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When pituitary tissue was subjected to Western blot analysis utilizing polyclonal antibody NIDDK-rLH-S-10, bands at 17 and 19 Kd representing LH subunits were identified. In addition, a high molecular weight 66 Kd band was seen. Surprisingly this high molecular weight band was also seen in rat cerebral cortex, brain stem, hypothalamus, spinal cord, lung, liver, pancreas, spleen, kidney, testis, and serum. Antibody preabsorbed with iodination grade rat LH antigen no longer recognized the 17 and 19 Kd bands in pituitary, but recognized the 66 Kd bands in pituitary and the other tissues examined. Since 66 Kd is the molecular weight of albumin, we found that antisera to rat albumin recognized this same high molecular weight band in the tissues examined. Preabsorption of LH antibody with albumin reduced the ability of that antibody to recognize this 66 Kd. A monoclonal antibody to bovine LH beta-subunit recognized only the LH protein in anterior pituitary, but no high molecular weight band in either pituitary or the other tissues studied. Finally, 10, 100, and 1000 micrograms of rat albumin caused no substantial interference under conditions of RIA. We conclude that the polyclonal antibody, provided by the NIH, is excellent for conditions of RIA, but caution must be exercised when it is used for Western analysis where some lots of this antibody may recognize other unrelated proteins.

Background: The risks and benefits of intensive therapy in non—insulin-dependent diabetes mellitus (NIDDM) need to be defined. In preparation for a long-term trial, a feasibility study of 153 men in 5 medical centers compared standard vs intensive insulin therapy. Objective: To assess the rate of development of new cardiovascular events and their correlates. Methods: Patients with a mean ± SD age of 60 ± 6 years and diagnosis of NIDDM for 7.8±4.0 years were randomly assigned to a standard (1 insulin injection every morning) or to an intensive treatment arm (stepped plan from 1 evening injection of insulin, alone or with glipizide, to multiple daily injections) designed to attain near-normal glycemia levels. A 2.07% separation of glycosylated hemoglobin (HbA 1c ) was sustained for a mean follow-up of 27 months ( P Results: Mild and moderate hypoglycemic events were more frequent in the intensive than in the standard treatment arm (16.5 vs 1.5 per patient per year, respectively). Mean insulin dose was 23% lower in the standard treatment arm ( P P =.10). There was no difference in total and cardiovascular mortality (n=5 and n=3 in the intensive and standard treatment arms, respectively) or in new events in patients with cardiovascular history (n=10 in each arm). In Cox regression analysis, the only significant correlate for new cardiovascular events was previous cardiovascular disease ( P =.04). Entering in the analysis any baseline cardiovascular abnormality, the regression model indicated a lower HbA 1c level prior to the event as the only correlate for new cardiovascular events ( P =.05). Conclusion: A long-term prospective trial is needed to assess the risk-benefit ratio of intensive insulin therapy for NIDDM in patients who require it. Arch Intern Med. 1997;157:181-188

We have recently reported that luteinizing hormone (LH) is present in the hypothalamus of rats. It has chromatographic and biologic characteristics similar to pituitary LH. In this report we focus on extrahypothalamic LH that is widely distributed in the rodent central nervous system. This material has a chromatographic profile similar to that of pituitary LH. Serial dilution of this material is parallel with dilutions of rat pituitary LH in the immunoassay. Brain extracts are active in the testis LH radioligand receptor assay and in the rat interstitial cell testosterone secretion bioassay. Prior incubation of extract with LH antibody significantly attenuated both of these activities. Thus, extrahypothalamic LH has immunologic, chromatographic, and biologic characteristics similar to hypothalamic and pituitary LH.

The question of whether ethanol's (ETOH's) known suppressive effect on serum luteinizing hormone (LH) could be mediated directly at the anterior pituitary level was addressed by examining the effects of ETOH in vitro on release of LH from cultured male rat pituitary cells. The impact of added ethanol concentrations ranging from 50 to 400 mg% on LH release was examined in the basal state and after stimulation by gonadotropin-releasing hormone (GnRH) at a dose of 5 x 10(-10) M. While ETOH did not significantly suppress basal LH release, secretion stimulated with GnRH was noted to be attenuated with higher doses of ETOH (greater than or equal to 100 mg%) compared to stimulated control cells. It is concluded that ETOH exposure in vitro alters stimulated LH secretion by acting directly on pituitary gonadotropes.

