1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.

Prolonged pregnancy following foetal hypophysectomy or adrenalectomy (Liggins, Kennedy & Holm, 1967;Drost & Holm, 1968) and premature parturition following injection of corticotrophin or corticosteroids into the ovine foetus (Van Rensburg, 1967;Halliday & Buttle, 1968;Liggins, 1968) implicate the foetal adrenal cortex in the initiation of parturition in sheep. Consequently, foetal plasma corticosteroid concentrations and corticosteroid secretion by the foetal adrenal should be greatly increased towards term. Observations during acute studies on anaesthetized sheep suggest this (Alexander, Britton, James, Nixon, Parker, Wintour & Wright, 1968), but there is no information about blood concentrations or secretion rates of corticosteroids in normal undisturbed sheep foetuses in utero.We cannulated the carotid artery and the facial branch of the jugular vein of four single Merino foetuses with polyvinyl chloride tubing (o.d. 1\m=.\27 mm., i.d. 0\m=.\86mm.) with the ewes under general anaesthesia (pentobarbitone sodium (20 mg./kg., i.v.) followed by halothane and oxygen). The catheters were brought to the exterior through the uterine incision and a separate stab incision in the flank of the ewe. Foetal blood samples (2-3 ml.) were drawn from the carotid catheter daily, followed by a sample from a jugular vein ofthe ewe. Plasma corticosteroid concentrations were determined by the protein-binding method of Bassett & Hinks (1969).One lamb was born after a gestation of normal length and survived ; parturition occurred 8-10 days early in the other three ewes. In these the foetuses were alive at the start of parturition, but two were dead when born.Maternal corticosteroid concentrations were greatly elevated for several days after surgery, but subsequently were generally less than 2 fig.flOO ml. (Fig. 1).Corticosteroid concentrations in the foetal circulation bore little relation to maternal concentrations. Despite the stress of surgery, foetal corticosteroid concentrations were generally below 2 fig./lOO ml. until 130 days gestation. However, commencing several days before birth, there was an increase in the plasma corticosteroid con¬ centration of all the foetuses, unrelated to changes in maternal corticosteroid con¬ centrations (Fig. 1). The highest concentrations, considerably above maternal levels, were reached at the time of birth. In the two lambs which survived birth there was a further temporary increase after birth. Corticosteroid concentrations in all foetuses at birth were similar to those of other newborn lambs (Bassett, Alexande & Oxborrow, 1968). In contrast, no comparable changes in maternal or foetal plasma concentrations of growth hormone, insulin, glucose and fructose or in the percentage oxygen satura-C.S.I.

The progesterone concentration in the peripheral plasma of ewes throughout pregnancy has been determined by a protein-binding method.Plasma progesterone concentrations during the first 50 days of pregnancy (2-3 ng./ml.) were not significantly higher than peak concentrations during the luteal phase in cycling non-pregnant ewes, but there was no decrease in the concentration 15-20 days after mating as occurs in non-pregnant ewes.Between 50 and 120 days after mating the plasma progesterone concentration increased steadily to values 2-5 times that found in early pregnancy. These high concentrations were maintained until lambing. A decrease in progesterone concentration during the week preceding lambing was usually, but not always, observed.Mean plasma progesterone concentrations during the last 50 days of pregnancy in ewes with twins were approximately twice those in ewes with a single foetus.

A sensitive method for the determination of corticosteroids in 0\m=.\1ml. or less of ovine plasma is described. The method uses the steroid-binding properties of corticosteroid-binding globulin (CBG) and gel filtration on small columns of Sephadex G-25 (fine) at 4\ s=deg\ for separation of CBG-bound and free steroids. Cortisol was found to be the predominant corticosteroid in ovine plasma and accounts for about 90% of the value determined by this method. The corticosteroid concentration in peripheral plasma of unstressed sheep was in the range 0\m=.\1\p=n-\1\m=.\0 \g=m\g./100 ml. In untrained animals, venipuncture increased corticosteroid concentration substantially; training reduced the effect. An infusion of cortisol sodium succinate (100 \g=m\g.cortisol/min.) increased the plasma corticosteroid level to 9\m=.\5 \m=+-\0\m=.\49\g=m\g./100 ml. Intravenous infusion of the synthetic adrenocorticotrophic preparation Synacthen at rates of 10 and 20 \g=m\g./hr. for 2 hr. increased peripheral corticosteroid concentrations to 8 \g=m\g./100 ml. Single i.v. injections of 0\m=.\2\p=n-\0\m=.\8 \g=m\g.Synacthen also significantly increased peripheral corticosteroid concentrations 7\p=n-\15 min. later. The injection of 0\m=.\05 and 0\ m=. \ 1\g=m\g.Synacthen significantly increased the corticosteroid concentration too, but the increase was not significantly greater than that produced by the injection of acidified saline diluent alone. Injection of insulin (0\m=.\25unit/kg. body weight, i.v.) caused a fivefold increase in the corticosteroid concentration 30\p=n-\60 min. later, in both adult sheep and lambs. Glucose (0\m=.\25g./kg. body weight, i.v.) had no effect on corticosteroid concentration.

