Different insulin concentrations in resuspended vs. unsuspended NPH insulin: Practical aspects of subcutaneous injection in patients with diabetes.

AIMS: This study measured the insulin concentration (Ins[C]) of NPH insulin in vials and cartridges from different companies after either resuspension (R+) or not (R-; in the clear/cloudy phases of unsuspended NPH). METHODS: Measurements included Ins[C] in NPH(R+) and in the clear/cloudy phases of NPH(R-), and the time needed to resuspend NPH and time for NPH(R+) to separate again into clear/cloudy parts. RESULTS: In vials of NPH(R+) (assumed to be 100%), Ins[C] in the clear phase of NPH(R-) was<1%, but 230±41% and 234±54% in the cloudy phases of Novo Nordisk and Eli Lilly NPH, respectively. Likewise, in pen cartridges, Ins[C] in the clear phase of NPH(R-) was<1%, but 182±33%, 204±22% and 229±62% in the cloudy phases of Novo, Lilly and Sanofi NPH. Time needed to resuspend NPH (spent in tipping) in vials was brief with both Novo (5±1s) and Lilly NPH (6±1s), but longer with all pen cartridges (50±8s, 40±6s and 30±4s from Novo, Lilly and Sanofi, respectively; P=0.022). Time required for 50% separation into cloudy and clear parts of NPH was longer with Novo (60±7min) vs. Lilly (18±3min) in vials (P=0.021), and affected by temperature, but not by the different diameter sizes of the vials. With pen cartridges, separation into clear and cloudy parts was significantly faster than in vials (P<0.01). CONCLUSION: Ins[C] in NPH preparations varies depending on their resuspension or not. Thus, subcutaneous injection of the same number of units of NPH in patients with diabetes may deliver different amounts of insulin depending on its prior NPH resuspension.

GLP-1 RAs as compared to prandial insulin after failure of basal insulin in type 2 diabetes: lessons from the 4B and Get-Goal DUO 2 trials.

The add-on of a prandial (short-acting) GLP-1 RA to basal insulin in subjects with T2DM who fail to control A1C on basal insulin, stems from the physiological principles of post-prandial glucose homeostasis, and it is based on evidence from clinical trials. The 4B and GetGoal DUO 2 studies are the first to establish in head-to-head comparison, the efficacy and safety of short-acting GLP-1 RAs vs prandial insulin, when added-on to basal insulin glargine. In the 4B study (exenatide 2/d vs lispro 3/d) exenatide demonstrated similar efficacy vs lispro in reducing A1C to ~7.2%. However, exenatide reduced also body weight and hypoglycemia incidence as compared to lispro. In GetGoal DUO 2, the head-to-head comparison was between lixisenatide 1/d vs glulisine either 1/d (at the main meal, basal-plus) or 3/d (basal-bolus). Like in 4B, in GetGoal DUO 2 the A1C decreased to similar values with lixisenatide or glulisine 1/d (~7.2%), or glulisine 3/d (~7.0%). Again, as in the 4B, body weight and hypoglycemia incidence were lower with lixisenatide. In both studies a similar percentage of subjects reached the A1C <7.0% on GLP-1 RA or prandial insulin. A higher percentage of subjects reported adverse events on GLP-1 RAs, primarily gastrointestinal related. The studies 4B and GetGoal DUO 2 suggest that after failure of basal insulin in T2DM, the add-on of prandial GLP-1 RA is as effective as prandial insulin in lowering A1C, with added benefits of reducing body weight and risk for hypoglycemia. In addition, the GLP-1 RA + basal insulin is a simpler therapeutic option as compared to basal-plus and basal-bolus regimens.

Glargine metabolism over 24 h following its subcutaneous injection in patients with type 2 diabetes mellitus: a dose-response study.

