Establishing the dose response curve for metabolic control with troglitazone, an insulin action enhancer, in type 2 diabetes patients.

Troglitazone is a novel once-daily oral antidiabetic agent for the treatment of type 2 diabetes patients. Here, we report the overall dose response characteristics of troglitazone, with respect to effects on metabolic control, using a pharmacodynamic model. Data from week 12 from two previously reported double-blind, randomized, parallel-group, placebo-controlled, dose-ranging multicentre studies examining once-daily doses of 10, 30, 100, 200, 400, 600 and 800 mg of troglitazone were combined for the analyses. The pharmacodynamic relationships for relevant parameters of metabolic control were modelled using a nonlinear regression modelling programme. The troglitazone dose-concentration relationship was linear over 10-800 mg. Using an inhibitory sigmoid Emax model, ED50 values of approximately 100 mg and 200 mg were found for fasting serum glucose and triglycerides, respectively. The 200 mg dose for HbA1c showed an inconsistent reduction compared with placebo between the two studies; this illustrates the difficulties associated with comparing results from different assay techniques. Insulin and nonesterified fatty acid reductions compared with placebo were not consistent between studies, and no pharmacodynamic modelling was possible. No changes in body weight were observed at any dose. Troglitazone was as well tolerated as placebo across the dose range investigated. This pharmacodynamic analysis has established that 200-600 mg once daily can be considered the therapeutic dose range of troglitazone that significantly improves metabolic control in type 2 diabetes patients.

Reproducibility and comparability of insulin sensitivity indices measured by stable-label intravenous glucose tolerance test.

We have investigated the reproducibility of (1) insulin sensitivity (S*I) and glucose effectiveness (S*G) as measured by the stable-label (one compartment) minimal model, and (2) insulin sensitivity (S*Ib), plasma clearance rate (PCR), basal hepatic output (HGOb), and total hepatic glucose output (HGO0-240) as measured by the novel stable-label two compartment model of glucose disappearance during labelled intravenous glucose tolerance test (IVGTT) using 6,6-(2)H-glucose. Ten normal male subjects were studied on two occasions one week apart. Both models provided estimates of all indices with acceptable precision (CV of parameter estimates < or =50%). The within subject CVs of S*I and S*Ib were comparable (17% vs 19%) as were the within subject CVs of S*G and PCR (13% vs 16%). A highly significant linear relationship was observed between S*Ib and S*I (0.303 +/- 0.046 ml kg(-1) min(-1) per mU l(-1) vs 13.04 +/- 1.89 10(-4) min(-1) per mU l(-1), y = 0.0037 x + 0.0002, r = 0.90, p < 0.001; mean +/- SE), but not between PCR and S*G (1.98 +/- 0.15 ml kg(-1) min(-1) vs 0.0089 +/- 0.0005 min(-1), rs = 0.34, NS). The two compartment model provided a plausible time-profile of hepatic glucose output during IVGTT, reproducible estimates of HGOb (1.96 +/- 0.18 mg kg(-1) min(-1), 15%; mean +/- SE, within subject CV), and a highly reproducible HGO0-240 (7%; within subject CV). We conclude that the stable-label (one compartment) minimal model and the stable-label two compartment model provide reproducible estimates of parameters of glucose kinetics in normal subjects. Insulin sensitivity indices estimated by the two models are strongly linearly related.

Constant infusion and bolus injection of stable-label tracer give reproducible and comparable fasting HGO.

We have investigated the reproducibility of fasting hepatic glucose output (HGO) estimates by use of isotope dilution methodology of stable-label tracers. Six normal subjects were studied on two occasions 1 wk apart. After an overnight fast, the subjects received a bolus injection of 7 mg/kg of [U-13C]glucose and, simultaneously, a primed constant infusion of 0.05 mg.kg-1.min-1 of [6,6(-2)H]glucose. The bolus injection provided one estimate of HGO (HGOBOL), and the constant infusion provided two estimates of HGO, namely, HGO at 2 h (HGOINF2) and HGO at 4 h (HGOINF4), both with the assumption of steady-state conditions. All estimates were similar in value; HGOBOL was highest, followed by HGOINF2 and HGOINF4 [2.30 +/- 0.11 (SE), 2.17 +/- 0.12, and 2.01 +/- 0.13 mg.kg-1.min-1]. The constant infusion gave highly reproducible results. In the case of HGOINF2, the within-subject coefficient of variation (CV) was only 3% compared with 5% of HGOINF4. The reproducibility of HGOBOL was comparable with the within-subject CV of 7%. We conclude that a constant infusion and a bolus injection of stable-label tracer give reproducible and comparable estimates of HGO.

