Ute Hochgeschwender

Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin.

Pro-opiomelanocortin (POMC)-derived peptides (the melanocortins adrenocorticotropin, α-, β- and γ-melanocyte stimulating hormone; and the endogenous opioid β-endorphin) have a diverse array of biological activities, including roles in pigmentation, adrenocortical function and regulation of energy stores, and in the immune system and the central and peripheral nervous systems. We show here that mice lacking the POMC-derived peptides have obesity, defective adrenal development and altered pigmentation. This phenotype is similar to that of the recently identified human POMC-deficient patients. When treated with a stable α-melanocyte-stimulating hormone agonist, mutant mice lost more than 40% of their excess weight after 2 weeks. Our results identify the POMC-null mutant mouse as a model for studying the human POMC-null syndrome, and indicate the therapeutic use of peripheral melanocortin in the treatment of obesity.

Integrated control of appetite and fat metabolism by the leptin-proopiomelanocortin pathway.

Leptin deficiency results in a complex obesity phenotype comprising both hyperphagia and lowered metabolism. The hyperphagia results, at least in part, from the absence of induction by leptin of melanocyte stimulating hormone (MSH) secretion in the hypothalamus; the MSH normally then binds to melanocortin-4 receptor expressing neurons and inhibits food intake. The basis for the reduced metabolic rate has been unknown. Here we show that leptin administered to leptin-deficient (ob/ob) mice results in a large increase in peripheral MSH levels; further, peripheral administration of an MSH analogue results in a reversal of their abnormally low metabolic rate, in an acceleration of weight loss during a fast, in partial restoration of thermoregulation in a cold challenge, and in inducing serum free fatty acid levels. These results support an important peripheral role for MSH in the integration of metabolism with appetite in response to perceived fat stores indicated by leptin levels.

Somatostatin Receptor Subtype 4 Couples to the M-Current to Regulate Seizures

The K+ M-current (I M, Kv7) is an important regulator of cortical excitability, and mutations in these channels cause a seizure disorder in humans. The neuropeptide somatostatin (SST), which has antiepileptic properties, augments I M in hippocampal CA1 pyramidal neurons. We used SST receptor knock-out mice and subtype-selective ligands to investigate the receptor subtype that couples to I M and mediates the antiepileptic effects of SST. Using pentylenetetrazole as a chemoconvulsant, SST2 , SST3 , and SST4 receptor knock-out mice all had shorter latencies to different seizure stages and increased seizure severity when compared with wild-type mice. However, the most robust differences were observed in the SST4 knock-outs. When seizures were induced by systemic injection of kainate, only SST4 knock-outs showed an increase in seizure sensitivity. We next examined the action of SST and subtype-selective SST agonists on electrophysiological parameters in hippocampal slices of wild-type and receptor knock-out mice. SST2 and SST4 appear to mediate the majority of SST inhibition of epileptiform activity in CA1. SST lacked presynaptic effects in mouse CA1, in contrast to our previous findings in rat. SST increased I M in CA1 pyramidal neurons of wild-type and SST2 knock-out mice, but not SST4 knock-out mice. Using M-channel blockers, we found that SST4 coupling to M-channels is critical to its inhibition of epileptiform activity. This is the first demonstration of an endogenous enhancer of I M that is important in controlling seizure activity. SST4 receptors could therefore be an important novel target for developing new antiepileptic and antiepileptogenic drugs.

Characterisation of [125I]-TyroDTrp8-somatostatin binding in sst1- to sst4- and SRIF-gene-invalidated mouse brain