BACKGROUND AND AIMS: After subcutaneous injection insulin glargine is rapidly metabolized to M1 and M2. In vitro, both M1 and M2 have metabolic effects and bind to IGF-1R similarly to human insulin, whereas glargine exhibits a higher affinity for the IGF-1R and greater mitogenetic effects. The present study was specifically designed to establish the dose-response metabolism of glargine over 24 h following s.c. injection in T2DM subjects on long-term use of glargine. METHODS AND RESULTS: Ten subjects with T2DM were studied during 24 h after s.c. injection of 0.4 (therapeutic) and 0.8 (high dose) U/kg of glargine on two separate occasions during euglycaemic clamps (cross-over design). Glargine, M1 and M2 over 24 h period were determined in appropriately processed plasma samples by a specific liquid chromatography-tandem mass spectrometry assay. Plasma M1 concentration (AUC0-24 h) was detected in all subjects and increased by increasing the glargine dose from therapeutic to high dose (p = 0.008). Glargine was detectable in 6 (therapeutic dose) and 9 (high dose) out of the 10 subjects and also increased by increasing the dose (p = 0.031). However, glargine concentration (AUC0-24 h--high dose) represented at most only 9.7% (4.6-15%) of the total amount of insulin measured in the blood. M2 was not detected at all. CONCLUSION: In T2DM people on long-term use of insulin glargine, even with higher doses (0.8 U/kg), glargine is nearly totally metabolized to the active metabolite M1. Glargine is often detectable in plasma, but its concentration remains well below that needed in vitro to potentiate IGF-1R binding and mitogenesis.

Effects of dietary protein restriction on albumin and fibrinogen synthesis in macroalbuminuric type 2 diabetic patients.

AIMS/HYPOTHESIS: Diabetic nephropathy is associated with hypoalbuminaemia and hyperfibrinogenaemia. A low-protein diet has been recommended in patients with diabetic nephropathy, but its effects on albumin and fibrinogen synthesis are unknown. METHODS: We compared the effects of a normal (NPD; 1.38 +/- 0.08 g kg(-1) day(-1)) or low (LPD; 0.81 +/- 0.04 g kg(-1) day(-1)) -protein diet on endogenous leucine flux (ELF), albumin and fibrinogen synthesis (L-[5,5,5,-2H3]leucine infusion), and markers of inflammation in nine type 2 diabetic patients with macroalbuminuria. Six healthy participants on NPD served as control participants. RESULTS: In comparison with healthy participants, type 2 diabetic patients on an NPD had similar ELF, reduced serum albumin (38 +/- 1.1 vs 42 +/- 0.8 g/l; p < 0.05), similar fractional synthesis rates (FSR) and absolute synthesis rates (ASR) of albumin, and both increased plasma fibrinogen concentration [10.7 +/- 0.6 vs 7.2 +/- 0.5 micromol/l (3.64 +/- 0.22 vs 2.45 +/- 0.18 g/l); p < 0.05] and fibrinogen ASR [11.03 +/- 1.17 vs 6.0 +/- 1.8 micromol 1.73 m(-2) day(-1) (3.7 +/- 0.4 vs 1.9 +/- 0.3 g 1.73 m(-2) day(-1)); p < 0.01]. After LPD, type 2 diabetic patients had the following changes in comparison with NPD: reduced proteinuria (2.74 +/- 0.4 vs 4.51 +/- 0.8 g/day; p < 0.05), ELF (1.93 +/- 0.08 vs 2.11 +/- 0.08 micromol kg(-1) min(-1); p < 0.05) and total fibrinogen pool; increased serum albumin (42 +/- 1 vs 38 +/- 1 g/l; p < 0.01) and albumin ASR (14.1 +/- 1 vs 9.9 +/- 1 g 1.73 m(-2) day(-1); p < 0.05); and reduced plasma IL-6 levels, which were correlated with albumin ASR (r = -0.749; p < 0.05). CONCLUSIONS/INTERPRETATION: LPD in type 2 diabetic patients with diabetic nephropathy reduces low-grade inflammatory state, proteinuria, albuminuria, whole-body proteolysis and ASR of fibrinogen, while increasing albumin FSR, ASR and serum concentration.

Effect of the amino acid alanine on glucagon secretion in non-diabetic and type 1 diabetic subjects during hyperinsulinaemic euglycaemia, hypoglycaemia and post-hypoglycaemic hyperglycaemia.