Immunoreactive and bioactive luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and growth hormone (GH) have been described by this and other laboratories to be present in discrete areas of the rodent and primate brain. In the present studies, LH and TSH concentrations in serum, pituitary, hypothalamus, brainstem, cerebellum, cerebral cortex and hippocampus were measured in male Sprague-Dawley rats at 2.5 months of age, when 11-12 months old and at 24 months of age. There was a significant decrease in hypothalamic TSH concentrations in the 11-12-month-old rats compared to younger animals (672 +/- 127 ng/mg vs 86 +/- 17 ng/mg, P less than 0.001). This was unaccompanied by any changes in TSH levels in serum, anterior pituitary, or in any of the extrahypothalamic brain areas that were examined. In contrast, there was a significant 50% drop in LH concentrations in the anterior pituitary gland of 11-12-month-old animals when compared with young controls (72 +/- 58 micrograms/mg vs 137.2 +/- 27 micrograms/mg, P less than 0.05) without any noted change in serum or brain LH concentrations. Similar decreases in LH concentration were also seen in the anterior pituitary glands of two-year-old animals. These discordant profiles between pituitary and brain LH and TSH provide additional circumstantial evidence that these brain peptides do not represent contaminants from the anterior pituitary. Further, these significant changes in TSH and LH concentrations that develop with aging may implicate these particular peptides in the development of certain features of senescence.

Suppressive effects of ethanol (ETOH) on in vivo serum growth hormone (GH) levels have been reported in both humans and animals. To determine whether this effect could be mediated directly at the pituitary level, we have designed a series of in vitro experiments utilizing pituitary cells from ETOH naive animals maintained in monolayer culture. We report that ETOH, in doses ranging from 50 to 400 mg%, caused a prompt and sustained reduction in basal GH secretion, as well as a significant fall in intracellular GH content. These data establish that the in vivo effects of ETOH on GH can be accounted for, at least in part, by a direct effect at the pituitary level, possibly due to reduced GH synthesis.

Exciting information is rapidly accruing about diabetes and suggests that maintenance of good metabolic control may prevent onset or delay progression of diabetic complications. Patients, as well as their families and health care providers, should be encouraged by the knowledge that tools are available for successful management of type II diabetes. Treatment consists of a diet-exercise-sulfonylurea regimen and, in a substantial number of patients, administration of insulin. Proper assessment of these modalities is essential if optimum metabolic control is to be achieved.

Prompted by immunohistochemical reports of prolactin-like immunoreactivity in cell bodies within the rat hypothalamus, a study was undertaken to quantitate the immunologic and biologic activity of this material. Hypothalamic concentrations of prolactin-like immunoreactivity averaged 402 +/- 23 pg/mg of protein (n = 30). 97% recovery of rat prolactin standards added to homogenates of hypothalamus insured that neuronal tissue, as prepared for these studies, did not interfere with the radioimmunoassay of rat prolactin. Examination of the elution profile from Sephadex G-75 columns of the prolactin-like immunoreactivity in hypothalamic extracts showed that the majority of hypothalamic prolactin-like substance was of a larger molecular size than pituitary prolactin. While increasing amounts of brain extract progressively displaced more I125 prolactin from antibody-binding sites, the displacement curve produced by adding hypothalamic extract was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Hypothalamic extracts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoactivity (B/I) ratios for hypothalamic extracts were significantly lower than the B/I ratios for pituitary prolactin (0.71 +/- 0.04 for pituitary, vs. 0.19 +/- 0.06 in the hypothalamus; p less than 0.001). Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in the hypothalamus, indicating that brain prolactin-like substance is regulated independently of pituitary prolactin and circulating serum prolactin levels.

