The earliest biological event in the action of ACTH upon its target tissue is thought to be an interaction of ACTH peptide with the cell membrane. This study was undertaken in order to clarify the relationship between ACTH's ability and its specific binding to fat cells. First of all, the lipolytic abilities of ACTH analogues were compared with ACTH (1-24) in order to determine what portion of the ACTH molecule is responsible for the lipolytic action of the hormone. Secondly, competitive displacement experiments were carried out in order to explain the action of the adrenergic blocking agent, phentolamine, on the lipolysis of ACTH.
Isolated fat cells were incubated with several kinds of ACTH peptides in the presence or absence of adrenergic blocking agents at 37°C for 2 hrs. Then the lipolytic action of ACTH was determined by measuring the free fatty acid levels in the medium. These lipolytic activities were expressed as the ratios of the amount of free fatty acid released to that of 1μg/ml epinephrine. On the other hand, 80μg/0.2ml of fat cell ghosts were incubated at 4°C for 40 min. with 3-4×10⁴ cpm of ¹²⁵ I-labelled ACTH (1-24) and unlabelled ACTH (1-24) in the presence or absence of adrenergic blocking agents. After centrifugation, the radioactivities of the fat cell ghosts were measured.
I. Dose dependent responses were found in the lipolysis stimulated with ACTH (1-24) at the concentration of 10^-10 -10¹⁷M. Then, lipolytic abilities of ACTH analogues were compared with ACTH (1-24). The relative lipolytic activity of ACTH (1-18) was 91.2 and 105.7% of ACTH (1-24) at the concentration of 10^-7 and 10^-6M, respectively. Therefore, ACTH (1-18) showed almost the same equivalent lipolytic activity as that of ACTH (1-24). The relative lipolytic activity of ACTH (1-14) was 17.3 and 69.9% at the concentration of 10-7 and 10^-6M, respectively. It was therefore concluded that ACTH (1-14) showed an apparent lipolytic activity. The relative lipolytic activity of ACTH (1-10) was 2.7 and 4.2 at the concentration of 10^-7 and 10^-6M, respectively. These values do not differ significantly from the control value (basal lipolytic activity in the absence of ACTH), however, ACTH (1 10) stimulated lipolysis at the concentration of 10^-5M. Therefore, it was ascertained that ACTH (1-10) had a slight lipolytic activity.
When fat cell ghosts were incubated with ¹²⁵ I-labelled ACTH (1-24), 18.0% of total radioactivities bound the fat cell ghosts. Displacement of ¹²⁵ I-labelled ACTH (1-24) occurred in the presence of unlabelled ACTH (1-24). Displacement of binding was not seen at the concentration under 10^-7M of ACTH. But the radioactivities of ACTH in the ghost cells showed a liner decrease according to an increase in the concentration of unlabelled ACTH (1-24) added. 6.3% of the bound radioactivity was not displaced over the concentration of 10^-4M of ACTH. Displacement of labelled ACTH binding the fat cell ghosts did not occur with the addition of insulin and epinephrine. It seemed that ACTH has no common receptor with those of insulin and epinephrine.
Second, influences of ACTH analogues on the binding of ACTH (1-24) with fat cell ghosts were examined. Unlabelled ACTH analogues caused also the displacement of labelled ACTH (1-24). Evaluation of binding experiments by the Scatchard method showed that not less than two receptor sites were present in adipocytes. The affinity constants were 1.0×10^-9M (low affinity constant was 1.3×10^-7M) for ACTH (1-24), 7.6×10^-10M (1.7×10^-7M) for ACTH (1-18) and 2.2×10^-9M (9.4×10^-8M) for ACTH (1-10). ACTH (1-18) showed the same extent of lipolytic activity and binding ability to the fat cells as ACTH (1-24).

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It is well known that estrone sulfate (E₁-S) is a major estrogen in human serum; however, the physiological role of E₁-S is not yet clarified. In order to elucidate this physiological role, the following experiments were performed.