Five somatostatin receptors (sst) have been cloned and mRNAs for the first four (sst1–4) are expressed in many brain regions. In the present work, we compared the distribution of the non-selective ligand [125I]-Tyr0-DTrp8-SRIF14 by autoradiography in 24 brain regions and pituitary in wild type, sst1- to sst4- or SRIF-gene invalidated (KO) mice. [125I]-Tyr0-DTrp8-SRIF14 binding was not significantly modified in sst1 KO mouse brain with the noticeable exception of the substantia nigra and only moderately decreased in pituitary. For sst2 KO mice, a general decrease (>75%) was observed in most regions, with the noticeable exception of the olfactory bulb and CA1 field of the hippocampus. SST3 KO brain displayed a decrease in binding in the external plexiform layer of the olfactory bulb only (−54%). For sst4 KO mice, [125I]-Tyr0-DTrp8-SRIF14 binding levels in the external plexiform (−35%) and glomerular (−39%) layers of the olfactory bulb as well as the hippocampus CA1 field (−68%) were significantly decreased. In SRIF KO mice, a significant increase in binding levels was observed in olfactory bulb, anterior olfactory nucleus, frontal cortex upper layers, lateral septum, CA1 field, zona incerta and lateral hypothalamus, substantia nigra, periaqueductal grey and parabrachial nucleus. Competition with selective ligands (CH275, octreotide or L-779,976, L-796,778, L-803,087, and octreotide or L-817,778, for sst1–5 receptors, respectively) was in accordance with these findings. Moreover, octreotide was still able to compete on residual [125I]-Tyr0-DTrp8-SRIF14 binding sites in sst2 KO pituitary. It is concluded that most [125I]-Tyr0-DTrp8-SRIF14 binding sites in mouse brain and pituitary belong to the sst2 subtype but for the olfactory bulb (sst3 and sst4 receptors), the CA1 of the hippocampus (sst4 receptors) and the pituitary (sst5 and sst1 receptors) in which other subtypes are also expressed. The overall increase in [125I]-Tyr0-DTrp8-SRIF14 binding in SRIF KO mice indicates that SRIF receptors, mostly from the sst2 subtype, are regulated by the endogenous ligand(s).

Null mutant mouse models of somatostatin and cortistatin, and their receptors

Somatostatin (somatotropin release inhibitory factor, SRIF) and the related cortistatin (CST) are multifunctional peptide molecules attributed with neurohormone, neurotransmitter/modulator, and autocrine/paracrine actions. The physiological responses of SRIF and CST are mediated by five widely distributed G protein-coupled receptors (sst1-5) which have been implicated in regulating numerous biological processes. Much of the information on the effects of somatostatin has been gained through pharmacological studies with analogs and antagonists. The possibility of targeted mutagenesis in the mouse has resulted, over the last 10 years, in the generation of mouse models which genetically lack somatostatin ligands or receptors. We will review here the mouse models generated, the studies undertaken with them, and what has been learned so far.

Failure of adrenal corticosterone production in POMC-deficient mice results from lack of integrated effects of POMC peptides on multiple factors

Production of corticosteroids from the adrenal gland is a multistep process in which corticosterone is enzymatically processed from its precursor cholesterol. The main hormone regulating the production of corticosterone is the proopiomelanocortin (POMC)-derived adrenocorticotropic hormone (ACTH). Adrenals of POMC-deficient (POMC(-/-)) mice do not produce corticosterone either at basal levels or in response to acute stimulation with ACTH. However, pharmacological amounts of ACTH delivered continuously elicit corticosterone production over time. To define the relative effects of ACTH on individual factors involved in corticosterone production, parameters of adrenal cholesterol metabolism and steroidogenesis were examined in POMC(-/-) mice compared with wild-type and ACTH-treated mutant mice. POMC(-/-) adrenals lack cholesterol esters (CE); adrenal CE is restored with ACTH treatment. However, discontinuation of ACTH treatment stops corticosterone production despite the presence of adrenal CE. Failure of corticosterone production by POMC(-/-) adrenals occurs despite the constitutive presence of transcripts of genes required for cholesterol metabolism and steroidogenesis. Levels of key proteins involved in selective cholesterol uptake and steroidogenesis were attenuated; ACTH treatment increased these protein levels, most significantly those of the receptor responsible for selective uptake of CE, scavenger receptor class B, type I (SR-BI). Our studies reveal that failure of corticosterone production of POMC(-/-) adrenal glands and its pharmacological reconstitution by ACTH are not mediated by any one individual protein, but rather as an integrated effect on multiple factors from import of the substrate cholesterol to its conversion to corticosterone.

