, proinflammatory cytokines, and nuclear factor kappaB, were measured, and all were increased by ethanol and/or burn. These increases were prevented by insulin. An antiinflammatory cytokine was reduced by ethanol and/or burn. Insulin prevented this decrease. Thus, insulin has a substantial antiinflammatory effect, and this may underlie its dramatic clinical benefit in critical illness.

Alterations in the protein kinase C (PKC) pathway may interrupt anterior pituitary luteinizing hormone (LH) synthesis and/or secretion, which may impair normal reproductive function. Work by our laboratory and others has shown that EtOH has profound deleterious effects on the regulation of the hypothalamic-pituitary-gonadal (HPG) axis. The present study focuses on PKC translocation from the cytosol to the membrane of anterior pituitary after acute EtOH exposure. Serum levels of LH were measured at three time points (15, 30, and 90 min) after an IP injection of either saline or 3 g/kg EtOH in adult castrated male rats. LH levels dropped significantly (p < 0.03) in EtOH-injected compared to saline-injected control animals. In the same animals, EtOH significantly suppressed PKC localization at its active site at the pituitary cell membrane (p < 0.05). These findings suggest that the mechanism of EtOH's suppression of LH is mediated, at least in part, through a decrease in PKC translocation to the anterior pituitary cell membrane.

Nearly 50% of the patients admitted to hospitals for burn injuries have detectable levels of alcohol (EtOH) in their circulation. In fact, EtOH is often a causal factor in their injury. It is well known that EtOH as well as burn injury disrupt function of the hypothalamic–pituitary–gonadal (HPG) axis. The cellular mechanisms by which EtOH and/or burn impacts on the HPG are not entirely understood. In the studies reported here, we tested the hypothesis that these injuries mediated their effects by local hypothalamic inflammation. Young adult male mice were subjected to either a 15% total body surface area, full thickness scald, to EtOH, or to both and compared to appropriate controls. They were sacrificed 48 h later. EtOH and burn, as well as the combined injury, consistently and impressively reduced serum testosterone, while increasing hypothalamic concentrations of all three of the pro-inflammatory cytokines, TNFα, IL-1β, and IL-6. In general, the increases induced by burn were greater than those caused by EtOH and the effect of the combined insult was not additive. Hypothalamic concentrations of LHRH were also increased. The data are consistent with the idea that EtOH and/or burn, as models of critical illness, medicate their hypothalamic suppressive effects via increase in pro-inflammatory cytokines.

Objective The Veterans Affairs Cooperative Study in Diabetes Mellitus Type 2 Feasibility Trial (VA CSDM) studied standard and intensive glycemic treatment groups, achieving and maintaining for 27 months a difference in HbA1c of 2.1% (9.2% vs. 7.1%, respectively). A substudy planned in advance examined health status as assessed by a health status questionnaire obtained at baseline and 24 months. Design and methods A randomized, prospective trial was carried out at five VA Medical Centers from 1990 to 1993. The sample involved 153 male veterans 40–69 years of age and with diabetes duration of 8±4 years, who were suboptimally controlled with standard glucose lowering treatment. The participants were randomized to intensive and standard treatment groups. In addition to a variety of indicators of glycemic control and complications, health-related qualify of life data were assessed using a 20-question version of the Medical Outcome Study instrument. Scores were evaluated at baseline and 24 months for changes between the treatment groups. Results The two groups were similar at baseline with respect to age, duration of diabetes, complications, comorbidities, and reported physical activity. The intensive treatment group had more frequent, mandatory self-glucose monitoring (vs. occasional measurement in the standard) and received two or more daily insulin injections (only one in the standard). This group had three times the number of clinic visits and 10-fold higher reported incidence of mild/moderate hypoglycemia. There were no significant changes in the health status over time in either the standard or intensive treatment groups, nor was there a difference between the two groups. Conclusions Intensive glucose control in advanced Type 2 diabetes mellitus (DM) has no effect on health status over 2 years. The successful lowering of glycemia does not improve health-related quality of life nor do the increased demands of an intensive therapy regimen worsen it.