1) The conjugation and metabolism of E₁-S in the Japanese monkey (Macaca fuscata)
Following an injection of the equimolar mixture of [4-¹⁴C] E₁ and [6, 7-³ H] E₁ -S into a femoral vein of the Japanese monkeys, urine, blood (from a femoral artery) and bile were collected at various time intervals over a period of 2 hs. Various tissues, that is, kidney, liver, lung, endometrium, fatty tissue, myometrium and skin, were taken after sacrifice. The metabolites were analyzed by DEAE Sephadex A-25 column chromatography, enzyme hydrolysis and TLC. E₁, E₁-glucosiduronate (E₁-G) and E₁-S were identified in the serum, and E₁, E₁-G, estradiol-17α-3G (E₂-17α-3G), estradiol-17β-3G (E₂ -3G), E₁ -S and E₂-17α-S were identified as urinary metabolites. In the bile, 16α-hydroxy-estrone-G was also present. A large amount of [³H] E₁ -S was present in the early collection of the serum, and [³H] E1-G gradually increased later. ¹⁴C, which was present less than 3 H in the serum was conjugated to [¹⁴ C] E₁-G and [¹⁴ C]E₁ -S later. Namely, E₁ -S was one of the conjugated forms of E₁ in the serum. There was much ¹⁴C and less ³H in the lung tissue. Therefore, one of the physiological roles of E₁-S is to pass through the pulmonary circulation as compared to E₁. E₁ -S and E₁ were conjugated into E₁ -G and E₁ -S in the general circulation. Glucosiduronate of estrogens was mainly excreted into the urine.
2) Renal conjugation and metabolism of E¹ and E₁-S
Following the injection of labelled estrogen into one of the renal arteries, urine was collected from both kidneys. Urinary metabolites were analyzed using the same methods as above.1 Injection of [4-¹⁴C] E₁ into one of the renal arteries and [6, 7-³H] E1 into a peripheral vein.
A larger amount of [¹⁴C] E₁ -G was identified in the injected side urine in the early period (5-10 min) rather than [³ H] E₁ -G. This denotes the formation of glucosiduronation in the kidney of the Japanese monkey in vivo.
2 When [4-¹⁴C] E₁ and [6, 7-³ F-³] E₁ -S were injected into one of the renal arteries of the monkey, El-S was partially filtered from the kidney directly, but no excretion of E₁ into the urine was detectable. It was shown that E₁-S was hydrolyzed and then was conjugated into E₁ -G in the general circulation, and after a while the E₁ -G was filtered from the kidney.
3 Following the injection of [4-¹⁴C] E₁ and [6, 7³H] E₁-G into one of the renal arteries, E₁-G was filtered very quickly from the kidney even compared to E₁-S.
3) Hydrolysis of E₁-S in the endometrium
The mixture of [6, 7³ FI] E₁ -S and [4-¹⁴C] E₁ was incubated with human endometrium.
The ethanol extract was analyzed by DEAE Sephadex A-25 column chromatography, enzyme hydrolysis and TLC. Myometrium, fatty tissue and muscle were also incubated as the control.
The results were as follows :
The hydrolysis of E₁ -S in the endometrium, target organ of estrogen, was 19.3% and those in the fatty tissue, muscle, and myometrium were 5.8, 3.3, 1.9% respectively.

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Previous animal experiments have given contradictory results concerning the effect of secretin on insulin release. The conflicting results are probably due to the fact that the injected hormone was administered in pharmacological amounts not usually attained under physiological conditions in vivo and that the preparation of the secretin used was an extraction which might have contained some other hormones. Moreover, small changes in insulin secretion are reported to remain undetected as the result of hepatic insulin clearance and of the dilution in the peripheral circulation. We have, therefore, investigated the effect of endogenous secretin released by the intraduodenal instillation of HC1 and 1-phenyl-1- hydroxy-n-pentane (PHP), and intravenously infused synthetic secretin in doses which raised portal plasma immunoreactive secretin (IRS) concentrations to those obtained after HC1 and PHP administration on portal plasma immunoreactive insulin (IRI), glucagon (IRG) and glucose concentration. In addition, the effect of somatostatin on IRI and IRG concentrations following acid and PHP administration was investigated by infusing synthetic cyclic somatostatin at the dose of 5 μg/kg/h.
Male Wistar rats weighing 240-270g and fasted overnight were used in all experiments. The rats were anesthetized with sodium pentobarbital by a subcutaneous injection. The body temperature was held at 37°C by a heating pad. One of the following solutions was infused into the duodenum at a rate of 2 ml/min for 2 min by means of a micro-tubing pump : 0.1 mo1/1 HC1,200 mg/kg/2 ml PHP and 2.5% arabic gum solution.
Blood samples were collected through an indwelling needle inserted into the portal vein in the direction of the venous blood flow at 0, 5, 10, 15, 20 and 30 min after the initiation of the infusion. The samples were centrifuged at 4°C, and plasma was stored at - 20°C until assayed.
IRI was measured by polyethylene glycol radioimmunoassay. IRG was determined by radioimmunoassay by means of a talc absorption technique using antiserum 30K. Rat insulin and porcine glucagon were used as standards in the IRI and IRG assay, respectively. Glucose concentrations were measured by the glucose oxidase method with a Toshiba LAC-02A glucose analyzer.
