Leptin is secreted from adipose tissue, and is thought to act as a 'lipostat', signalling the body fat levels to the hypothalamus resulting in adjustments to food intake and energy expenditure to maintain body weight homeostasis. In addition, plasma leptin concentrations have been shown to be related to insulin sensitivity independent of body fat content, suggesting that the hyperleptinemia found in obesity could contribute to the insulin resistance. We investigated the effects of leptin on insulin binding by isolated adipocytes. Adipocytes isolated from Sprague-Dawley rats exhibited a dose-dependent reduction in the uptake of 125I-labelled insulin when incubated with various concentrations of exogenous leptin. For example, addition of 50 nM leptin reduced total insulin binding in isolated adipocytes by 19% (P < 0.05). Analysis of displacement curve binding data suggested that leptin reduced maximal insulin binding in a dose-dependent manner, but had no significant effect on the affinity of insulin for its binding site. We conclude that leptin directly inhibited insulin binding by adipocytes, and the role of leptin in the development of insulin resistance in obese individuals requires further investigation.

Acid phosphatase (EC 3.1.3.2) activity was examined for its possible utilization as a biochemical marker for the profound changes that occur in the prostate gland after castration. Tissue filtrates were prepared from the prostate glands of mature male rats at various times after castration. The acid phosphatase activity was characterized by polyacrylamide gel electrophoresis and the percentage inhibition in the presence of tartrate. Prostatic acid phosphatase from mature rats has been shown to have two bands of activity, a lysosomal acid phosphatase and a secretory acid phosphatase. After castration, there was a loss of the secretory acid phosphatase from gel electrophoresis patterns by day 5 and a corresponding rise in the percentage inhibition by tartrate from the normal value of 43·2% to a maximum of 55·4% on day 7. Between days 7 and 15 there was a linear decrease in the percentage inhibition by tartrate, but after day 15 the value did not change significantly from 31·1% After castration, the specific activity of the uninhibited enzyme increased from a normal basal level of 2·16 μmol h⁻¹ mg protein⁻¹ to a maximum on day 7 of 8·10 μmol h⁻¹ mg protein⁻¹. After this time, the specific activity decreased slowly until it reached a normal level on day 21. Intraperitoneal administration of testosterone, 5α-dihydrotestosterone or 5α-androstane-3α,17β-diol at a dose of 2 mg/day and starting immediately after castration prevented the changes in percentage inhibition by tartrate and the loss of the secretory band of acid phosphatase. Administration of these androgens from day 7 after castration led to a decrease in the percentage inhibition from 50·1% to a minimum of 31·5% before the level returned to the normal value found in the mature rat. The secretory band of acid phosphatase, which was not present in gels at day 7, reappeared after 8–11 days of treatment with androgens. Of the androgens used,5α- androstane-3α,17β-diol was the most potent.

Polyacrylamide gel electrophoresis of filtrates from adult rat prostatic tissue showed two bands of acid phosphatase activity. These corresponded to the lysosomal and secretory acid phosphatases. After castration the secretory acid phosphatase disappeared. The specific activity of the enzyme increased from the time of castration to a maximum on day 7 before declining steadily, while the percentage inhibition by tartrate of acid phosphatase increased from control levels to a maximum on day 7 and then decreased to a new steady state by day 15. When 5α-androstane-3β,17β-diol was administered i.p. at a dose of 2 mg/day, starting immediately after castration, the secretory acid phosphatase was retained but the percentage inhibition and the specific activity were both raised above control levels. When this steroid was administered daily starting 7 days after castration the secretory acid phosphatase band on the gels returned more rapidly than with the classical androgens, but the percentage inhibition and specific activity were once again raised.

Intraperitoneal administration of 5β-dihydrotestosterone, at a dose of 2 mg/day, did not maintain the secretory acid phosphatase activity which disappeared by day 5. However, the specific activity of acid phosphatase and the percentage inhibition by tartrate were both raised throughout the experiment. If this steroid was given 7 days after castration, the percentage inhibition by tartrate did not respond and fell to the level seen in castrated rats. The specific activity, however, remained significantly above the level found in castrated control rats.