Background: The female liver is more sensitive to the toxic effect of chronic alcohol intake than the male liver. The aim of the study was to compare the influence of gender and sex hormonal status on apoptosis and cell proliferation following chronic ethanol intake. Methods: Male and female rats were pair fed for 8 weeks a liquid diet containing 36% of their total daily calories as ethanol (ETOH group) or sucrose (control group). Liver samples were analyzed for apoptosis and hepatocyte proliferation by immunohistochemistry. The hepatic production of factors able to influence cell death and proliferation, such as tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) were determined. Results: In both male and female rats, ethanol intake promoted apoptosis in the liver. This effect of ethanol was more evident in female than male rat livers. Hepatic TNFα levels, which promote apoptosis, are significantly more elevated in female than in male livers. Hepatic IL-6 production, which promotes hepatocyte proliferation, was induced by ethanol only in males, but not female animals. Conclusion: This observed difference in cytokine responses may contribute to the enhanced sensitivity of female liver to EtOH-induced injury.

The major drug of abuse among teenagers in the United States continues to be ethanol (EtOH), but use is seen in children as young as nine. In the studies reported here, the impact of EtOH on biologic and hormonal parameters of puberty was assessed in female rats. Rats were fed a liquid diet containing EtOH, pair fed an identical liquid diet containing dextrimaltose instead of EtOH, or fed a liquid diet not containing EtOH ad libitum. Feeding was started at 21, 25, or 28 d of age. EtOH markedly delayed the age at vaginal opening (34.5 +/- 0.5 d in controls vs 48.5 +/- 2.4 d in EtOH animals; p0.001), delayed the age at first estrous (40.9 +/- 0.6 d in controls vs 61.2 +/- 2.6 d in EtOH animals; p0.001), increased the length of the estrous cycle, and decreased the number of proestrous days. EtOH, concomitant with reduced ovarian and uterine weight, decreased serum estradiol and progesterone. Associated with these changes in ovarian hormones there was a selective increase in follicle-stimulating hormone, but not luteinizing hormone. EtOH consistently reduced insulin-like growth factor-1. In general, EtOH-induced disruption was more severe the younger the animals were at the start of feeding. Opiate receptor blockade with naltrexone completely prevented the EtOH-induced delay in vaginal opening. The impact of EtOH on female puberty is dramatic, is an emerging public health problem, and deserves more study.

Cancers that metastasize to the thyroid gland are uncommon. Metastasis to the thyroid gland has been reported in renal cell carcinoma (RCC), breast cancer, lung cancer, gastrointestinal malignancies, malignant melanoma, sarcoma, hematologic malignancies, and other genitourinary cancers. A computer search of the records of the department of pathology at Loyola University Medical Center was done to determine the number of thyroidectomies performed between 1988 and 1998. A detailed review of the clinical records of patients with metastasis to the thyroid gland from RCC was done. A total of 941 thyroidectomies were performed between 1988 and 1998. Metastasis to the thyroid gland was seen in six cases (0.64%). Three of these six cases had metastasis from RCC. The interval between the diagnosis of the primary RCC and the thyroid metastasis was 2 to 10 years. Two of these three patients had an adenomatous thyroid gland. Metastases to the thyroid, though relatively rarely diagnosed clinically as a cause of thyroid nodule, must be considered in the differential diagnosis of thyroid nodule, particularly in patients who have a history of RCC.

To determine whether a difference in HbA(1c) could be safely sustained between a standard therapy (STD) arm and an intensive therapy (INT) arm, while maintaining HbA(1c) levels in both arms within a range acceptable in community practice. The effects of intensive treatment on various parameters were studied in this feasibility trial. We report here the results of 24 months of INT on peripheral and autonomic neuropathy.A prospective trial was conducted in five medical centers in 153 men of 60 +/- 6 years of age who had a known diagnosis of diabetes for 7.8 +/- 4 years. They were randomly assigned to a standard insulin treatment group (one morning injection per day) or to an intensive therapy group designed to attain near-normal glycemia and a clinically significant separation of glycohemoglobin from the standard arm. A four-step plan was used in the intensive therapy group along with daily self-monitoring of glucose: (1) an evening insulin injection, (2) the same injection adding daytime glipizide, (3) two injections of insulin alone, and (4) multiple daily injections. Peripheral neuropathy was diagnosed clinically by a history and physical examination, and by abnormal autonomic neuropathy Valsalva ratio (VR < 1.2) and RR variation (RRV < 10). An average HbA(1c) separation of 2.07% was achieved with INT, having HbA(1c) at or below 7.3% (p = 0. 001 versus STD). Baseline prevalence of peripheral neuropathy was 53% in STD, and 48% in INT. By 24 months, the prevalence increased to 69% in STD (p = 0.005 versus baseline), and to 64% in INT (p = 0. 008 versus baseline, but no different than STD). Though INT did not reverse all elements of peripheral neuropathy, there was a decreased prevalence of cranial neuropathy (p = 0.053 versus STD) and more frequent preservation of touch sensation in the upper extremities (p = 0.03 versus STD) in INT. At baseline, an abnormal Valsalva ratio and/or RR variation was seen in 38% of STD and 31% of INT. By 24 months in STD, the prevalence rose to 55% (p = 0.0067 versus baseline), and in INT, to 48% (p = 0.012 versus baseline and no different from STD). The prevalence of erectile dysfunction increased from 53% at baseline to 73% at 2 years, p = 0.002 in STD, and from 51% to 73% at 2 years (p = 0.003 versus baseline) and no different from STD. There was no change in the frequency of abnormal gastrointestinal or sweating symptoms. Our conclusion was that 2 years of meticulous glycemic control did not decrease overall prevalence of peripheral or autonomic neuropathy. In fact, the prevalence rose equivalently and significantly in both treatment arms. There was some benefit, however, in decreased frequency of cranial neuropathy and better preservation of touch sensation in INT.