A luteinizing hormone (LH)-like molecule has been described in the rodent central nervous system and has been shown to have immunologic, chromatographic, and biologic activity similar to that of pituitary LH. In this communication we report that a depolarizing stimulus, high potassium concentration in the presence of calcium, causes in vitro release of LH from hypothalamic explants containing the pars tuberalis. Release from other brain areas containing LH was not seen nor was release of LH, in vitro, significantly provoked from dorsal segments of the hypothalamus, thus suggesting that most, if not all, of the releasable LH from intact hypothalamic explants may come from cells of the pars tuberalis.

Immunoreactive and bioactive luteinizing hormone (LH) has been shown to be widely distributed in the rodent central nervous system (CNS), particularly in the hypothalamus. Subcellular localization of this LH in fractions rich in synaptosomes and in vitro release by potassium-induced depolarization suggests that this peptide may act in trans-synaptic neuromodulatory roles. Furthermore, a variety of experiments have proved that this brain-based LH is not of pituitary origin. In the in vitro studies reported here characterization of brain-based LH release, in response to gonadotropin-releasing hormone (GnRH), beta-endorphin, and biogenic amines, was examined. Adult male rats were sacrificed by decapitation, hypothalami removed, quartered, and incubated in Krebs Ringer's Bicarbonate (KRB). High potassium concentration and GnRH induced release of LH from these hypothalamic explants in short-term culture and serotonin significantly inhibited release of LH from these explants. In contrast, however, beta-endorphin, norepinephrine, dopamine, and acetylcholine, agents known to modulate pituitary LH release, had no effect on the release of LH from hypothalamic tissues in vitro. Furthermore, beta-endorphin did not alter potassium-induced release of LH from the hypothalamus. Whereas there are some similarities between LH release from the pituitary and from the CNS, the differences reported here suggest that hypothalamic LH does not simply serve as a supplemental source for LH in the general circulation but more likely subserves an entirely different role(s) than its pituitary counterpart, presumably acting in a neuromodulatory fashion within the brain.

We have recently reported immunoassayable luteinizing hormone (LH) in several areas of the rat brain and conspicuously present in the hypothalamus. In this report, we focus on the presence of LH in the hypothalamus and its potential role in regulation of pituitary LH release. In adult female rats, examined during the course of the estrous cycle, a significant fall in hypothalamic LH coincides with the surge in pituitary and serum LH at the time of proestrus, signaling ovulation. Ovariectomized adult rats show no change in hypothalamic LH at a time when there is a dramatic rise in both anterior pituitary and serum LH. These data support the concept that hypothalamic LH is not of pituitary origin and that it may play a role in the short-loop negative feedback system controlling the surge in anterior pituitary LH release, and event initiating ovulation.

Pharmacologic therapy reduces cardiovascular risk in a variety of primary and secondary prevention clinical situations in addition to lifestyle modifications. Low density lipoprotein cholesterol (LDL-C) is a key mediator of atherogenesis. Most cholesterol guidelines propose specific LDL-C while recently the ACC/AHA recommends statin therapy without a specific LDL-C target. Proprotein convertase subtilisin kexin 9 (PCSK9) is a serine protease that leads to LDL receptor degradation. The decreased availability of LDL receptors results in decreased clearance and an increase of circulating LDL-C particles. Monoclonal antibodies that inhibit PCSK9 (PCSK9 abs) reduce LDL-C levels and may be especially helpful in familial hypercholesterolemia and statin-intolerant patients. PCSK9 inhibition is an exciting and promising new therapy for protection against macrovascular events. Keywords: LDL, LDL receptor, Atherothrombosis, Familial hyperlipidemia, Alirocumab, Evolocumab

Alphaxalone is considered the anaesthetic of choice in neuroendocrine reproductive studies in female rats, since it appears to have little, if any, effect on release of gonadotrophin-releasing hormone. There has been less study of the effects of this anaesthetic on the male reproductive neuroendocrine axis, however. Accordingly, the time-dependent effects of alphaxalone, as well as of urethane and ketamine, on the increased levels of LH in castrated rats were determined. Each anaesthetic was administered i.p. and each depressed LH levels significantly compared with those in castrated unanaesthetized rats killed by decapitation (controls). The effect of the anaesthetics was noted 15 min after administration and persisted at 30 and 60 min in animals anaesthetized with alphaxalone and urethane. Only in ketamine-anaesthetized animals did serum concentrations of LH finally rise to concentrations not significantly different from those in control rats. Thus alphaxalone, though useful in female neuroendocrine studies, is as profoundly disruptive as other anaesthetics on the male rat hypothalamic-pituitary reproductive unit. J. Endocr. (1987) 115, 221–223