Abstract

We have previously demonstrated that 1·9 kb of ovine LHβ promoter fused to bacterial chloramphenicol transferase (CAT) coding sequence is sufficient to target expression of the transgene specifically to the gonadotroph cells of the anterior pituitary in mice with no expression being observed in other tissues. However, it is not known if this region of the ovine LHβ promoter contains the necessary elements that confer transcriptional regulation by gonadal steroids and GnRH. Following gonadectomy, both endogenous pituitary LH and CAT activity significantly (P>0·001) increased as did plasma LH. This postgonadectomy increase in CAT, pituitary and plasma LH could be suppressed in females by treatment with oestradiol alone or oestradiol and progesterone, with an additional significant (P<0·05) reduction in CAT activity being observed in one line following the combined steroid treatment. In castrated males, testosterone suppressed CAT activity in one line. Treatment of transgenic ovariectomized females with oestradiol alone significantly suppressed plasma LH (P<0·01) with no change in pituitary LH content. There was no difference in pituitary LH between oestradiol-treated ovariectomized transgenic and non-transgenic females. Treatment of intact females from both lines with either GnRH antiserum or agonist demonstrated a decrease in pituitary CAT activity whereas similar treatment in intact males had no effect. While endogenous pituitary LH concentrations were variable, plasma LH was lower in all treated animals irrespective of line, sex or expression of the transgene. In conclusion, these results indicate that (1) the presence of CAT protein is not toxic and does not compromise either endogenous LH synthesis, storage and secretion and (2) the ovine LHβ–CAT gene is regulated in a similar but more variable manner to the endogenous LHβ gene. This may relate to the use of CAT as a reporter where its release is not necessarily related to that of the endogenous hormone whose synthesis, storage and release may differ.

Journal of Endocrinology (1996) 151, 481–489

Growth hormone insensitivity syndrome (GHIS) has been reported in a family homozygous for a point mutation in the GH receptor (GHR) that activates an intronic pseudoexon. The resultant GHR (GHR1–656) includes a 36 amino-acids insertion after residue 207, in the region known to be important for homodimerization of GHR. We have examined the functional consequences of such an insertion in mammalian cells transfected with the wild type (GHRwt) and mutated GHR (GHR1–656). Radio-ligand binding and flow cytometry analysis showed that GHR1–656 is poorly expressed at the cell surface compared with GHRwt. Total membrane binding and Western blot analysis showed no such difference in the level of total cellular GHR expressed for GHR1–656 vs GHRwt. Immunofluorescence showed GHR1–656 to have different cellular distribution to the wild type receptor (GHRwt), with the mutated GHR being mainly perinuclear and less vesicular than GHRwt. Western blot analysis showed GH-induced phosphorylation of Jak2 and Stat5 for both GHR1–656 and GHRwt, although reduced Stat5 activity was detected with GHR1–656, consistent with lower levels of expression of GHR1–656 than GHRwt at the cell surface. In conclusion, we report that GHIS, due to a 36 amino-acids insertion in the extracellular domain of GHR, is likely to be explained by a trafficking defect rather than by a signalling defect of GHR.

Distal-less 3 (Dlx3) is a homeobox factor that functions as a placental-specific transcriptional regulator. Dlx3 null mice (−/−) have compromised placental development and do not survive in utero past embryonic day (E) 9.5. The current studies were undertaken to examine the expression of Dlx3 in mouse placenta during gestation, and to determine whether Dlx3 was involved in placental progesterone production. Dlx3 was not detectable at E8.5 but was detected in E9.5 placenta with continuing but diminished expression through E15.5. Dlx3 immuno-localization was restricted to the labyrinth, was nuclear and was found in cytokeratin-positive cells. Previous studies in choriocarcinoma cell lines support the conclusion that Dlx3 is required for expression of 3′-hydroxysteroid dehydrogenase VI (3βHSD VI), an obligate enzyme in the production of progesterone by trophoblast giant cells. In a rat trophoblast stem cell line (Rcho-1), Dlx3 expression was non-detectable in Rcho-1 cells induced to differ-entiate using mitogen withdrawal. In vitro progesterone production in placental cultures and 3βHSD VI mRNA from Dlx3 (+/+), (+/−) and (−/−) mice were equivalent. In situ hybridization for 3βHSD VI revealed mRNA expression restricted to trophoblast giants cells with no detectable expression in the labyrinth suggesting that Dlx3 and 3βHSD VI were not colocalized within the placenta. These studies support the conclusion that Dlx3 protein expression is restricted to the labyrinth region of the murine placenta into late gestation and that Dlx3 does not appear to be expressed in trophoblast giant cells. Further, loss of Dlx3 was not correlated with synthesis of progesterone from E9.5 mouse placentas.

Melanocortin receptor accessory protein 2 (MRAP2) is a transmembrane accessory protein predominantly expressed in the brain. Both global and brain-specific deletion of Mrap2 in mice results in severe obesity. Loss-of-function MRAP2 mutations have also been associated with obesity in humans. Although MRAP2 has been shown to interact with MC4R, a G protein-coupled receptor with an established role in energy homeostasis, appetite regulation and lipid metabolism, the mechanisms through which loss of MRAP2 causes obesity remains uncertain. In this study, we used two independently derived lines of Mrap2 deficient mice (Mrap2 ^tm1a/tm1a ) to further study the role of Mrap2 in the regulation of energy balance and peripheral lipid metabolism. Mrap2 ^tm1a/tm1a mice have a significant increase in body weight, with increased fat and lean mass, but without detectable changes in food intake or energy expenditure. Transcriptomic analysis showed significantly decreased expression of Sim1, Trh, Oxt and Crh within the hypothalamic paraventricular nucleus of Mrap2 ^tm1a/tm1a mice. Circulating levels of both high-density lipoprotein and low-density lipoprotein were significantly increased in Mrap2 deficient mice. Taken together, these data corroborate the role of MRAP2 in metabolic regulation and indicate that, at least in part, this may be due to defective central melanocortin signalling.