Pursuant to our report of an immunoreactive and bioactive luteinizing hormone-releasing hormone (LHRH)-like molecule in rat spleen lymphocytes, we sought to determine whether these cells were capable of synthesizing LHRH by determining whether lymphocytes contain LHRH mRNA. To do this, total RNA was extracted from hypothalamic tissue, anterior pituitaries and from lymphocytes, and then this was reverse transcribed to cDNA and amplified via the polymerase chain reaction (PCR) utilizing synthetic oligonucleotides bracketing a portion of the LHRH gene. Following gel electrophoresis a discrete band of the expected size of 375 base pairs was found in the hypothalamus (positive control), and in lymphocytes, but not in the anterior pituitary (negative control). Furthermore, after Southern blotting, a 32P-labelled LHRH cDNA, hybridized to the 375 base pair, was amplified in hypothalamus fragments and in lymphocytes, but not in anterior pituitary tissue. These data strongly suggest that LHRH, in addition to being an important neuropeptide, is an immune cell synthesized immunomodulator.

Teenage drinking continues to be a major problem in the United States as well as abroad. A significant depression in serum testosterone in adolescents who consume EtOH has been well described. In the male rodent model, a similar fall in testosterone has been reported, and prevention with the opiate blocker naltrexone has been demonstrated. To explore further the impact of chronic EtOH exposure on the reproductive axis in peripubertal rats, we designed this study specifically to define whether or not there was recovery after abstinence by examining reproductive hormones and their genes during and after EtOH exposure. Peripubertal male rats 35 d old were fed an EtOH-containing diet or a calorically matched control diet for 60 d. A third group was fed the control liquid diet ab libitum. EtOH was then withdrawn and all animals were fed standard rat chow and water ad libitum for an additional 3 mo. The EtOH-imbibing animals were found consistently to weigh less than their pair-fed mates and liquid diet ad libitum animals. Serum testosterone levels and testicular weights were significantly decreased by EtOH whereas serum estradiol levels were higher, suggesting enhanced peripheral conversion by EtOH. Spermatogenesis, assessed by histological parameters, was unaltered by EtOH. Serum luteinizing hormone levels were not different among the groups. Hypothalamic luteinizing hormone-releasing hormone mRNA levels were unaffected by EtOH. During the 3-mo recovery period, all the changes reversed, with a significant increase noted in testosterone. All other parameters remained the same among the groups. Thus, although chronic EtOH exposure in the peripubertal age period results in significant reproductive alterations, there is complete recovery on withdrawal.