We have previously described the presence and wide distribution of a luteinizing hormone (LH)-like peptide, widely distributed in the rat central nervous system, with highest levels in the hypothalamus. We have found that intracerebroventricular injection of 100 micrograms colchicine causes a significant rise in hypothalamic LH, from 549 +/- 170 pg/mg protein (n = 12) in controls to 1,679 +/- 279 pg/mg protein (n = 13) in treated animals, p less than 0.01. There was no associated change in levels of LH in the pituitary. Since colchicine stops axoplasmic flow, these findings of colchicine-induced increase in hypothalamic LH indicate that at least a portion of hypothalamic LH is present in long-axoned neuronal elements with cell bodies within the hypothalamus and axons extending to the extrahypothalamic brain. Thus, hypothalamic LH does not solely represent measurement of LH in cells of the contignous pars tuberalis.

The reproductive alterations induced by ethanol (ETOH) in the male rodent have been intensively investigated. Although gonadal effects are well characterized, the impact of ETOH on the hypothalamic peptide luteinizing hormone-releasing hormone (LHRH) has been less well defined. The releasability of hypothalamic LHRH in the presence of ETOH has not been directly studied. We report here that ETOH in concentrations of 50 mg% to 400 mg% failed to inhibit LHRH release in vitro.

In order to study the impact of short-term ethanol feeding on pituitary luteinizing hormone (LH) levels, LH release and hormone microheterogeneity, 28 female oophorectomized rats were fed either a diet containing 36% ethanol or an isocaloric diet without ethanol for 16 days. On the 14th day of the experiment, all rats were given 50 micrograms of estradiol subcutaneously to provide a uniform steroid milieu and to induce an LH surge, and sacrificed 48 hours later. Total levels of pituitary and serum immunoreactive LH were identical between the ethanol and control groups. However, when pituitary LH was analyzed by isoelectric focusing on agarose gel a distinct difference was found. Four distinct immunoreactive peaks at pI's 3.50-4.55, 5.20-6.50, 7.00-7.50, 8.15-9.30 were noted. Ethanol-treated animals exhibited an 8-fold increase in the acidic LH subspecies in the pI range 3.50-4.55 and similarly more LH was determined at pI 7.00-7.50 compared to controls (P less than 0.01). More LH was found at the basic pI's from control pituitaries, compared to LH from the ethanol-exposed animals (P less than 0.01). In summary, short-term ethanol feeding in the female rats does not cause quantitative changes in pituitary LH levels or in the estrogen-induced LH surge. However, qualitative changes are present in isoelectric focusing patterns. A significant shift to the more acidic species of the pituitary LH isohormone was noted for ethanol-fed animals as compared to controls. These qualitative changes in the LH molecule, seen before quantitative changes could be appreciated, may help to explain why excessive exposure to alcohol results in reproductive impairments both in rats and in humans.

The effect of exposure to ethanol on hypothalamic LH-releasing hormone (LHRH) release in vivo was investigated in rats both acutely (i.p. injection) and after 3 days of administration, utilizing a permanent gastric cannula. In both designs, the animals were castrated before being given ethanol and, in both experiments, ethanol successfully lowered the post-castration LH rise compared with control castrated animals. In both the acutely and chronically treated groups, basal LHRH release was not impaired, despite the documented decrease in LH levels. Finally, stimulated LHRH release was investigated with depolarizing concentrations of potassium and, again, no change was noted between the hypothalamic release of this decapeptide in the ethanol-exposed compared with the ethanol-naive animals. Thus, ethanol failed to inhibit basal or stimulated LHRH secretion in the acutely and chronically treated animal. This lack of effect on LHRH occurred despite a concomitant lowering of serum concentrations of LH. Journal of Endocrinology (1989) 121, 37–41