AIMS/HYPOTHESIS: The aim of our study was to establish whether the well-known defective or absent secretion of glucagon in type 1 diabetes in response to hypoglycaemia is selective or includes lack of responses to other stimuli, such as amino acids. MATERIALS AND METHODS: Responses of glucagon to hypoglycaemia were measured in eight patients with type 1 diabetes and six non-diabetic subjects during hyperinsulinaemic (insulin infusion 0.5 mU kg(-1) min(-1)) and eu-, hypo- and hyperglycaemic clamp studies (sequential steps of plasma glucose 5.0, 2.9, 5.0, 10 mmol/l). Subjects were studied on three randomised occasions with infusion of low- or high-dose alanine, or saline. RESULTS: With saline, glucagon increased in hypoglycaemia in non-diabetic subjects but not in diabetic subjects. Glucagon increased further with low-dose (181 +/- 16 ng l(-1) min(-1)) and high-dose alanine (238 +/- 20 ng l(-1) min(-1)) in non-diabetic subjects, but only with high-dose alanine in diabetic subjects (area under curve 112 +/- 5 ng l(-1) min(-1)). The alanine-induced glucagon increase in diabetic subjects paralleled the spontaneous glucagon response to hypoglycaemia in non-diabetic subjects not receiving alanine. The greater responses of glucagon to hypoglycaemia with alanine infusion were offset by recovery of eu- or hyperglycaemia. CONCLUSIONS/INTERPRETATION: In type 1 diabetes, the usually deficient responses of glucagon to hypoglycaemia may improve after increasing the concentration of plasma amino acids. Amino acid-enhanced secretion of glucagon in response to hypoglycaemia remains under physiological control since it is regulated primarily by the ambient plasma glucose concentration. These findings might be relevant to improving counter-regulatory defences against insulin-induced hypoglycaemia in type 1 diabetes.

A randomized controlled trial to evaluate the efficacy of ultrasound-guided laser photocoagulation for treatment of benign thyroid nodules.

This randomized controlled study was designed to test the efficacy and safety of percutaneous ultrasound (US)-guided laser photocoagulation (PLP) for treatment of subjects with compressive symptoms due to benign thyroid nodules and/or at high surgical risk. Twenty six subjects were randomized to the intervention (no. 13, age 68+/-3 yr, mean+/-SEM) or observation (no. 13, age 71+/-2 yr) groups. In the control group, the volume of nodules did not significantly change over the 30 week period of observation. In the intervention group, median nodule volume at baseline was 8.2 ml (range 2.8-26.9) and was not significantly different from that of the control group. Nodules decreased significantly (p<0.0001) by 22% after 2 weeks (6.5 ml; range 2.4-16.7) and by 44% after 30 weeks (4.6 ml; range 0.69-14.2). Energy given was correlated (p<0.05) with the reduction of thyroid nodule volume. All patients tolerated the treatment well and reported relief from compressive and cosmetic complaints (p<0.05). At the time of enrolment 7/13 (54%) and 6/13 (46%) of patients in the intervention and control groups, respectively, had sub clinical hyperthyroidism. PLP normalized thyroid function at 6 and 30 weeks after treatment. In conclusion, PLP is a promising safe and effective procedure for treatment of benign thyroid nodules in patients at high surgical risk.

Effects of whole-body vibration exercise on the endocrine system of healthy men.