The Veterans Affairs Cooperative Study in Type II Diabetes Mellitus prospectively studied insulin-treated patients with type 2 (non-insulin-dependent) diabetes mellitus, achieving 2.1% glycosylated hemoglobin separation between intensive- and standard-treatment arms (P.001) for 2 years.To assess the effect of intensive therapy on serum fibrinogen and lipid levels, compared with standard treatment.One hundred fifty-three male subjects with type 2 diabetes mellitus and who required insulin treatment were recruited from 5 Veterans Affairs medical centers. The subjects were divided into intensive- and standard-treatment arms for a randomized prospective study. Dyslipidemia was managed identically in both arms (diet, drugs). Fibrinogen levels and lipid fractions were measured in the full cohort. Lipid fractions are separately reported in patients not treated with hypolipidemic agents.There were no baseline differences between arms. Fibrinogen levels rose in the intensive-treatment arm at 1 year (from 3.34+/-0.12 to 3.75+/-0.15 g/L; P.001) but returned to baseline at 2 years (3.47+/-0.12 g/L). There was no change in the standard-treatment arm. Triglyceride levels decreased in the intensive-treatment arm from 2.25+/-0.27 to 1.54+/-0.14 mmol/L (199+/-24 to 136+/-12 mg/ dL) at 1 year (P = .004) and to 1.74+/-0.18 mmol/L (154+/-16 mg/dL) at 2 years (P = .03); there was no change in the standard-treatment arm. Cholesterol levels decreased in the intensive-treatment arm at 1 year from 5.4+/-0.21 to 4.99+/-0.13 mmol/L (207+/-8 to 193+/-5 mg/dL) (P = .02); there was no change in the standard-treatment arm. Levels of low- and high-density lipoprotein cholesterol decreased in the standard-treatment arm only by 2 years, from 3.44+/-0.13 to 3.16+/-0.10 mmol/L (133+/-5 to 122+/-4 mg/ dL) (P =.02) and from 1.10+/-0.03 to 1.00+/-0.03 mmol/L (42+/-1 to 38+/-1 mg/dL) (P.001) for low-density and high-density lipoprotein cholesterol, respectively. Levels of apolipoprotein B decreased in both treatment arms (P.001), and apolipoprotein A1 levels decreased in the standard-treatment arm (P.01). Lipoprotein (a) levels did not change in either treatment arm. Lipid results were essentially identical whether examined in the full cohort or excluding those patients receiving hypolipidemic agents.Intensive insulin therapy led to a potentially beneficial reduction in serum triglyceride levels and preservation of high-density lipoprotein cholesterol and apolipoprotein A1 levels. However, it caused transient elevation in plasma fibrinogen levels, a possible thrombogenic effect.

OBJECTIVE The Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus was conducted in NIDDM patients to determine if a significant difference in HbA1c could be achieved between groups receiving standard and intensive treatment. We observed differences in the response to exogenous insulin between African-Americans and other intensively treated patients. Therefore, we assessed the variations of response and correlated factors that might explain such differences. RESEARCH DESIGN AND METHODS One hundred fifty-three men aged 40–69 years with NIDDM for ≤15 years were randomized to either the standard therapy (n = 78) or the intensive therapy (n = 75) arm. Of the 75 patients in the intensive therapy group, 57 completed the study on insulin therapy alone. Of these, 18 were African-Americans and 39 were non-African-Americans. We conducted an analysis of the data collected to determine differences in baseline characteristics, glycemic response, insulin requirement, body weight, exercise, and basal C-peptide level, factors that may explain a difference in response to insulin therapy. RESULTS Glycemic control improved in all patients with intensive insulin therapy. African-Americans achieved a greater improvement in HbA1c compared with non-African-Americans with a similar increment in insulin. This difference could not be explained by differences in body weight, activity, concomitant use of other medicines, or insulin-secretory capacity of the pancreas. CONCLUSIONS We conclude that ethnic differences may exist in the response to insulin therapy. A knowledge of such differences may aid in achieving good glycemic control, especially since minorities have a greater prevalence of and burden from the microvascular complications of diabetes.