Although there was a rapid rise in serum IRS concentrations at 5 min after the administration of HCl and PHP, no significant changes in IRI concentrations were elicited by these stimuli. By contrast, IRG concentrations were significantly increased at 30 min after the intraduodenal infusion of HC1.
Constant infusion of somatostatin did not influence either IRI or IRG levels during intraduodenal stimulation with HCl and PHP, but it profoundly inhibited the HC1-induced IRG increase.
When synthetic secretin was infused to elevate the portal plasma IRS levels up to the same levels as those after HCl or PHP, no changes in the levels of IRI and IRG in the portal venous blood were observed.
The present observations with direct intraduodenal instillation of acid and PHP in quantitites sufficient to produce significant elevations of portal venous IRS concentrations did not result in an elevation of portal plasma IRI levels. In addition, the administration of physiological quantities of synthetic secretin failed to elicite any effect on portal IRI levels. It is therefore unlikely that secretin is responsible for entero-insular axis.

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To evaluate the relationship between the histological features of the thyroid and its functional state, and also to evaluate the chronological course of those patients with a focal type of chronic thyroiditis according to Woolner's classification, a needle biopsy of the thyroid and an estimation of serum levels of thyroxine (T₄) and triiodothyronine (T₃), and of thyrotropin (TSH) levels to thyrotropin-releasing-hormone (TRH) administration (500 μg, i.v.) were performed on 100 patients with the disease (97 females, 3 males; average age of 41.6).
(1) The patients were subgrouped according to the degree of small round cell infiltration area to total area of the thyroid slide specimen of each case (Factor C); namely : into CI (less than 10%, n=22), C_II (10-50%, n=28), C_III (50-75%, n=27) and C_IV (75% nearly diffuse, n=23) groups.
The serum levels of T₄ were in 40 normals (11 females, 29 males; average age of 32.6), CI, CH, CHI and qv subgroups were 9.9 ± 0.3, 8.7 ± 0.9*, 7.5 ± 0.4*, 7.4 ± 0.6* and 7.0±0.5*/μ1g/100ml (mean±SE); those of T₃ were 133±3,154±17,195±20*, 156±15 and 166 ± 11* ng/100ml; the levels of basal TSH (b-TSH) were 2.3± 0.3, 4.3 ± 0.9, 6.4 ± 1.1*, 12.4 ± 6.3* and 13.6 ± 8.0*μU/ml; the maximal levels of TSH to TRH (max-TSH) were 18.0 ± 1.6, 34.9 ± 5.7*, 37.7 ± 5.4*, 61.6 ± 13.6* and 60.9 ± 22.7* AU/ml; the incremental TSH to TRH (ATSH) were 15.7 ± 1.6, 30.5 ± 5.3*, 31.3 ± 4.5*, 49.2 ± 8.8* and 47.3 ± 14.9* μU/ml, respectively. (Asterisks indicate significant difference from normals.) There were tendencies to decrease in serum T₄ levels, to increase in serum T3 levels, and to increase levels of b-TSH, max-TSH, and _??_TSH in accordance with the increase of cell infiltration area. The significant reverse correlations between the levels of serum T₄ and T₃ and those of b-TSH (r=- 0.3175 and r=- 0.2715, respectively) were observed, even though the mean levels of serum T₄ and T₃ of the subgroup were within the normal range (mean ±2SD of normals).
(2) The same tendencies of levels of serum T₄, T₃, b-TSH, max-TSH, and ATSH were observed in subgroups with the advancement of a factor (Factor M) which was classi fied according to the ratio of the microfollicular area to the total follicular one in the slide, and in subgroups with a factor (Factor O) which was determined in accordance with the degree of prevalence of the oxyphilic follicular epithelium.
(3) Repeated needle biopsies were performed on 29 of the patients with the disease at an interval of 12 to 31 (mean of 17) months.
i) Those cases which had had high b-TSH (more than 10 μU/ml) levels at the first biopsy subsequently showed the aggravation of the histological features expressed as Factors C, M and 0.
ii) It appeared likely that the thyroid replacement therapy with desiccated thyroid had not shown any effect on the subsequent histological features of the disease expressed as Factors C, M and O.
iii) There was hardly any definitive tendency between the changes of the histological features and those of the thyroid function in the time course of the two biopsies; however, there did exist a small number of cases (3 out of 8) which showed a more exaggerated maxTSH elevation to TRH at the second biopsy than at the first, and in whom no remarkable histological changes were observed during the period.
From the above mentioned results, it is suggested that pathohistological thyroid changes have a close correlation with the thyroid functional state in chronic focal thyroidi tis,

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