Evidence for arginine vasopressin as the primary activator of the HPA axis during adjuvant-induced arthritis.

1. Adjuvant-induced arthritis (AA) is an experimental inflammation of the joints that results in chronic activation of the hypothalamo-pituitary-adrenal (HPA) axis. 2. In this study the role of hypothalamic corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of the HPA axis in this condition both in Sprague-Dawley (SD), and Piebald-Viral-Glaxo (PVG) rats has been further characterized. 3. The increase in AVP peptide content of portal blood (as early as day 11), just prior to the onset of arthritis is confirmed and further increases, peaking at day 16 are shown, coincident with the progression of inflammation in the PVG rats. 4. The increase in AVP is associated with a significant increase in the expression of AVP but not CRF mRNAs in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN) of arthritic SD rats. 5. In the presence of maximal inflammation of SD rats there was a significant decrease in the maximum binding of [125I]-Tyr-oCRF to anterior pituitary membranes, whereas AVP receptor concentration in anterior pituitary membranes from both PVG and SD rats showed a significant increase with respect to controls. 6. The basal adrenocorticotrophin (ACTH) secretion in vitro was similar in both control and arthritic SD rats but that from arthritic PVG rat pituitaries was significantly greater than the respective controls (436 +/- 91 v 167 +/- 23 pg/tube). The ACTH response of pituitaries of arthritic PVG rats to CRF or the combination of CRF and AVP was significantly higher compared with the controls, although the ACTH response of arthritic SD rat pituitaries was unchanged. 7. The results are consistent with the view that activation of the parvocellular vasopressin system has an important role in the adaptation of the HPA axis to experimentally-induced chronic stress of arthritis.

The effects of oral sumatriptan, a 5-HT1 receptor agonist, on circulating ACTH and cortisol concentrations in man.

1. The effects of oral sumatriptan (50, 100 and 200 mg), a 5-HT1 receptor agonist, and placebo, on circulating adrenocorticotrophic hormone (ACTH) and cortisol concentrations were determined over 24 h after dosing, in 26 healthy male subjects. ACTH was measured by immunoradiometric assay and cortisol by radioimmunoassay. 2. After sumatriptan all subjects displayed a normal diurnal rhythm for circulating ACTH and cortisol compared with placebo. 3. There was a reduction in the trough circulating ACTH concentration over 0-4 h which was 18% with 100 mg (P = 0.002), and 25% with 200 mg (P < 0.001). The 5 h, post-prandial, peak ACTH concentration was reduced by 21% with 100 mg (P = 0.018) and by 20% with 200 mg (P = 0.024). The weighted mean ACTH over 24 h was reduced by 8% with 100 mg (P = 0.029) and by 8% with 200 mg (P = 0.018). The nadir concentration of ACTH over the 24 h and the ACTH concentration 24 h after sumatriptan were not, however, significantly reduced. All results are compared with placebo. 4. There was a reduction in the trough circulating cortisol concentration over 0-4 h which was 15% with 50 mg (P = 0.015), 14% with 100 mg (P = 0.022) and 24% with 200 mg (P < 0.001). The 5 h, post-prandial, peak cortisol concentration was reduced by 16% with 100 mg (P = 0.012) and by 15% with 200 mg (P = 0.017).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of food deprivation and altered thyroid status on the hypothalamic-pituitary-thyroid axis in the rat.

Propylthiouracil (PTU) was administered to rats for different lengths of time with or without food deprivation on the last 2 days. Within 4 days of PTU treatment peripheral 3,5,3'-tri-iodothyronine (T3) fell to low levels and beta-subunit of thyroid-stimulating hormone (beta-TSH) mRNA increased significantly in the anterior pituitary. Pro-thyrotrophin-releasing hormone (pro-TRH) mRNA in the hypothalamic paraventricular nucleus (PVN) increased significantly in the control group of animals by 8 days and in the food-deprived group by day 12; the increment of pro-TRH mRNA in the food-deprived group on day 12 was significantly less than that in the control group. In a second study, animals were treated with intraperitoneal injections of T3 with or without the food deprivation. After 4 days of T3 treatment, peripheral T3 levels were markedly increased and pro-TRH mRNA in the PVN and beta-TSH mRNA in the anterior pituitary were significantly reduced. Food deprivation had no additional suppressive effect. These studies confirm that the predominant effect of food deprivation on the thyroid axis is at the hypothalamic or suprahypothalamic level and that it can, at least in part, overcome the increase in TRH mRNA due to diminished T3 feedback.