OBJECTIVE The feasibility study for the VA Cooperative Study on Glycemic Control and Complications in Type 2 Diabetes (VA CSDM) prospectively studied 153 insulin-requiring type 2 diabetes patients, randomized between an intensively treated arm and a standard treatment arm during a mean follow-up of 27 months. The glycemic response to each of the progressive, sequential phases of insulin treatment was assessed, along with the incidence of hypoglycemic reactions and the relative efficacy of different doses of glipizide in combination with fixed doses of insulin. RESEARCH DESIGN AND METHODS Five medical centers participated; half of the patients were assigned to the intensive treatment arm aiming for normal HbA1c levels. Age of patients was 60 ± 6 years, duration of diabetes 8 ± 3 years, and BMI 30.7 ± 4 kg/m2. A fourstep management technique was used, with patients moving to the next step if the operational goals were not met: Phase I, evening intermediate or long-acting insulin; phase II, added daytime glipizide; phase III, two injections of insulin alone; and phase IV multiple daily insulin injections. Home glucose monitoring measurements were done twice daily and at 3:00 A.M. once a week. Hypoglycemic reactions and home glucose monitoring results were recorded and counted in each of the treatment phases. RESULTS Baseline HbA1c was 9.3 ± 1.8%, and fasting plus serum glucose was 11.4 ± 3.3 mmol/1. Fasting serum glucose fell to near normal in phase I, and remained so in the other treatment phases. An HbA1c separation of 2.1% between the arms was maintained during the course of the study, while the intensive arm kept HbA1c levels below 7.3% (P = 0.001). Most of the decrease in HbA1c occurred with one injection of insulin alone (phase I, − 1.4%) or adding daytime glipizide (phase II, −1.9% compared with baseline). HbA1c did not decrease further after substituting two injections of insulin alone, with twice the insulin dose. Multiple daily injections resulted in an additional HbA1c fall (−2.4% compared with baseline). However, two-thirds of the patients were still on one or two injections a day at the end of the study Changes in home glucose monitoring levels paralleled those of the HbA1c, as did the increments in number of reported hypoglycemic reactions, virtually all either “mild” or “moderate” in character. For the combination of glipizide and insulin (phase II), the only significant effect was obtained with daily doses up to 10 mg a day; there were no significant additional benefits with up to fourfold higher daily doses, and HbA1c levels had an upward trend with doses >20 mg/day. CONCLUSIONS A simple regime of a single injection of insulin, alone or with glipizide, seemed sufficient to obtain clinically acceptable levels of HbA1c for most obese, insulin-requiring type 2 diabetes patients. Further decrease of HbA1c demanded multiple daily injections at the expense of doubling the insulin dose and the rate of hypoglycemic events. In combination therapy, doses of glipizide >20 mg/day offered no additional benefit.

Objective To present two new cases of gynecomastia as the initial manifestation of hyperthyroidism. Methods We describe detailed case reports of two men with breast enlargement who were found to have hyperthyroidism, and we review the related literature. Results Two men sought medical assistance because of unilateral tender gynecomastia. In one of these patients, thyroid, gonadal, and prostate examinations showed normal findings at the time of initial assessment, and symptoms of hyperthyroidism developed later. In our other patient with gynecomastia, other symptoms of hyperthyroidism—for example, nervousness, irritability, palpitations, and fatigue—had been present for a prolonged period but had been considered “normal” by the patient. In both patients, the hyperthyroidism was treated with radioiodine. Breast pain disappeared in both patients, and breast enlargement disappeared in one patient and was decreased in the other patient after euthyroidism was achieved. Review of the literature disclosed only two similar cases. Conclusion Because of the rarity of gynecomastia as the initial symptom of hyperthyroidism, we believe that thyroid function tests are not indicated in the workup of patients whose major complaint is gynecomastia. (Endocr Pract. 1997; 3:80-81)

The hormones responsible for regulating the hypothalamic-pituitary-gonadal axis are essential for proper reproductive function. Ethanol (EtOH) has been shown to exert its effect at all three levels of this axis. The present study defines striking differences in the time course of recovery of luteinizing hormone (LH) in gonadally intact, compared with, castrated male rats after acute EtOH administration. Serum levels of LH and testosterone were measured at various time points up to 2 weeks (1.5, 3, 24, 48, 72, 96, 168, and 336 hr) after a single intraperitoneal injection of either saline or 3 g/kg of EtOH in intact adult male rats. One EtOH injection significantly suppressed testosterone levels as low as 20% (p < 0.01) of saline-injected intact rats. This occurred as early as 1.5 hr after EtOH administration (the first measured time point), and statistically significant suppression was sustained for 96 hr. Similarly, LH levels showed a significant decrease. However, this significant fall in LH did not begin until 3 hr (p < 0.05) and continued up to 96 hr (p < 0.01), with a gradual return to control levels at 168 and 336 hr after treatment. Despite the significant and prolonged fall in testosterone levels in the EtOH-treated intact rats, beta-LH mRNA levels were inappropriately not elevated, as would be expected in the context of low circulating testosterone. However, at 168 and 336 hr, steady-state levels of beta-LH mRNA were significantly higher than seen in saline-injected controls (p < 0.05 and p < 0.01, respectively), temporally correlating with the return of serum LH to control. LH levels in the castrated animals were significantly suppressed at 1.5 hr (p < 0.05) and 3 hr (p < 0.01) after EtOH treatment, compared with controls, yet they returned much more quickly by 24 hr after treatment. beta-LH mRNA levels of castrated animals also showed a significant depression at 1.5 and 3 hr, and returned to control levels by 24 hr. In these rats, the hypothalamic LH-releasing hormone mRNA levels were not altered by a single EtOH injection at any time point. However, in the intact animals, there was a transient increase in LH-releasing hormone mRNA at 72 and 96 hr (p < 0.01 and p < 0.05, respectively) that may lead to the upregulation of beta-LH mRNA expression. These studies indicate that EtOH causes prolonged decreases in important serum hormones that are essential to the reproductive axis of the adult male rat.