Our laboratory has previously described the widespread distribution of an immunoreactive and bioactive rat growth hormone (rGH)-like protein in rat brain. It has also been demonstrated that regulation of pituitary rGH secretion is at least partly mediated by a short-loop negative feedback system. In such a system, increased levels of rGH, acting at a suprapituitary locus, would decrease pituitary GH secretion. Thus, the present study has dealt with attempts to further investigate the hypothesis that one function of brain-based rGH might be as a mediator of the short-loop negative feedback system controlling pituitary rGH release. If brain-based rGH were to function as a mediator of such a system, then in situations where serum rGH levels are decreased, brain rGH concentrations should increase, indicating activation of a negative feedback loop. In the present communication we report that significantly decreased serum GH levels in oophorectomized and in thyroidectomized rats were coupled with a significant increase in rGH concentrations in the hypothalamus and in the amygdala. By contrast, adrenalectomy, which was not associated with any changes in levels of GH in serum caused no perturbations in levels of rGH in the brain. These discordant changes in serum and brain-based rGH are findings compatible with the hypothesis that one function of brain-based rGH is as a mediator of the short-loop negative feedback system regulating the release of pituitary GH.

Brain and anterior pituitary growth hormone (GH), thyroid-stimulating hormone (TSH) and luteinizing hormone (LH) were measured during fetal, neonatal, and pubertal life and into adulthood. Immunoassayable GH and TSH could be found in the fetal whole brain before their detection in the fetal pituitary. Developmental patterns of pituitary and brain hormones differed in that pituitary hormones showed a gradual rise in levels from birth to puberty at approximately 20 days of age. Biochemically similar, brain-based peptides demonstrated a remarkable preparturitional surge in concentrations that was limited to a few days immediately preceding birth. Twenty-four hours after birth, brain GH, TSH, and LH had dropped to levels equal to or less than concentrations in the neonatal pituitary and subsequently rose to adult levels around the time of puberty. In these studies it could be shown that both the placental-fetal barrier and the neonatal blood-brain barrier were intact. These observations indicate the presence of two biochemically and immunologically similar but topographically distinct pools of peptides present in the developing brain and in the anterior pituitary gland.

Exposure to ethanol is recognized to cause reproductive impairment in man and animals. Since elevated levels of prolactin will interfere with normal functioning of the hypothalamic-pituitary-gonadal axis, and since ethanol has been shown by others to lead to increased prolactin secretion in vivo , the present in vitro study was undertaken to determine whether there is a direct effect of ethanol (ETOH) on prolactin release. Prolactin release from anterior pituitary cells maintained in monolayer culture and exposed to either no ethanol or media containing ethanol at concentrations of 50, 100, 200, or 400 mg% was measured at 1, 4, 24, 48, 72 hours in incubation. Ethanol added directly to pituitary cells stimulated prolactin release at all time points examined. Significant stimulation occurred with addition of low and mid-range ethanol concentrations (50–200 mg%); no augmented prolactin secretory response was seen with the highest ethanol concentration used (400 mg%). This pattern of response was maintained throughout the entire 72 hour incubation period. Thus, the effect of ethanol on prolactin secretion is mediated, at least in part, at the anterior pituitary level.

Pursuant to our identification of prolactin-like immunoreactivity (PLI), widely distributed in rat brain, the spinal cord was examined for the presence of this pituitary-hormone-like protein. PLI was present in all spinal cord extracts examined and averaged 500 +/- 53 pg/mg protein. Hypophysectomy, causing a fall in serum prolactin to undetectable levels, was not associated with any change in levels of PLI in spinal cord. Recovery of rat prolactin standards added to spinal cord homogenates was 97.6 +/- 3.9%. When increasing concentrations of spinal cord extract were assayed in a prolactin radioimmunoassay, displacement of rat 125I-Prolactin from antiserum was parallel to that displacement produced by increasing concentrations of rat anterior pituitary standards. Upon subjection to gel permeation chromatography, the elution profiles of immunoreactive prolactin from spinal cord were different from the profiles of anterior pituitary prolactin. In addition to an immunoreactive prolactin peak eluting with pituitary prolactin, spinal cord extracts showed a large void volume peak and late eluting low-molecular-weight materials not seen with anterior pituitary. In the Nb2 lymphoma cell assay, all spinal cord extracts demonstrated prolactin-like bioactivity with a bioactivity/immunoreactivity ratio of 1.05 +/- 0.13. We conclude: (1) PLI, widely distributed in rat brain, is also present in spinal cord; (2) spinal cord prolactin levels are independent of levels in pituitary and peripheral circulation; (3) this immunoreactive prolactin is bioactive, and (4) differing gel permeation chromatographic elution profiles indicate that there may be some molecular differences between pituitary and spinal cord prolactin.