Whole-body vibration is reported to increase muscle performance, bone mineral density and stimulate the secretion of lipolytic and protein anabolic hormones, such as GH and testosterone, that might be used for the treatment of obesity. To date, as no controlled trial has examined the effects of vibration exercise on the human endocrine system, we performed a randomized controlled study, to establish whether the circulating concentrations of glucose and hormones (insulin, glucagon, cortisol, epinephrine, norepinephrine, GH, IGF-1, free and total testosterone) are affected by vibration in 10 healthy men [age 39 +/- 3, body mass index (BMI) of 23.5 +/- 0.5 kg/m2, mean +/- SEM]. Volunteers were studied on two occasions before and after standing for 25 min on a ground plate in the absence (control) or in the presence (vibration) of 30 Hz whole body vibration. Vibration slightly reduced plasma glucose (30 min: vibration 4.59 +/- 0.21, control 4.74 +/- 0.22 mM, p=0.049) and increased plasma norepinephrine concentrations (60 min: vibration 1.29 +/- 0.18, control 1.01 +/- 0.07 nM, p=0.038), but did not change the circulating concentrations of other hormones. These results demonstrate that vibration exercise transiently reduces plasma glucose, possibly by increasing glucose utilization by contracting muscles. Since hormonal responses, with the exception of norepinephrine, are not affected by acute vibration exposure, this type of exercise is not expected to reduce fat mass in obese subjects.

Meal intake similarly reduces circulating concentrations of octanoyl and total ghrelin in humans.

UNLABELLED: ABSTRACT. Several data show that meal intake and nutritional status regulate circulating ghrelin concentrations in humans. Ghrelin mainly circulates in two different forms: octanoyl and des-octanoyl ghrelin. Most circulating ghrelin is des-octanoyl ghrelin which is considered inactive because it lacks endocrine activity. However, recent evidence suggests that des-octanoyl ghrelin exerts biological activity such as stimulation of adipogenesis, cardiovascular effects and control of cell growth. In healthy humans, although the total ghrelin concentration is known to peak before meals and to be reduced by food intake, no data are available about the octanoyl ghrelin response in the absorptive state. Therefore, after an overnight fast, we compared the effects of a mixed meal ingestion (meal study) or of additional 240 min fasting (control study) on plasma concentrations of octanoyl and total ghrelin in 6 healthy subjects (body mass index: 23 +/- 0.7). At baseline, octanoyl-ghrelin accounted for 3.15 +/- 0.2% of total circulating ghrelin without differences between the two sessions. A similar ratio was maintained in the absorptive state with no differences between the studies and basal values. Compared with control, meal intake significantly suppressed (nadir at 90 min) octanoyl and total ghrelin by 38 +/- 3 and 40 +/- 3% of basal values, respectively. In the meal study, multivariate analysis of variance showed that serum insulin best predicted plasma octanoyl-ghrelin concentrations accounting for 97% of its variation (r2 = -0.97,p = 0.0016). IN CONCLUSION: in healthy humans, octanoyl-ghrelin represents about 3-4% of total circula-ting ghrelin and this ratio is closely maintained in post-absorptive and absorptive states.

Circulating ghrelin levels of visceral obese men are not modified by a short-term treatment with very low doses of GH replacement.

Ghrelin, the endogenous ligand for the GH secretagogue-receptor (GHS-R), in addition to its GH-releasing action, has orexigenic and adipogenic properties. These characteristics make ghrelin a potential hormone involved in the pathogenesis of obesity. Ghrelin levels are decreased in obese humans and it is unknown whether this decrease is responsible for the blunted GH secretion reported in visceral obesity. Since only few data are available on the potential feedback regulation by GH on systemic ghrelin concentrations, it remains to be established whether the correction of circulating GH concentrations in obese individuals affects ghrelin concentrations. To answer this question, we measured plasma ghrelin levels after a week of administration of low doses of recombinant human GH (rhGH) in a randomized, double-blind, placebo (PL)-controlled trial. This study was originally designed to evaluate the effects of GH replacement on lipid kinetics in visceral obese men. Six adult men with abdominal/visceral obesity (age 42+/-3 yr, body weight 107 +/- 10 kg, BMI 33 +/- 1 kg/m2, waist circumference 111 +/- 3 cm, mean +/- SE) were evaluated in the basal state (BS) and after one week of treatment with subcutaneous bedtime injections of either PL, 2.5 (GH2.5) or 3.3 (GH3.3) pg/kg/die of rhGH. In comparison to BS either PL, GH2.5 or GH3.3 did not significantly modify circulating ghrelin concentrations (p = 0.77). In contrast, a significant increase of serum GH (p = 0.0028), IGF-I (p = 0.0033) and whole body rate of lipolysis (p = 0.038, GH2.5; p = 0.009, GH3.3) occurred, in comparison to BS or PL, after GH2.5 and GH3.3, without differences between the two treatments. These data demonstrate that in abdominal/visceral obese men a short-term treatment with very low doses of rhGH replacement, sufficient to augment the rate of lipolysis, do not modify circulating ghrelin levels.

