Maxine K. Turney

Rictor/mTORC2 facilitates central regulation of energy and glucose homeostasis

Insulin signaling in the central nervous system (CNS) regulates energy balance and peripheral glucose homeostasis. Rictor is a key regulatory/structural subunit of the mTORC2 complex and is required for hydrophobic motif site phosphorylation of Akt at serine 473. To examine the contribution of neuronal Rictor/mTORC2 signaling to CNS regulation of energy and glucose homeostasis, we utilized Cre-LoxP technology to generate mice lacking Rictor in all neurons, or in either POMC or AgRP expressing neurons. Rictor deletion in all neurons led to increased fat mass and adiposity, glucose intolerance and behavioral leptin resistance. Disrupting Rictor in POMC neurons also caused obesity and hyperphagia, fasting hyperglycemia and pronounced glucose intolerance. AgRP neuron specific deletion did not impact energy balance but led to mild glucose intolerance. Collectively, we show that Rictor/mTORC2 signaling, especially in POMC-expressing neurons, is important for central regulation of energy and glucose homeostasis.

Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPARδ) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

Central nervous system (CNS) lipid accumulation, inflammation and resistance to adipo-regulatory hormones, such as insulin and leptin, are implicated in the pathogenesis of diet-induced obesity (DIO). Peroxisome proliferator-activated receptors (PPAR α, δ, γ) are nuclear transcription factors that act as environmental fatty acid sensors and regulate genes involved in lipid metabolism and inflammation in response to dietary and endogenous fatty acid ligands. All three PPAR isoforms are expressed in the CNS at different levels. Recent evidence suggests that activation of CNS PPARα and/or PPARγ may contribute to weight gain and obesity. PPARδ is the most abundant isoform in the CNS and is enriched in the hypothalamus, a region of the brain involved in energy homeostasis regulation. Because in peripheral tissues, expression of PPARδ increases lipid oxidative genes and opposes inflammation, we hypothesized that CNS PPARδ protects against the development of DIO. Indeed, genetic neuronal deletion using Nes-Cre loxP technology led to elevated fat mass and decreased lean mass on low-fat diet (LFD), accompanied by leptin resistance and hypothalamic inflammation. Impaired regulation of neuropeptide expression, as well as uncoupling protein 2, and abnormal responses to a metabolic challenge, such as fasting, also occur in the absence of neuronal PPARδ. Consistent with our hypothesis, KO mice gain significantly more fat mass on a high-fat diet (HFD), yet are surprisingly resistant to diet-induced elevations in CNS inflammation and lipid accumulation. We detected evidence of upregulation of PPARγ and target genes of both PPARα and PPARγ, as well as genes of fatty acid oxidation. Thus, our data reveal a previously underappreciated role for neuronal PPARδ in the regulation of body composition, feeding responses, and in the regulation of hypothalamic gene expression.

An ACTH-producing small cell lung cancer expresses aberrant glucocorticoid receptor transcripts from a normal gene

ACTH production by non-pituitary tumors is generally not suppressible by exogenous glucocorticoid administration. We had postulated that defects in the glucocorticoid receptor (GR) signaling system might be responsible for this apparent glucocorticoid resistance and had previously demonstrated that DMS-79 cells, derived from an ectopic ACTH-producing tumor, express an abnormal GR mRNA. In this DMS-79 cell GR the sequence normally derived from exons 8 and 9 is replaced by sequence unmatched in the DNA databases. The protein encoded by this mRNA lacks the steroid-binding domain and does not function as a ligand-activated transcription factor. In the present work, we sought to identify the origin of the novel GR mRNA sequence. Southern blot analysis of DMS-79 genomic DNA showed no major structural alteration of the GR gene. Southern blotting of cosmid clones of the normal GR gene revealed that the novel DMS-79 GR mRNA sequence is derived from intron G, between exons 7 and 8. No splice site mutations were found in PCR-amplified DMS-79 DNA fragments surrounding the downstream splice junctions. Further sequencing indicated that the aberrant GR transcript appears to be generated by use of a consensus cleavage/polyadenylation signal found 3650 base pairs into the normal intron G. We conclude that abnormal GR pre-mRNA processing rather than a GR gene mutation confers glucocorticoid resistance on DMS-79 cells.

High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17 beta-bromoacetate.

Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptors ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17 beta-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80% based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17 beta-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.

Expression of 11β-hydroxysteroid dehydrogenase type 2 in an ACTH-producing small cell lung cancer