The effects of ethanol (EtOH) on the male hypothalamic pituitary reproductive axis are multiple and varied. Although direct gonadal toxicity has been reported, hypothalamic-pituitary perturbations have also been noted. The difficulty of sampling the hypothalamus has made direct investigation of EtOH-induced alterations on luteinizing hormone-releasing hormone (LHRH) fraught with interpretation problems. To circumvent this, we have conducted a series of experiments exploring the effect of 200 mg% EtOH in vitro on GT1-7 cells, a newly developed LHRH secreting neural cell line. Cell lines were treated with EtOH-containing or EtOH-free media for 2, 6, 24, or 48 hr. EtOH caused no significant change in LHRH secretion at any time point, although there was a trend to increased secretion after 2 hr EtOH exposure when compared with control. Significantly increased total (i.e., cellular plus secreted) pro-LHRH coupled with significantly reduced cellular LHRH after 6 hr only of EtOH exposure suggested that EtOH caused a transient decrease in processing from bioinactive pro-LHRH to bioactive LHRH. However, even at this time point, LHRH secretion from these EtOH-exposed cells was no different than from control cells. Steady-state LHRH mRNA levels were not changed by EtOH at any time point. These findings are concordant with previous in vitro data using hypothalamic tissue that has similarly demonstrated no effect of EtOH on LHRH secretion. Taken together with the in vivo demonstration that EtOH reduces hypothalamic-pituitary portal blood levels of LHRH, these data indicate that EtOH exerts its effect either at an extrahypothalamic locus and/or on non-LHRH-producing cells within the hypothalamus.

The impact of ethanol on the male reproductive axis are multiple and varied, with both gonadal and control hypothalamic-pituitary pertubations being reported. There appears to be a discrepancy, however, between the in vivo and in vitro effects of ethanol on hypothalamic luteinizing hormones releasing hormone (LHRH) and the pituitary gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). While in vivo data suggests a decrease in LHRH release after EtOH, in vitro studies find no effect on secretion. Similarly, in vivo acute EtOH profoundly diminishes LH synthesis and secretion, while in vitro impaired release with no alteration in the transcription of beta LH has been found. A potential exploration for these discrept results could be the in vivo metabolism of EtOH into acetaldehyde and acetate, or the subsequent formation of salsolinol, a product of acetate combining with dopamine. To test this possibility, a series of in vitro experiments were conducted exposing dispensed anterior pituitary cells from male rats to different doses of acetaldehyde, acetate or salsolinol for varying amounts of time for which gonadotropin secretion and beta LH mRNA levels were assessed. The results demonstrated no effect of either acetaldehyde or acetate on basal or LHRH stimulated LH release, FSH release or steady-state beta LH mRNA levels. These data suggest that the metabolites of EtOH, which occur in vivo but not in vitro, are not responsible for the discrepant gonadotropin changes reported between the in vivo and in vitro setting. Other potential mechanisms to explain this phenomenon include differences in the molarity of EtOH, hyperprolactinemia and suprapituitary influences including hypothalamic LHRH, catecholamines, excitatory amino acids, substance P and beta endorphin.

