Development of a method for the determination of pulsatile growth hormone secretion in mice

Measures of pulsatile GH secretion require frequent collection and analysis of blood samples at regular intervals. Due to blood volume constraints, repeat measures of circulating levels of GH in mice remain challenging. Consequently, few observations exist in which the pulsatile pattern of GH secretion in mice have been characterized. To address this, we developed a technique for the collection and analysis of circulating levels of GH at regular and frequent intervals in freely moving mice. This was achieved through the development of a sensitive assay for the detection of GH in small (2 μl) quantities of whole blood. The specificity and accuracy of this assay was validated following guidelines established for single-laboratory validation as specified by the International Union of Pure and Applied Chemistry. We incorporated an established method for tail-clip blood sample collection to determine circulating levels of GH secretion in 36 whole blood samples collected consecutively over a period of 6 h. Resulting measures were characterized by peak secretion periods and interpulse stable baseline secretion periods. Periods characterized by elevated whole blood GH levels consisted of multicomponent peaks. Deconvolution analysis of resulting measures confirmed key parameters associated with pulsatile GH secretion. We show a striking decrease in pulsatile GH secretion in mice after 12-18 h of fasting. This model is necessary to characterize the pulsatile profile of GH secretion in mice and will significantly contribute to current attempts to clarify mechanisms that contribute to the regulation of GH secretion.     

Altered multihormone synchrony in obese patients with polycystic ovary syndrome

Luteinizing hormone (LH) concentrations and pulsatility are increased in obese women with polycystic ovary syndrome (PCOS). In addition, patients have hyperandrogenemia and insulin resistance. The mechanisms involved in aberrant hormone regulation in PCOS are still unclear. We investigated 15 obese PCOS women with a body mass index between 30 and 54 kg/m² and 9 healthy obese controls (body mass index, 31-60 kg/m²) with regular menstrual cycles. Subjects underwent 24-hour blood sampling at 10-minute intervals for later measurements of LH, leptin, testosterone, and insulin concentrations. Data were analyzed with a new deconvolution program, approximate entropy (and bivariate approximate entropy), and a cross-correlation network. Patients had increased LH pulse frequency and more than 2-fold greater daily LH secretion, with diminished pattern regularity. Testosterone secretion was increased 2-fold, but pattern regularity was similar to that in controls. In the network construct, insulin was correlated positively with LH, whereas leptin and testosterone were correlated negatively with LH. Bivariate synchrony of LH with insulin was decreased. Short-term caloric restriction paradoxically increased LH secretion by 1.5-fold and pattern irregularity, and reduced interpulse variability. Testosterone secretion and fasting concentrations of estradiol and sex hormone-binding globulin levels remained unchanged. Correlations between LH and insulin, leptin, and calculated free testosterone decreased. This study demonstrates marked alterations in the control of LH secretion in PCOS in the fed and calorie-restricted states. The ensemble results point to abnormal feedback control of not only the GnRH-gonadotrope complex, but also LH's relationships with leptin, insulin, and testosterone.     

Loss of inverse relationship between pulsatile insulin and glucagon secretion in patients with type 2 diabetes

OBJECTIVE

In patients with type 2 diabetes, glucagon levels are often increased. Furthermore, pulsatile secretion of insulin is disturbed in such patients. Whether pulsatile glucagon secretion is altered in type 2 diabetes is not known.

RESEARCH DESIGN AND METHODS

Twelve patients with type 2 diabetes and 13 nondiabetic individuals were examined in the fasting state and after mixed meal ingestion. Deconvolution analyses were performed on insulin and glucagon concentration time series sampled at 1-min intervals.

RESULTS

Both insulin and glucagon were secreted in distinct pulses, occurring at ∼5-min intervals. In patients with diabetes, postprandial insulin pulse mass was reduced by 74% (P < 0.001). Glucagon concentrations were increased in the patients during fasting and after meal ingestion (P < 0.05), specifically through an increased glucagon pulse mass (P < 0.01). In healthy subjects, the increase in postprandial insulin levels was inversely related to respective glucagon levels (P < 0.05). This relationship was absent in the fasting state and in patients with diabetes.