Using a protein-binding technique, progesterone concentrations in peripheral plasma (jugular vein) were measured throughout the oestrous cycle of 24 ewes. Examination of the specificity of the method by thin-layer chromatography indicated that interference from other steroids was not significant in sheep plasma. During the first 4 days of the cycle (days 0-3), plasma progesterone concentrations were below 0\m=.\4 ng./ml., increasing to a mean level of 1\m=.\5-2\m=.\5 ng./ml. between days 4 and 9, and remaining at this level for approximately 5 days, before declining rapidly on days 14 and 15 to reach a low level on the day before oestrus. The progesterone concentration on the day of oestrus was extremely low (0\m=.\1ng./ml.), and was of the same order as that found in the plasma of wethers and anoestrous or ovariectomized ewes. Three ewes, superovulated with pregnant mare serum gonadotrophin, showed marked elevation of peripheral progesterone concentration during the luteal phase of the cycle, the concentration being proportional to the number of corpora lutea formed.

SUMMARY Progesterone concentrations in the peripheral plasma of cows were measured by a protein-binding radioassay method. The mean concentration was lowest at oestrus (0·44 ng/ml) and then increased to a maximum of 6·8 ng/ml about day 14 of the 21-day cycle. The concentration decreased rapidly during the last 4 days of the cycle, reaching low levels on the day before oestrus. There were no significant changes in progesterone concentration during oestrus. After ovariectomy the plasma progesterone concentration decreased to a very low level (< 0·4 ng/ml). After hysterectomy, progesterone concentrations remained high for longer than in a normal cycle. At puberty, plasma progesterone concentrations indicated cyclic ovarian activity before the first observed oestrus. Daily treatment of cows with oxytocin (0·4 u./kg body weight) from day 2 reduced the oestrous cycle length to 9 days, but did not significantly alter the slow increase in plasma progesterone concentration during the first 5 days of the cycle. Plasma progesterone concentrations decreased again after day 5 to low values. Plasma progesterone concentration during early pregnancy was similar to the luteal phase value (4–6 ng/ml), declined during mid-pregnancy and then increased to a maximum (7–8 ng/ml) at about 240 days gestation. The concentration declined 2–3 weeks before calving. During lactation progesterone concentrations were very low until the resumption of cyclic ovarian activity. The first post-partum cycle, whether accompanied by observed oestrus or not, was usually preceded by a small increase in plasma progesterone concentration 3–5 days before the start of the cycle. Undernutrition significantly increased plasma progesterone concentrations in mid- and late pregnancy. Undernutrition of non-pregnant cows increased progesterone concentrations during the luteal phase of the first cycle, but reduced it in later cycles.

To determine the effects of feeding on plasma concentrations of insulin and growth hormone (GH) in sheep during the full 24 h of the normal feeding cycle, two experiments have been conducted on a group of 18 crossbred wethers.The eating once daily of 800 g of a lucerne chaff-oat grain (1 : 1) diet resulted in rapid increases in plasma insulin and rapid decreases in plasma GH concentrations, with maxima and minima respectively 2-4 h after feeding, in groups of six sheep in both experiments. After this, insulin and GH levels returned towards prefeeding values. The eating of larger amounts of the same diet did not greatly alter the magnitude of changes in insulin, but tended to prolong the period when they were high. An extra 800 g of food 12 h after the usual feed resulted in a repetition of the normal changes after feeding, an increase in insulin and a decline in GH concentrations. In wethers fed 1200 g of food daily or fed ad libitum, GH levels tended to be lower during the full 24 h. The withholding of food for 24 h caused a steady decline in insulin levels, but GH levels did not increase further above prefeeding values in sheep fed 800 g daily. There were large oscillations in GH levels of sheep that were fasted or fed 800 g daily.Corticosteroid concentrations in plasma fluctuated during the day, but there was no influence of feeding on them. There was no evidence for a circadian rhythm.Maximum plasma insulin and minimum GH concentrations occurred at about the same time as maximum concentrations of volatile fatty acids and minimum concentrations of free fatty acids in plasma after feeding, but there was no close correspondence between changes in metabolite and hormone concentrations. Maximum glucose concentrations occurred later after feeding than did those of insulin. There were only small changes in a-amino nitrogen concentrations related to feeding.It is doubtful whether changes in plasma metabolite levels after feeding play a major role in determining the changes in insulin or GH release during the day.

The in vitro effects of glucose and analogs of glucose on insulin secretion have been reinvestigated with rat and mouse islets. Curves relating rates of insulin secretion to glucose concentration were sigmoid with a K m of 6-8 mM and tending to V max at approximately 20 mM glucose. Mannose and glucosamine were the only analogs of glucose which stimulated secretion in the absence of glucose. Fructose, N-acetyl glucosamine and galactose (rat only) potentiated secretion at 2.5 or 5 mM glucose but not at 20mM glucose. Other analogs of glucose were inactive. Mannoheptulose inhibited glucose effects at all concentrations of glucose; glucosamine was inhibitory at 5 mM and 20 mM glucose. It is concluded that there may be quantitatively or qualitatively two distinct types of receptor mechanism in islets: (1) an initiator unit sensitive to effects of glucose, mannose, glucosamine, mannoheptulose and leucine and (2) a potentiator unit sensitive to effects of fructose, N-acetyl glucosamine and galactose (and also glucose and mannose?). Rates of glucose utilization measured in islets with [5-3 H] glucose were linearly related to rates of insulin secretion with varying glucose concentrations, with mannoheptulose and with glucosamine. It is concluded that evidence currently available does not determine whether initiation of insulin secretion by glucose, mannose or glucosamine involves a direct effect of the sugar or of a metabolite. DIABETES 21 (Suppl. 2): 538-45, 1972.