We have previously described the presence of and the immunologic, chromatographic and biologic characteristics of a thyroid-stimulating hormone (TSH)-like peptide, widely distributed in the rat and monkey central nervous system (CNS). In order to test the hypothesis that brain TSH, specifically hypothalamic TSH, participates in the turnover of pituitary TSH, we have assessed the effect on hypothalamic TSH of two endocrine manipulations, castration and adrenalectomy, known to significantly decrease pituitary and/or serum TSH in the rat. Orchidectomy led to a significant decline of pituitary and serum TSH while a significant increase in hypothalamic TSH concentrations was seen. Adrenalectomy also led to decreased pituitary concentration of TSH linked with a significant increase in hypothalamic TSH levels. Neither manipulation led to changes in TSH in the extrahypothalamic brain. The inverse relationship wherein serum and/or pituitary TSH decrease is accompanied by an increase in hypothalamic TSH is compatible with a role for hypothalamic TSH in pituitary TSH regulation. The possible significance of these findings in terms of TSH triiodothyronine interactions in the CNS is discussed.

Prompted by interest in immunohistochemical reports of prolactin-like immunoreactivity (PLI) in the rat hypothalamus, we investigated and have reported that an immunoreactive and bioactive prolactin-like material can be extracted from the rat hypothalamus. In the present communication the subcellular distribution of this protein is reported. Using a sensitive and specific radioimmunoassay for rat prolactin and a standardized procedure for subcellular fractionation of neuronal tissue, we have found that 90% of hypothalamic PLI is particulate-bound with only 10% remaining in the S4 or cytosolic fraction. Almost 80% of the particulate-bound PLI is found in the P2 fraction containing myelin, synaptosomes and mitochondria. When P2 is further fractioned on a discontinuous sucrose density gradient, approximately 66% of the P2-associated PLI was found in subfractions rich in synaptosomes and poor in myelin and mitochondria. Such findings support the probability that hypothalamic PLI functions trans-synaptically as a neuromodulator in the brain.

Ultracentrifugation and sucrose gradient separation techniques have been applied to study of the subcellular distribution of hypothalamic luteinizing hormone (LH). Subcellular particle-bound LH accounted for 42% of the total LH while cytoplasm contained 58% of the total immunoassayable LH in these extracts. 70% of particle-bound LH was present in the P2 fraction and most of the particle-associated LH was found in subfractions of P2 rich in synaptosomes but poor in myelin and mitochondria. While actual amounts of LH in the P3 and P4, the microsomal fractions, were relatively small, the concentration of LH, expressed as nanograms per milligram of protein was quite high. Both content and concentration of LH in the nuclear pellet, P1, were substantially lower. These findings provide further framework for speculation on the role of brain-based LH.

This study describes the finding of an immunoassayable and bioassayable luteinizing hormone (LH) in the rat hypothalamus. Validation of the immunoassay forLH inhypothalamicextractsis presented. Chromatographic patterns of hypothalamic LH were found to be identical with those of rat pituitary LH. Radioimmunoassay values of serial dilutions of hypothalamic extract paralleled dilution values of rat pituitary LH. Furthermore, such homogenates promoted release of testosterone from dispersed rat testis cells in vitro. This effect was largely abolished by prior incubation of homogenate with an antiserum to rat LH. Following removal of the anterior pituitary, LH levels in the hypothalamus persist but drop significantly, signifying that this particular brain-based LH is partially dependent on the presence of a functioning pars distalis. Immunoassayable LH measured in other areas of the rodent central nervous system did not change following hypophysectomy.