CONCLUSIONS

Glucagon and insulin are secreted in a coordinated, pulsatile manner. A plausible model is that the postprandial increase in insulin burst mass represses the corresponding glucagon pulses. Disruption of the insulin–glucagon interaction in patients with type 2 diabetes could potentially contribute to hyperglucagonemia.The pathogenesis of type 2 diabetes involves multiple metabolic defects, the most important ones likely being β-cell dysfunction and insulin resistance (1,2). In addition, abnormal regulation of glucagon secretion contributes to the hyperglycemia in diabetic patients (3–5), and a number of studies have reported elevated fasting glucagon concentrations in patients with type 2 diabetes as well as in individuals with impaired glucose tolerance (3,6,7). Furthermore, whereas glucagon levels typically decline after oral or intravenous glucose administration in healthy individuals, the glucose-induced suppression of glucagon secretion is markedly impaired in patients with type 2 diabetes, and mixed meal–induced glucagon excursions are typically exaggerated in such patients (3,6). The inappropriately elevated glucagon levels may contribute to the exaggerated hepatic glucose production that characterizes patients with type 2 diabetes (8).The mechanistic reasons underlying increased glucagon secretion in such patients are less well understood. Thus, some studies have reported increased numbers of α-cells in the diabetic pancreas (9,10). An alternative hypothesis is the lack of α-cell inhibition by insulin in diabetic patients (11,12). Indeed, suppression of glucagon secretion by insulin has been well established in various in vitro and in vivo models (13), and a selective loss of β-cells has been associated with the development of hyperglucagonemia (14). It has also been demonstrated that the glucagon response to hypoglycemia is lost in the absence of insulin (11,15).Secretion of insulin from pancreatic islets in nondiabetic individuals is regulated in a pulsatile manner, with distinct bursts of insulin release occurring approximately every 5 min (16–18). In contrast, the amplitude and the orderliness of insulin secretion are markedly reduced in patients with type 2 diabetes (19–24). Impaired insulin pulsatility has been suggested to contribute to the development of insulin resistance in such patients (18,20,25).For glucagon, a pulsatile secretion pattern has been reported in different large animal models (26,27). Based on these studies, a close interaction between insulin and glucagon secretion has been suggested. To examine this relationship in more detail, previous studies have examined insulin and glucagon levels in pigs before and after a selective β-cell reduction induced by the β-cytotoxin alloxan (14). It is noteworthy that there was a significant inverse relationship between postprandial insulin and glucagon secretion in healthy animals, but this pulsatile intra-islet inhibition of glucagon secretion by insulin was lost after reduction of β-cell mass, leading to overt hyperglucagonemia. Such studies have prompted speculation that reduction of intra-islet insulin secretion might also cause insufficient suppression of glucagon in patients with type 2 diabetes (14). However, to date, a pulsatile pattern of glucagon secretion has not been established in humans.Therefore, in the present studies we addressed the following questions. (1) Is there evidence of a pulsatile pattern of glucagon secretion in humans? (2) Is there an inverse relationship between insulin and glucagon secretion? (3) Are the time patterns of glucagon secretion and its interactions with insulin secretion different in normal subjects and patients with type 2 diabetes?     

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell‐intrinsic and ‐extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP‐based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time‐consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell‐intrinsic and ‐extrinsic factors. Although the latter can be achieved by cleared fat‐pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild‐type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat‐pad transplantations with MMECs isolated from K14cre;Cdh1^F/F; Trp53^F/F mice expressing Cre recombinase under control of the cytokeratin‐14 promoter and carrying conditional null alleles for p53 and E‐cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell‐intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Effects of metformin on inappropriate LH release in women with polycystic ovarian syndrome and insulin resistance

The role of hyperinsulinaemia in neuroendocrine abnormalities in polycystic ovarian syndrome (PCOS) is controversial. The present study applied frequent blood sampling to assess the response of LH to metformin treatment in insulin-resistant women with PCOS. Thirteen predominantly overweight women with PCOS were studied before and after treatment with 1.5 g/day metformin for 3 months. Serum LH and testosterone were measured every 10 min for 10 h; LH was measured for an additional 2 h after gonadotrophin-releasing hormone (GnRH) administration. LH pulses were characterized by cluster analysis, secretory LH episodes by a deconvolution procedure, and synchronicity of paired LH-testosterone concentrations by lag-specific cross-correlation. After treatment, basal LH concentrations, amplitude of LH pulses, LH secretory amplitude, response to exogenous GnRH, and basal testosterone concentrations significantly decreased in seven patients, whereas in the remaining women these parameters remained unaltered. Before treatment, decreased coordinate LH and testosterone release was manifested by all patients; metformin treatment led to re-establishment of the feed-back control of testosterone on LH secretory rates by -20 to 0 min. Treatment did not modify the glucose:insulin ratio or serum insulin concentrations. In conclusion, administration of metformin allowed the identification of two subsets of PCOS women in whom neuroendocrine abnormalities may improve independently of the presence of insulin resistance or hyperinsulinaemia.