Alcohol abuse in men has been long known to result in testicular atrophy, impotence, and reduced reproductive function, and only recently have the mechanisms responsible for this clinical picture been elucidated. Although direct gonadal toxicity after ethanol (ETOH) exposure has been well documented, the failure of serum luteinizing hormone (LH) and serum follicle-stimulating hormone (FSH) levels to rise in the presence of blunted testosterone values and impaired spermatogenesis supports a central neuroendocrine defect as well. The neuroendocrine target of ethanol’s action appears to be mediated both at a pituitary and a supra-pituitary level. Alcohol also appears to be disruptive to the male growth hormone axis, resulting in suppression of both peripheral growth hormone (GH) and insulin-like growth factor I (IGF-I) levels, in addition to altering pituitary GH synthesis. This chapter, which will deal with male reproductive and growth dysfunction, will attempt to summarize the available data on this topic.

It has been previously shown that acute ethanol (EtOH) exposure in vivo resulted in suppression of serum LH and pituitary beta-LH subunit mRNA levels in castrated male rats. While serum FSH levels also were noted to fall after in vivo, the mRNA for beta-FSH was not altered. The aim of the present studies was to determine whether these effects could be accounted for by a direct EtOH effect at pituitary level. To this end we examined the direct effect of EtOH on LH and FSH synthesis and secretion utilizing dispersed anterior pituitary cells from gonadectomized adult male rats. After a 72 hour post dissociation healing period, the cells were exposed to media containing 0 or 200 mg% EtOH for one hour. The media was removed and the cells incubated with EtOH-free media for an additional 1, 3 or 6 hrs. In the EtOH exposed cells, secretion of both LH and FSH increased to300% (p0.001) of control. At 6 hrs after withdrawal of EtOH a significant reduction in both LH and FSH secretion was seen. Intracellular content of LH and FSH was unchanged before and after withdrawal of EtOH. Steady state levels of beta LH and beta FSH mRNA were unchanged at all time points. In a separate series of experiments, pituitary cells from gonadectomized adult male rats were continuously exposed to different concentrations of EtOH ranging from 0-400 mg% for 3 hrs. LH secretion was stimulated by 400 mgm% EtOH only, while the intracellular content of LH was significantly reduced with the 400 mg% dose. The secretion of FSH was stimulated by 200 mg% and 400 mg% high dose EtOH after 3 hours, with concomitant reduction in FSH pituitary content at both these EtOh dose levels. The mRNA for both beta-LH and beta FSH was not different with any dose of EtOH compared to levels of control, non-EtOH exposed cells. We conclude that though there were similarities between in vivo and in vitro LH and FSH responses to EtOH, the differences reported here indicate that the in vivo responses are not totally explained by a direct EtOH effect at pituitary level. Rather, they must, in addition, reflect action at suprapituitary site(s), pituitary effects of EtOH metabolites or condensation products, and/or alterations in LH and FSH clearance.

Continuous administration of LHRH agonist suppresses the pituitary-gonadal axis, achieving chemical castration. Thus, LHRH agonist has been used as an alternative (to surgical castration) for the treatment of steroid-dependent prostate cancer. However, recent reports have demonstrated that LHRH agonist had a direct inhibiting effect on prostate cancer cell proliferation and that cancerous prostate tissue contained a LHRH-like peptide. In this paper we are reporting for the first time that the normal rat ventral prostate contained immunoactive and bioactive LHRH as well as its precursor molecule, pro-LHRH. Our investigation showed that the LHRH concentration in prostate increased 2 weeks after castration from 1.68 +/- 0.09 to 3 +/- 0.2 pg/mg tissue (P < 0.001). At the same time, the concentration of pro-LHRH decreased from 149 +/- 6.5 to 68 +/- 6.8 pg/mg tissue (P < 0.001). Furthermore, intact rat prostate expressed LHRH mRNA, which increased 13-fold 2 weeks after castration. In summary, the prostate of intact Sprague-Dawley rats has the capacity to produce the LHRH precursor and process it to the mature decapeptide, and this production/processing increases significantly after castration.

The effects of ethanol (EtOH) on GH and PRL have been previously explored, and a dicotomy in results noted. While serum GH levels appear to fall after EtOH exposure, PRL levels rise. We have attempted to expand these studies by examining the impact of acute or "binge" EtOH in vivo on GH and PRL synthesis and secretion. At 0.5, 1.5, and 3.0 h after one dose of ip EtOH, serum GH levels fell significantly compared with those seen in saline-injected controls. This correlated with a fall in GH mRNA levels, but no change in pituitary GH content. Conversely, serum PRL levels rose significantly, while the mRNA for PRL decreased by approximately 20%. There was no change in pituitary PRL content. Interestingly, the mRNA for pit-1 (GHF-1), a transcription factor important to both GH and PRL gene expression, was unchanged at any time point. Despite the fall in GH and PRL mRNA levels, the pituitary cAMP content was markedly elevated at 0.5 h, with no change at any other time point. In summary, acute EtOH exposure in vivo appears to dampen both GH and PRL synthesis, while serum levels behave dissimilarily. Possible explanations for these findings are discussed.