Corticotrophin-releasing factor and arginine vasopressin in the hypothalamo-hypophyseal portal blood of rats following high-dose glucocorticoid treatment and withdrawal.

Hypothalamo-hypophyseal portal blood was obtained from rats treated with chronic, high-dose prednisolone in the drinking water and after subsequent withdrawal of the steroid for up to 7 days. Corticotrophin-releasing factor (CRF) immunoreactivity in portal blood was reduced by treatment with prednisolone but not completely abolished. There was a rapid recovery of CRF to control values within 3 days of withdrawal of prednisolone. There was no significant change in arginine vasopressin (AVP) in portal blood over the time-course of the experiment.

Inhibition of hypothalamic thyrotropin-releasing hormone messenger ribonucleic acid during food deprivation.

Food deprivation in laboratory rats induces profound changes in the neuroendocrine system. We have investigated the hypothalamic and pituitary responses of the hypothalamo-pituitary thyroid axis to 48-h food deprivation in Sprague-Dawley rats. Peripheral T3 and hypophysial portal TRH were measured by RIA, and TSH beta, PRL, and pro-TRH mRNA were measured using in situ hybridization histochemistry. Peripheral total T3 was greatly reduced in food-deprived rats. Hypothalamic portal blood TRH levels declined significantly with time in control animals. The initial level of TRH in the portal blood of food-deprived rats was significantly reduced compared to that in controls, but did not fall further with time. In situ hybridization histochemistry revealed significantly lower pro-TRH mRNA in the paraventricular nucleus of food-deprived animals, while pro-TRH mRNA in the reticular nucleus remained unaltered. Furthermore, in the anterior pituitary, TSH beta mRNA decreased significantly in food-deprived animals, while PRL mRNA was unaltered. We conclude that the reduction in circulating T3 after food deprivation appears to be due primarily to decreased hypothalamic TRH synthesis and release.

Effect of a long-acting somatostatin analogue (BIM23014) on proliferative diabetic retinopathy: a pilot study.

A pilot study on the use of a continuous infusion of somatostatin, by subcutaneous pumps in the management of proliferative diabetic retinopathy is reported. Two patients out of eight with proliferative retinopathy demonstrated improvement. One patient demonstrated regression of disc new vessels and the other a reduced area of retinal capillary non-perfusion, both demonstrated by fluorescein angiography. Control patients showed worsening of fluorescein leakage over the observation period of four to six weeks whereas the other six patients given the somatostatin infusion did not demonstrate any deterioration. The mechanism of action of somatostatin in this study is unknown but it is thought to have direct anti-angiogenic properties as well as inhibiting growth hormone secretion.

Osmotic regulation of hypothalamo-neurointermediate lobe corticotrophin-releasing factor-41 in the rat.

We have detected significant amounts of corticotrophin-releasing factor-41 (CRF-41) in the rat hypothalamo-neurointermediate lobe system using a radioimmunoassay and reversed-phase high-performance liquid chromatography. Total amounts of CRF-41 in extracts of median eminence (ME), supraoptic nucleus (SON), paraventricular nucleus (PVN) and neurointermediate lobe (NIL) from control animals were 1076 +/- 132, 196 +/- 44, 22 +/- 7 and 147 +/- 50 fmol respectively (means +/- S.E.M., n = 6). In animals given 340 mmol NaCl/l instead of tap water to drink for 12 days, no significant changes occurred in the CRF-41 content of the ME, SON or PVN, but CRF-41 content increased more than twofold in the NIL (362 +/- 58 fmol). Plasma concentrations of CRF-41 and ACTH in control animals were 23 +/- 6 and 51 +/- 8 pmol/l respectively. After saline treatment no significant change in plasma CRF-41 was detected (20 +/- 8 pmol/l) but concentrations of circulating ACTH were decreased (15 +/- 2 pmol/l). The CRF-41 content of both the ME and the NIL was significantly depleted after intracerebroventricular injection of colchicine (414 +/- 81 and 34 +/- 7 fmol respectively). These data suggest that NIL CRF-41 is of hypothalamic origin and can be regulated by an osmotic stimulus.