Metabolic response to exercise.

At the beginning, the survival of humans was strictly related to their physical capacity. There was the need to resist predators and to provide food and water for life. Achieving these goals required a prompt and efficient energy system capable of sustaining either high intensity or maintaining prolonged physical activity. Energy for skeletal muscle contraction is supplied by anaerobic and aerobic metabolic pathways. The former can allow short bursts of intense physical activity (60-90 sec) and utilizes as energetic source the phosphocreatine shuttle and anaerobic glycolysis. The aerobic system is the most efficient ATP source for skeletal muscle. The oxidative phosporylation of carbohydrates, fats and, to a minor extent, proteins, can sustain physical activity for many hours. Carbohydrates are the most efficient fuel for working muscle and their contribution to total fuel oxidation is positively related to the intensity of exercise. The first metabolic pathways of carbohydrate metabolism to be involved are skeletal muscle glycogenolysis and glycolysis. Later circulating glucose, formed through activated gluconeogenesis, becomes an important energetic source. Among glucose metabolites, lactate plays a primary role as either direct or indirect (gluconeogenesis) energy source for contracting skeletal muscle. Fat oxidation plays a primary role during either low-moderate intensity exercise or protracted physical activity (over 90-120 min). Severe muscle glycogen depletion results in increased rates of muscle proteolysis and branched chain amino acid oxidation. Endurance training ameliorates physical performance by improving cardiopulmonary efficiency and optimizing skeletal muscle supply and oxidation of substrates.

Diabetes and exercise.

Physical activity has acute and chronic effects on glucose, lipid and protein metabolism. Long-term effects of regular exercise are particularly advantageous for Type 2 diabetic patients. Regular aerobic exercise reduces visceral fat mass and body weight without decreasing lean body mass, ameliorates insulin sensitivity, glucose and BP control, lipid profile and reduces the cardiovascular risk. For these reasons, regular aerobic physical activity must be considered as an essential component of the cure of Type 2 diabetes mellitus. In this regard, individual behavioral strategies have been documented to be effective in motivating sedentary Type 2 diabetic subjects to the adoption and the maintenance of regular physical activity. In Type 1 diabetic subjects, the lack of the physiological inhibition of insulin secretion during exercise results in a potential risk of hypoglycemia. On the other hand, exercise-induced activation of counter-regulatory hormones might trigger an acute metabolic derangement in severe insulin-deficient subjects. Thus, diabetic patients, before starting exercise sessions, must be carefully educated about the consequences of physical activity on their blood glucose and the appropriate modifications of diet and insulin therapy.

VLA-6 integrin distribution and calcium signalling in capacitated boar sperm.