Prolactin (PRL), or a PRL-like molecule has been identified in the central nervous system and other tissues by numerous investigators. The previous finding of PRL in brain persisting for weeks following hypophysectomy led us, and others, to conclude the brain and central nervous system PRL is synthesized locally. Also, our previous results showing PRL mRNA in hypothalamic and extra-hypothalamic brain regions using reverse transcription-polymerase chain reaction (RT-PCR) techniques, along with this report that the sequence of the PRL message in the brain is identical to that found in the anterior pituitary solidifies our, and others, hypothesis that PRL is synthesized in many locations other than the traditional one (anterior pituitary). The actual sequencing of hypothalamic PRL cDNA produced from RT-PCR of mRNA from intact or hypophysectomized rats demonstrates unequivocally that brain PRL mRNA is identical to anterior pituitary prolactin mRNA.

Previous work by our laboratory has described the presence and widespread distribution of a PRL-like immunoreactive protein in brain. The persistence of this PRL in brain after hypophysectomy provided substantial evidence that brain PRL represented the product of a synthetic pool separate from that of the anterior pituitary PRL. To pursue this concept of independent synthesis further, we sought to determine whether brain tissue expressed PRL mRNA. Although we were easily able to detect a single species of PRL mRNA in pituitary by Northern hybridization, we could not visualize message in hypothalamus or extrahypothalamic brain by this technique. Therefore, we performed the polymerase chain reaction on cDNAs from anterior pituitary, hypothalamus, discrete extrahypothalamic brain regions, and other tissues. Hypothalamus and extrahypothalamic brain parts, including the cerebellum, caudate, brain stem, amygdala, thalamus, cortex, and hippocampus, were all positive to varying degrees. Lung and liver were negative, and anterior pituitary was consistently positive. All positive tissues, including anterior pituitary, expressed two hybridization signals: the expected amplified product and another smaller one. The smaller amplified product is presumably the result of an alternatively spliced transcript that is missing part of the PRL gene. Hypophysectomized animals did not express PRL message in brain, but expression was restored in hypophysectomized animals treated with testosterone. Transcripts for Pit-1 (GHF-1), a transcription factor important in regulation of pituitary PRL, were not detected in hypothalamus or any of the extrahypothalamic brain parts. The finding of testosterone stimulation of brain PRL message and undetectable levels of Pit-1 (GHF-1) in hypothalamic and extrahypothalamic brain regions indicates that the transcriptional regulation of PRL in the brain is different from that in the anterior pituitary.

The suppressive effects of ethanol (EtOH) on the male rodent reproductive axis, especially on serum luteinizing hormone (LH) levels, are well known. In this study we examined, in coordinate fashion, the effects of EtOH on LH secretion and on steady-state levels of the mRNA for the two genes that direct LH synthesis, namely alpha- and beta-LH. A single intraperitoneal (IP) injection of EtOH given to castrated male rats produced a statistically significant fall in serum LH levels at 1.5 (p less than 0.05) and 3 hours (p less than 0.01) after injection compared to saline-injected controls. This effect had dissipated by 24 and 72 hours after treatment. Intrapituitary LH content was significantly increased in EtOH-compared to saline-treated animals 1.5 hours after injection (p less than 0.05) at the same time there was a significant decrease in serum LH. Steady-state levels of beta-LH mRNA were significantly diminished in EtOH-treated animals at 1.5 (p less than 0.05) and 3 hours (p less than 0.001) after injection, but returned to control levels thereafter. Since the half-life of beta-LH mRNA is greater than 8 hours, this fall is most likely due to decreased beta-LH mRNA stability and may also involve decreased beta-LH gene transcription. alpha mRNA levels were unchanged at all time points investigated. These findings were verified in four repetitions of this experiment. These data suggest that the EtOH-induced fall in serum LH is due to impaired secretion of LH and to decreased LH synthesis as indicated by diminished steady-state levels of beta-LH subunit mRNA, secondary mainly to altered mRNA stability.