Somatostatin and Thyrotrophin-Releasing Hormone Response and Receptor Status of a Thyrotrophin-Secreting Pituitary Adenoma: Clinical and in vitro Studies.

Abstract We have described a patient with a thyrotrophin-secreting pituitary adenoma and correlated a detailed physiological and anatomical investigation of the surgically resected tumour with its in vivo regulation. Thyrotrophin secretion was inhibited by circulating thyroid hormones, dopaminergic agonists and the somatostatin analogue SMS 201-995 but could not be stimulated by thyrotrophin-releasing hormone or further inhibited by exogenous triiodothyronine. Prolonged treatment with SMS 201-995 caused tumour shrinkage as shown by successive computed tomography scans but was accompanied by tumour desensitization and the development of diabetes mellitus. This is the first thyrotroph adenoma in which somatostatin receptors have been directly demonstrated and shown to completely block thyrotrophin-releasing hormone-induced inositol phospholipid accumulation when occupied. In addition, preincubation with triiodothyronine significantly inhibited thyrotrophin-releasing hormone-induced inositol phospholipid turnover in dispersed pituitary cells indicating that in this tumour, circulating thyroid hormones were exerting feedback inhibition at the level of the pituitary either by reducing the number of thyrotrophin-releasing hormone receptors and/or their coupling to second messenger pathways. In keeping with this hypothesis, the acute reduction in intrapituitary triiodothyronine by sodium ipodate in vivo had no effect on peripheral thyrotrophin over 6 h suggesting that the onset of the effect of triiodothyronine withdrawal on thyrotrophin secretion was suitably delayed. The importance of the inositol phospholipid second messenger pathway in transducing the secretory response in this tumour was further corroborated by electrophysiological studies which demonstrated thyrotrophin-releasing hormone-induced changes in K(+) currents which are dependent on intracellular Ca(2+) ions, presumably mobilized via the inositol phospholipids. In addition to thyrotrophin and alpha subunit, growth hormone mRNA and growth hormone were found throughout the tumour as were two populations of cells distinguished electron microscopically by the size of their secretory granules. Although acromegalic features are not unusual in thyrotroph adenomas, our patient showed no evidence of inappropriate growth hormone secretion during surgery or in response to pre- or post-operative insulin stress tests.

The TSH, T4, T3 and prolactin responses to consecutive infusions of a potent and stabilized thyrotrophin releasing hormone analogue, RX77368, in man.

The endocrine manifestations of a stabilized thyrotrophin releasing hormone (TRH) analogue, RX77368, have been investigated in six male volunteers. Infusions were given on two occasions, with a 5-day interval between infusions. On the second exposure to RX77368, there was a significant reduction in the TSH response, despite normal basal T3 and T4 levels, while the response of prolactin to RX77368 was unaltered. Domperidone administered during the infusion of RX77368 caused a further elevation of prolactin levels, whilst TSH levels were unchanged. This study shows the differential regulation of thyrotrophs and lactotrophs in response to stimulation by a TRH analogue, and shows, for the first time, down-regulation of the TSH response in vivo, in the presence of normal peripheral thyroid hormone levels. The T3 response to infusions of RX77368 was less than to a bolus injection of TRH, despite a greater TSH response to the analogue, suggesting impaired biological activity of TSH released in response to an infusion of the analogue.

Differential effects of hypothalamic catecholamine depletion on the release of arginine vasopressin and CRF-41 into hypothalamo-hypophyseal portal blood.

The effects of 6-hydroxydopamine lesions of the ventral noradrenergic bundle (VNAB) and of intracerebroventricular 6-hydroxydopamine on hypothalamo-hypophyseal portal blood (HPB) concentrations of arginine vasopressin (AVP), corticotrophin releasing factor (CRF-41), and noradrenaline have been investigated. VNAB lesions and intracerebroventricular 6-hydroxydopamine caused a reduction of HPB CRF-41 concentration, while AVP remained unchanged. HPB noradrenaline concentration was reduced in animals treated with lateral ventricular 6-hydroxydopamine, but was unchanged in VNAB lesioned animals. Our results suggest differential effects of noradrenaline on the release of AVP and CRF-41 into HPB.