Pro-opiomelanocortin peptides and the adrenal gland

The adrenal gland regulates a number of essential biological functions through production of steroids and catecholamines. Pro-opiomelanocortin (POMC)-derived peptides have been implicated in all aspects of generating, maintaining, and functioning of the adrenal glands. An appreciation for the roles of POMC-derived peptides with respect to the adrenal has been gained from experiments in vitro, and in vivo in different animal models which surgically, pharmacologically, or genetically decrease or increase the amount of POMC peptides available. We recently produced a mouse model with a deletion of the entire coding region of the POMC gene, thus lacking all POMC-derived peptides, from all sources, and at all times. Here we will summarize and discuss the results of traditional in vivo studies on the role of POMC peptides in adrenal development, maintenance, and function in the context of findings in a mouse model genetically lacking all POMC-derived peptides.

The Role of Melanocyte- Stimulating Hormone in Insulin Resistance and Type 2 Diabetes Mellitus

In humans, mice, and other mammals, the melanocortin system consists of four peptide hormones with a core amino acid sequence of histidine-phenylalanine-arginine-tryptophan and five melanocortin receptors. Both the melanocortin hormones and their receptors are produced in diverse tissues throughout the body. The ligand of primary interest for treatment of insulin resistance is α-melanocyte-stimulating hormone (α-MSH), which is derived, as are all melanocortins, from tissue-specific post-translational proteolytic processing of the proopiomelanocortin (POMC) precursor protein. Recent results have shown that α-MSH is the complement of leptin in the endocrine circuit, regulating bodyweight, food intake, and metabolic rate. α-MSH can decrease bodyweight, weight gain, and food intake in mice with diet-induced and genetic obesity. As obesity is a major risk factor for type 2 diabetes mellitus, it was reasonable to investigate the endocrine agents involved in obesity for their involvement in diabetes. α-MSH analogs have also been shown to affect blood glucose levels in some mouse models of obesity. For instance, the POMC null mouse is extremely sensitive to insulin in an insulin tolerance test, while being otherwise euglycemic. The results from rodent studies with α-MSH suggest reciprocal effects: α-MSH appears to increase sensitivity to insulin when present in the CNS, while α-MSH in the periphery is necessary for insulin resistance. Should these trends be validated in humans, α-MSH-based therapeutics specifically active in the CNS or peripheral circulation may be promising for the treatment of type 2 diabetes.

Isolation and characterization of the mouse homolog of the preprodynorphin (Pdyn) gene

We have isolated and sequenced the mouse preprodynorphin gene (Pdyn). The Pdyn gene can encode for six biologically active dynorphin peptides. The predicted mouse preprodynorphin has 90%, 67%, and 66% identity with the predicted rat, porcine, and human preprodynorphins, respectively. Using an RT-PCR technique, we show that the Pdyn gene starts being expressed at embryonic day 12.5, with a steep increase of expression by embryonic day 14.5; in the adult mouse it is expressed in the brain, but not in liver, heart, spleen, or kidney.

Plasma α–melanocyte-stimulating hormone: sex differences and correlations with obesity

Rodent experiments raise the possibility of a regulatory role of peripheral alpha-melanocyte-stimulating hormone (alpha-MSH) in obesity and metabolism, but human data on peripheral alpha-MSH levels remain fragmentary. Because of the possible relationship between alpha-MSH and obesity, we endeavored to test the hypothesis that higher levels of alpha-MSH in obese patients would correlate with leptin levels and with other markers of obesity. Sixty normal-weight to obese healthy men and women participated. Weight, measures of body composition, and diet diaries were obtained; fasting blood was analyzed for alpha-MSH, lipids, glucose, insulin, leptin, and adiponectin. To begin to understand the source of peripherally measured hormones, alpha-MSH was also measured in serum samples from 5 individuals with untreated Addison disease. Levels of alpha-MSH were higher in men vs women (10.1 +/- 4.3 vs 7.6 +/- 3.4 pmol/L, P = .019), and alpha-MSH levels were higher in patients with Addison disease vs controls (17.7 +/- 2.3 vs 8.7 +/- 0.52 pmol/L, P < .001). Measures of adiposity correlated with insulin and leptin in men and women, and with adiponectin in women. alpha-Melanocyte-stimulating hormone levels did not correlate significantly with any parameter of adiposity or diet composition. The elevated alpha-MSH levels in patients with untreated Addison disease suggest possible pituitary secretion of alpha-MSH to the periphery. The lack of correlation between peripheral alpha-MSH and parameters of adiposity suggests that endogenous plasma alpha-MSH levels are not a metric for body composition per se.