ABSTRACT

Seventeen human subjects fasted without electrolyte replacement for 3 days and hormone levels were measured before, during and after the fast. Immediate consequences of the fasting state in healthy human subjects include a marked increase in plasma cortisol, ACTH, β-endorphin, β-lipotrophic hormone, adrenaline, noradrenaline and dopamine. Levels of all these hormones were much greater on the first morning of the fast than in the post-prandial state, even though the plasma glucose level was no lower than that observed on the morning before the fast began. A clear fall in TSH and tri-iodothyronine (T₃) levels was observed, but thyroxine levels did not change significantly. Insulin levels fell whereas proinsulin levels did not fall during the fast, though they did rise markedly upon re-feeding. An increase in GH levels was particularly apparent in male subjects, but was also seen in females when evening samples were compared. Pancreatic glucagon showed a modest rise during the fast, but fell again on refeeding; total glucagon also rose as the fast proceeded, but increased markedly upon re-feeding. Levels of gastrin and peptide YY remained low during the fast. Plasma electrolyte levels were unchanged. The following were closely correlated: cortisol with ACTH, T₃ with log₁₀TSH, dopamine with noradrenaline, and (negatively, during the fast) pancreatic glucagon with glucose.

Journal of Endocrinology (1989) 120, 337–350

Type 2 diabetes is characterized by reduced insulin secretion from the pancreas and overproduction of glucose by the liver. Glucagon-like peptide-1 (GLP-1) promotes glucose-dependent insulin secretion from the pancreas, while glucagon promotes glucose output from the liver. Taking advantage of the homology between GLP-1 and glucagon, a GLP-1/glucagon hybrid peptide, dual-acting peptide for diabetes (DAPD), was identified with combined GLP-1 receptor agonist and glucagon receptor antagonist activity. To overcome its short plasma half-life DAPD was PEGylated, resulting in dramatically prolonged activity in vivo. PEGylated DAPD (PEG-DAPD) increases insulin and decreases glucose in a glucose tolerance test, evidence of GLP-1 receptor agonism. It also reduces blood glucose following a glucagon challenge and elevates fasting glucagon levels in mice, evidence of glucagon receptor antagonism. The PEG-DAPD effects on glucose tolerance are also observed in the presence of the GLP-1 antagonist peptide, exendin(9–39). An antidiabetic effect of PEG-DAPD is observed in db/db mice. Furthermore, PEGylation of DAPD eliminates the inhibition of gastrointestinal motility observed with GLP-1 and its analogues. Thus, PEG-DAPD has the potential to be developed as a novel dual-acting peptide to treat type 2 diabetes, with prolonged in vivo activity, and without the GI side-effects.

Type 2 diabetes (T2DM) is associated with pancreatic islet dysfunction. Loss of β-cell identity has been implicated via dedifferentiation or conversion to other pancreatic endocrine cell types. How these transitions contribute to the onset and progression of T2DM in vivo is unknown. The aims of this study were to determine the degree of epithelial-to-mesenchymal transition occurring in α and β cells in vivo and to relate this to diabetes-associated (patho)physiological conditions. The proportion of islet cells expressing the mesenchymal marker vimentin was determined by immunohistochemistry and quantitative morphometry in specimens of pancreas from human donors with T2DM (n = 28) and without diabetes (ND, n = 38) and in non-human primates at different stages of the diabetic syndrome: normoglycaemic (ND, n = 4), obese, hyperinsulinaemic (HI, n = 4) and hyperglycaemic (DM, n = 8). Vimentin co-localised more frequently with glucagon (α-cells) than with insulin (β-cells) in the human ND group (1.43% total α-cells, 0.98% total β-cells, median; P < 0.05); these proportions were higher in T2DM than ND (median 4.53% α-, 2.53% β-cells; P < 0.05). Vimentin-positive β-cells were not apoptotic, had reduced expression of Nkx6.1 and Pdx1, and were not associated with islet amyloidosis or with bihormonal expression (insulin + glucagon). In non-human primates, vimentin-positive β-cell proportion was larger in the diabetic than the ND group (6.85 vs 0.50%, medians respectively, P < 0.05), but was similar in ND and HI groups. In conclusion, islet cell expression of vimentin indicates a degree of plasticity and dedifferentiation with potential loss of cellular identity in diabetes. This could contribute to α- and β-cell dysfunction in T2DM.