Previous investigations showed that VLA-6 integrin present on boar sperm membrane can induce acrosome reaction upon exposure to laminin accumulated in expanded cumuli (Mattioli et al., 1998. To further investigate this novel sperm egg-recognition system, the authors studied the distribution of VLA-6 integrin on the membrane of boar sperm throughout capacitation and following acrosome reaction, and analyzed intracellular Ca(2+) changes occurring in spermatozoa exposed to laminin. Immunofluorescent localisation of VLA-6 revealed a low proportion (nearly 22%) of positive cells in freshly ejaculated sperm, with integrin mainly concentrated in clustered spots. After 3 hr incubation most of the spermatozoa showed integrin molecules on the membrane, with three different labeling patterns: fluorescence localised on the edge of the acrosome (58.2 +/- 14.2% of the cells); fluorescence uniformly spread over the whole sperm head (5.0 +/- 1.9%) and finally fluorescence concentrated in clustered spots (7.6 +/- 5.6%), as recorded in freshly ejaculated sperm. Twenty-nine percent of cells did not show any distinct fluorescence. Following acrosome reaction sperm with fluorescence on the acrosomal region virtually disappeared and the proportion of unstained cells rose from 29.2 +/- 9.2 to 69.0 +/- 10.1%. Electron microscopy demonstrated that VLA-6 integrin was exclusively located on the sperm membrane of intact spermatozoa. Confocal analysis showed that laminin triggers distinct Ca(2+) raises, and that sperm exposed and kept in the presence of laminin fully retained their ability to rise intracellular Ca(2+) in response to zona pellucida proteins. These data indicate that boar sperm accumulate VLA-6 integrin on the membrane and concentrate it on the acrosomal region as capacitation progresses. Probably due to this compartmentalisation, sperm exposed to laminin experience a Ca(2+) raise that originates in the anterior sperm head where it is more adequate for the induction of acrosome reaction. Mol. Reprod. Dev. 59:322-329, 2001.

Ram-induced ovulation to improve artificial insemination efficiency with frozen semen in sheep.

Ram effect, defined as shortening of seasonal anestrus in ewes by exposure to the ram, is now well recognized but the underlying mechanisms are still unclear. Little information also exists whether the ram is able to influence the estrus cycle and ovulation. Three experiments were conducted to investigate endocrine response, time of ovulation and pregnancy rate of ewes in proestrus, exposed to the ram (treated) or an adult ewe (control). In the first experiment, ewes (n = 20) were treated with fluorgestone acetate pessaries for 12 days and were given eCG and cloprostenol one day before withdrawal of pessaries. On the day after removal of the pessaries ewes in the treated group (n = 10) were exposed to the ram and those in the control group (n = 10) were exposed to an adult ewe. Blood samples were taken for LH assay every 20 min from 2 h before to 24 h after ram exposure. In the second experiment, ewes (n = 120) were induced into proestrus and on the day after removal of the pessaries were exposed to either a ram (n = 60) or a ewe (n = 60) as described above and were laparoscoped 50, 60 or 70 h after pessary withdrawal (n = 20 at each time interval). In the third experiment ewes (n = 90) were induced and exposed to the ram (n = 45) or an adult ewe (n = 45) and inseminated via a laparoscope whit frozen-thawed semen at 50 or 60 h after pessary removal, respectively. Exposure to the ram was followed in 2 h by a marked rise in LH, equivalent to a preovulatory surge in duration and amplitude. It was also followed by concentrated ovulation within 25 to 30 h and by an increased pregnancy rate in exposed ewes (73.3 vs. 53.3%).

Effects of dietary protein restriction on fibrinogen and albumin metabolism in nephrotic patients.

BACKGROUND: Nephrotic syndrome (NS) is characterized by profound changes in albumin and fibrinogen levels. Dietary protein restriction has been advocated in the treatment of patients with NS, but its effects on albumin and fibrinogen metabolism have not been fully elucidated. METHODS: We evaluated the effects of dietary protein restriction on endogenous leucine flux (ELF), fibrinogen and albumin metabolism in seven patients with NS who consumed either a normal protein diet (NPD; 1.20 +/- 0.06 g/kg/day), or a low protein diet (LPS; 0.66 +/- 0.04 g/kg/day) for four weeks. Seven normal subjects served as controls. The postabsorptive ELF value, fractional synthesis rate (FSR) and absolute synthesis rate (ASR) of both albumin and fibrinogen were evaluated during the last 120 minutes of a five-hour 5,5,5-D3-L-leucine infusion. RESULTS: During the NPD regimen. ELF was increased, serum albumin was reduced, plasma fibrinogen was increased, albumin FSR and ASR were both increased, fibrinogen FSR was normal, and fibrinogen ASR was greater in patients with NS compared to controls. In patients with NS the LPD regimen reduced proteinuria, ELF, albumin FSR and ASR, plasma fibrinogen levels, fibrinogen ASR, and increased serum ulbumin levels. Dietary-induced changes in albumin and fibrinogen synthesis were significantly correlated (r = 0.719, P < 0.05). CONCLUSIONS: Patients with NS treated with LPD show: (1) a reduction of proteinuria, albumin ASR and FSR, with an increase in serum albumin levels and its intravascular pool; (2) a decrease of fibrinogen ASR, with a reduction in both plasma fibrinogen levels and intravascular pool; and (3) a reduced rate of whole body proteolysis.