Immunoreactive vasopressin and oxytocin in hypothalamo-hypophysial portal blood of the Brattleboro and Long-Evans rat: effect of adrenalectomy and dexamethasone.

Immunoreactive vasopressin and oxytocin were measured in the hypothalamo-hypophysial portal blood of both Long-Evans and homozygous Brattleboro rats. Adrenalectomy caused an increase in vasopressin immunoreactivity in portal blood in the Long-Evans strain, whilst administration of dexamethasone to these adrenalectomized animals resulted in a reduction in portal vasopressin immunoreactivity to levels below those seen in sham-operated animals. This vasopressin immunoreactivity co-eluted with synthetic vasopressin on high-pressure liquid chromatography (HPLC), and diluted in parallel in radio-immunoassay. In Brattleboro rats, however, although vasopressin-like immunoreactivity was detected, the portal concentration did not vary with the adrenal status of the animal, nor did it show the characteristics of standard vasopressin on HPLC or in immunoassay. Oxytocin was present in the portal blood of both Long-Evans and Brattleboro rats at similar very high concentrations, but did not vary in response to adrenalectomy. These results are consistent with a role for vasopressin, but not oxytocin, in the hypothalamic response to adrenalectomy and glucocorticoid feedback. Neither vasopressin immunoreactivity nor oxytocin appear to subserve this role in the homozygous Brattleboro rat.

Hypothalamo-hypophyseal portal blood sampling from laboratory rats. The effects of endocrine manipulations on portal blood catecholamine concentrations.

We have obtained hypothalamo-hypophyseal portal blood using a modified Worthington-Fink technique, and measured the concentrations of catecholamines with a high pressure liquid chromatography - electrochemical detection system. The concentrations of noradrenaline, adrenaline and dopamine have been measured in portal blood after various endocrine manipulations, including adrenalectomy, orchidectomy and chemically induced hypothyroidism. After the induction of hypothyroidism, dopamine concentrations in portal blood increased. After orchidectomy, there was an increase in portal adrenaline concentration, though noradrenaline and dopamine were unchanged.

Electrophysiological observations in patients with motor neuron disease receiving a thyrotropin releasing hormone analogue (RX77368).

Twenty nine patients with motor neuron disease receiving a thyrotropin releasing hormone analogue showed acute 25-30% increase in mean corrected fibre density and mean macro EMG median amplitude and area in brachial biceps muscle. The data are consistent with a direct or indirect action of the drug on anterior horn cells.

Controlled acute trial of a thyrotrophin releasing hormone analogue (RX77368) in motor neuron disease.

Twenty five patients with motor neuron disease completed a double blind randomised cross over trial of RX77368, a stabilised TRH analogue, iv over 2 hours against saline. Temporary improvement in bulbar symptoms including speech, respiratory parameters, tongue movements and swallowing were seen. Fasciculations increased and spasticity decreased. Change in muscle force with drug was different from placebo but both increase and decrease in force were seen and did not result in detectable changes in function. Side effects were clinically significant in 50% of the patients and cleared within 12 hours. Prolonged rise of thyroxine and an increase in plasma levels of prolactin, thyroid stimulating hormone and growth hormone were seen and followed characteristic patterns.

Neurotensin in hypothalamo-hypophyseal portal blood.

The control of hypothalamo-hypophyseal neurotensin secretion was studied using a modification of the Worthington-Fink portal sampling technique. Portal concentrations of neurotensin were significantly greater than the concentrations found in peripheral plasma, and HPLC characterisation demonstrated that both portal and peripheral plasma neurotensin were very similar. Two different anaesthetic agents, urethane and althesin, did not differ in the resulting portal concentrations of neurotensin. Electrical stimulation of the median eminence resulted in a very marked increase in portal neurotensin concentration.

Hypothalamo-hypophysial blood flow: a novel control mechanism in pituitary function?

The flow rate of portal plasma was measured during hypothalamo-hypophysial portal sampling in the rat using a modified Worthington-Fink technique. In rats rendered hypothyroid with propylthiouracil, there was a significant 140% increase in portal plasma flow. Median eminence stimulation also increased portal plasma flow by 41%. Orchidectomy and adrenalectomy had no effects on plasma flow. Modification of flow rate in the hypothalamo-hypophysial vascular bed may represent a further mechanism involved in the control of pituitary function.