Abstract Non-pituitary tumors that produce adrenocorticotropic hormone (ACTH) exhibit resistance to the normal feedback effects of glucocorticoids on proopiomelanocortin (POMC) gene expression. This glucocorticoid resistance is typically complete, although some tumors show only relative glucocorticoid resistance in the clinical setting. The molecular mechanisms responsible for these clinical pathophysiologic observations are unknown, but might include glucocorticoid receptor defects or aberrant expression of enzymes or transporters that exclude glucocorticoids from access to their intracellular receptors. We examined whether ACTH-producing non-pituitary tumor cells might express 11β-hydroxysteroid dehydrogenase (11β-HSD), the principal ‘gatekeeper’ enzyme known to metabolize glucocorticoids. 11β-HSD mRNA and enzyme activity were assessed in DMS-79 cells, a line derived from an ACTH-producing small cell lung cancer. RT-PCR studies showed expression of mRNA encoding 11β-HSD2 but not 11β-HSD1 in DMS-79 cells. Control human fibroblasts expressed predominantly 11β-HSD1 but also had detectable 11β-HSD2 mRNA, while HepG2 hepatoma cells also expressed only 11β-HSD2 mRNA. Whole cell assays in DMS-79 cells revealed 11β-HSD activity with a K m for cortisol of 26.1 ± 9.0 nM and V max of 57.0 ± 5.9 pmol/h/mg protein. HepG2 cells expressed a similar high affinity enzyme activity, while control fibroblasts expressed 11β-HSD activity with a K m for cortisol of 652 nM. Conversion of cortisol to cortisone in DMS-79 cells was inhibited to 7% of baseline by addition of 10 μM glycyrrhetinic acid. Dexamethasone (20 nM) was converted to a single product in DMS-79 cells at a rate of 17.2 pmol/h/mg protein; this activity was also inhibited by glycyrrhetinic acid. We conclude that DMS-79 cells express 11β-HSD2. While DMS-79 cells harbor additional defects in glucocorticoid signaling, these data suggest that expression of 11β-HSD2 might contribute to the development of the glucocorticoid-resistant phenotype of some ACTH-producing tumors.

Comparison of insulins detemir and glargine: effects on glucose disposal, hepatic glucose release and the central nervous system

Aims: The effects of insulins detemir (Det) and glargine (Glar) on endogenous glucose production (EGP) and net hepatic glucose output (NHGO) were compared.

A Duplexed High-Throughput Screen to Identify Allosteric Modulators of the Glucagon-Like Peptide 1 and Glucagon Receptors:

Injectable, degradation-resistant peptide agonists for the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R), such as exenatide and liraglutide, activate the GLP-1R via a complex orthosteric-binding site and are effective therapeutics for glycemic control in type 2 diabetes. Orally bioavailable orthosteric small-molecule agonists are unlikely to be developed, whereas positive allosteric modulators (PAMs) may offer an improved therapeutic profile. We hypothesize that allosteric modulators of the GLP-1R would increase the potency and efficacy of native GLP-1 in a spatial and temporally preserved manner and/or may improve efficacy or side effects of injectable analogs. We report the design, optimization, and initial results of a duplexed high-throughput screen in which cell lines overexpressing either the GLP-1R or the glucagon receptor were coplated, loaded with a calcium-sensitive dye, and probed in a three-phase assay to identify agonists, antagonists, and potentiators of GLP-1, and potentiators of glucagon. 175,000 compounds were initially screened, and progression through secondary assays yielded 98 compounds with a variety of activities at the GLP-1R. Here, we describe five compounds possessing different patterns of modulation of the GLP-1R. These data uncover PAMs that may offer a drug-development pathway to enhancing in vivo efficacy of both endogenous GLP-1 and peptide analogs.

An androgenic affinity ligand covalently binds to cytosolic aldehyde dehydrogenase from human genital skin fibroblasts.

A 56 kDa protein expressed in human genital skin fibroblasts was first identified by independent laboratories on the basis of its specific expression in androgen target cells and its ability to covalently bind androgenic affinity ligands. Recently, immunoscreening of a cDNA library with antisera directed against this protein resulted in the isolation of a partial cDNA clone identical to human cytosolic aldehyde dehydrogenase (ALDH1). We report here the preparation of a full-length cDNA encoding ALDH1 from human genital fibroblasts. Translation of the encoded protein in a cell-free system yields a 56 kDa product that can be covalently radiolabeled with [3H]dihydrotestosterone 17 beta-bromoacetate (DHT-BA). Expression of the full-length clone in mammalian cells also results in expression of a 56 kDa DHT-BA binding protein. The covalent binding of DHT-BA by ALDH1 is an intrinsic property of the enzyme and is not dependent on androgen receptor expression.

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17β-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.

Gene expression phenotyping of an ACTH-producing small cell lung cancer line

DNA microarray techniques were used to compare gene expression in an adrenocorticotropin (ACTH)-producing human small cell lung carcinoma line (DMS-79) with six other small cell lung cancer (SCLC) lines that do not produce ACTH. Twelve genes were expressed at more than five-fold higher levels in DMS-79 cells. Two transcription factors were the genes that exhibited the most remarkable over-expression: T-box 3 mRNA was detected at levels 19.37 +/- 3.78 times those observed in the SCLCs. Thyroid transcription factor (TTF-1, T/ebp, Nkx2.1) was expressed at 14.24 +/- 3.41-fold higher in DMS-79 cells. Seven genes were identified whose expression levels were at least five-fold lower in the ACTH-producing cell line. Variation in culture medium formulation did not significantly affect the gene expression profile of DMS-79 cells and expression data observed in microarray experiments were corroborated by northern blot analysis of RNA from the same cell lines. These experiments reveal new candidate genes that could be involved in the dysregulation of POMC gene expression manifested by ACTH-producing nonpituitary tumors.