Administration of recombinant human growth hormone on alternate days is sufficient to increase whole body protein synthesis and lipolysis in growth hormone deficient adults.

OBJECTIVE: At present, the duration of the effect of recombinant human growth hormone (rhGH) on the rates of protein synthesis and lipolysis in GH deficient (GHD) adults is unknown. This study was designed to establish the frequency of rhGH administration necessary to provide the beneficial metabolic effects of the hormone in GHD adults. DESIGN AND PATIENTS: Two different studies (A and B) were performed in two groups of five GHD men. In study A, whole body protein and lipid kinetics was determined in the basal state (Bas), 12 (GH12h) and 36 (GH36h) h after the last of seven injections of rhGH (3.3 microg/kg), given at bedtime on alternate days. In study B, the same parameters were determined in the basal state (Bas), 60 (GH60h) and 84 (GH84h) h after the last of seven injections of rhGH (3.3 microg/kg), given at bedtime at 3 day intervals. MEASUREMENTS: The rates of protein metabolism were estimated by infusing [1-13C]leucine, and those of lipolysis by infusing [1,1,2,3, 3-D5]glycerol. RESULTS: Leucine oxidation decreased (P < 0.01) by approximately 30% after GH12h and GH36h but did not change after GH60h and GH84h. Non-oxidative leucine disposal increased after GH12h and GH36h by approximately 13% (P < 0.05) whereas it did not change after GH60h and GH84h. Glycerol appearance increased (P < 0. 01) by approximately 45% after GH12h and GH36h but did not change after GH60h and GH84h. CONCLUSIONS: The effects on protein and lipid metabolism following the injection of rhGH last longer than 36 and less than 60 h. In fact, rhGH administration on alternate days induced a sustained increase in the rates of protein synthesis and lipolysis of GHD adults, whereas a longer interval of administration (3 days) had no effect by 60 h.

Nerve growth factor production in sheep antral follicles.

Despite their continuous turnover, sheep antral follicles are always regularly innervated. The local production of neurotrophins is probably involved in the control of ovarian innervation. In this context the present investigation was designed to evaluate the ability of sheep antral follicles to produce neurotrophic factors. In the first part of the paper neurotrophic activity was measured in follicular fluid of sheep antral follicles of different size. Using an in vitro model the effect of gonadotrophins on neurotrophin production was then evaluated. The levels of neurotrophic activity in conditioned medium or follicular fluid and the kind of neurotrophin produced were determined by using the chicken embryo dorsal root ganglia test combined with an immunoneutralization step. Follicular fluid from medium-large follicles (>4 mm) contains high levels of NGF (240-250 ng/ml), whereas the factor is nearly undetectable in small follicles (<3 mm) and in early atretic follicles. Experiments in vitro based on the culture of follicle shells for 12 hr confirmed that medium-large follicles can produce NGF. The production is strictly dependent on gonadotrophin stimulation. When gonadotrophins were not added or were added separately, no detectable levels of neurotrophic activity accumulated in medium. By contrast, in the presence of both LH and FSH the production of NGF became apparent showing a clear dose-response behavior. In addition, this production increased progressively with increasing follicle size from 4 to >5 mm up to values of about 60 ng/follicle, whereas follicles with a diameter of less than 3 mm were insensitive to gonadotrophins stimulation and did not produce significant amount of NGF. The data presented demonstrate that sheep follicles produce relevant amounts of NGF as long as the correct hormonal milieu is provided. Under these conditions the production of the NGF increases with increasing follicle size. This may be responsible for the rapid innervation of the wall of growing follicles and/or take part in other non-neural processes that are generally attributed to gonadotrophin stimulation.