Cardiac effects of moxonidine in spontaneously hypertensive obese rats.

Moxonidine, an imidazoline receptor agonist that acts centrally to inhibit sympathetic activity, has been shown to reduce effectively blood pressure, fasting insulin levels, and free fatty acids. In this study, we investigated the long-term effects of moxonidine treatment on cardiac natriuretic peptides (ANP and BNP) in Spontaneously Hypertensive Obese Rats (SHROBs), a rat model that resembles human Syndrome X. SHROBs expressing spontaneous hypertension, insulin resistance, and genetic obesity (weight 590 +/- 20 g, at 30 weeks) received moxonidine in chow at 4 mg/kg/day for 15 days. Moxonidine significantly reduced not only systolic blood pressure (187 +/- 6 versus 156 +/- 5 mm Hg, P < 0.05) but also plasma ANP (1595 +/- 371 versus 793 +/- 131 pg/mL, P < 0.05) and BNP (22 +/- 3 versus 14 +/- 1 pg/mL, P < 0.04), without influencing cardiac content of either peptide. Semi-quantitative PCR revealed that atrial ANPmRNA/GAPDHmRNA decreased to 39% 6 10% of pair-fed controls, P < 0.03. In left ventricles, moxonidine also decreased ANP mRNA to 69% +/- 7% and BNP mRNA to 74% +/- 6% of control, P < 0.02, but right ventricular ANP and BNP mRNA were not affected. These findings indicate that chronic inhibition of sympathetic activity with moxonidine in SHROB is associated with decreased ventricular natriuretic peptide transcription, consistent with the cardioprotective effects of moxonidine given the role of ANP and BNP as markers of cadiac disease. Moxonidine also improves the metabolic profile in these rats, thus it may be considered the drug of choice in treatment of metabolic syndrome X.

IRAS splice variants.

The human I(1)-imidazoline receptor candidate gene, iras, has previously been cloned and mapped to locus 3p21.1-9 (also known as Nischarin; accession No. AC006208). By comparison to a database of expressed sequence tags (ESTs), three alternatively spliced transcripts have been deduced. A map of 21 exons was constructed for the medium-length transcript (IRAS-M) containing 5,232 base pairs (bp) and encoding 1,504 amino acids (aas). Introns 13B and 13C are inserted into the two alternative transcripts, forming IRAS-S and IRAS-L mRNA (short and long isoforms). Northern blots confirmed the existence of these mRNA isoforms. In most brain regions the order of mRNA abundance was IRAS-M > IRAS-L > IRAS-S mRNA. Although aas 1 through 510 are theoretically identical, truncated proteins could be derived from IRAS-S (2,678 bp transcript yields 515 aas) and IRAS-L (9,457 bp transcript yields 583 aas). Because exon-16 of the iras gene has been proposed to encode the functional domains of imidazoline and a-5 integrin binding, only IRAS-M is expected to possess I(1) receptor properties. Subtype-selective cDNA expression constructs were therefore generated and used to transfect CHO cells. High-affinity I(1) binding was endowed by IRAS-M and IRAS-L, but not by IRAS-S transfection.

The I1-imidazoline receptor in PC12 pheochromocytoma cells activates protein kinases C, extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK).

We sought to further elucidate signal transduction pathways for the I1-imidazoline receptor in PC12 cells by testing involvement of protein kinase C (PKC) isoforms (betaII, epsilon, zeta), and the mitogen-activated protein kinases (MAPK) ERK and JNK. Stimulation of I1-imidazoline receptor with moxonidine increased enzymatic activity of the classical betaII isoform in membranes by about 75% and redistributed the atypical isoform into membranes (40% increase in membrane-bound activity), but the novel isoform of PKC was unaffected. Moxonidine and clonidine also increased by greater than two-fold the proportion of ERK-1 and ERK-2 in the phosphorylated active form. In addition, JNK enzymatic activity was increased by exposure to moxonidine. Activation of ERK and JNK followed similar time courses with peaks at 90 min. The action of moxonidine on ERK activation was blocked by the I1-receptor antagonist efaroxan and by D609, an inhibitor of phosphatidylcholine-selective phospholipase C (PC-PLC), previously implicated as the initial event in I1-receptor signaling. Inhibition or depletion of PKC blocked activation of ERK by moxonidine. Two-day treatment of PC12 cells with the I1/alpha2-agonist clonidine increased cell number by up to 50% in a dose related manner. These data suggest that ERK and JNK, along with PKC, are signaling components of the I1-receptor pathway, and that this receptor may play a role in cell growth.

The in vitro pharmacology of the beta-adrenergic receptor pet ligand (s)-fluorocarazolol reveals high affinity for cloned beta-adrenergic receptors and moderate affinity for the human 5-HT1A receptor.

RATIONALE: s-Fluorocarazolol [(S)-FCZ] is the major positron emission tomography (PET) ligand currently used to visualize central beta-adrenergic receptors in vivo, although its pharmacology is incompletely known. OBJECTIVE: Our objective was to comprehensively characterize the in vitro pharmacology of (S)- and (R)-FCZ to determine its suitability for study of central and peripheral beta-adrenergic receptors. METHODS: We characterized the in vitro pharmacology of (S)-FCZ at 42 biogenic amine receptors and transporters in vitro using the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. RESULTS: As expected (R)- and (S)-FCZ had high affinities for beta-adrenergic receptors (Ki values=0.08-0.45 nM) and negligible affinities (Ki values>100 nM) for nearly all other tested receptors and transporters with the exception of the h5-HT1A receptor for which (S)-FCZ had high affinity (Ki=34 nM). Interestingly, (R)-FCZ had low affinity for the h5-HT1A receptor (Ki=342 nM). CONCLUSION: The high affinity of (S)-FCZ for the h5-HT1A receptor is not likely to interfere with studies of peripheral beta-adrenergic receptors, since 5-HT1A receptors are expressed at very low levels outside the central nervous system. Indeed, computer simulations predict that even at low ligand concentrations, 5-HT1A binding in brain regions like hippocampus are likely to be substantial. Thus, (S)-FCZ may not be a suitable PET ligand for studies of central nervous system beta-adrenergic receptors unless the contribution by 5-HT1A sites can be shown to be negligible.

Differential cardiorespiratory control elicited by activation of ventral medullary sites in mice.

We studied the respiratory and blood pressure responses to chemical stimulation of two regions of the ventral brainstem in mice: the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). Stimulation of the RVLM by microinjections of the excitatory amino acid L-glutamate induced increases in diaphragm activity and breathing frequency, elevation of blood pressure (BP), and a slight increase in heart rate (HR). However, activation of the CVLM induced a decrease in breathing frequency, mainly due to prolongation of expiratory time (TE), and hypotension associated with a slight slowing of HR. Because adrenergic mechanisms are known to participate in the control of respiratory timing, we examined the role of alpha(2)-adrenergic receptors in the RVLM region in mediating these inhibitory effects. The findings demonstrated that blockade of the alpha(2)-adrenergic receptors within the RVLM by prior microinjection of SKF-86466 (an alpha(2)-adrenergic receptor blocker) significantly reduced changes in TE induced by CVLM stimulation but had little effect on BP responses. These results indicate that, in mice, activation of the RVLM increases respiratory drive associated with an elevation of BP, but stimulation of CVLM induces prolongation of TE via an alpha(2)-adrenergic signal transduction pathway.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

alpha(2)-adrenergic receptors are not required for central anti-hypertensive action of moxonidine in mice.

In the mouse medulla oblongata, we characterized binding properties and functional responses of two recognition sites for imidazoline compounds: I(1)-imidazoline and alpha(2)-adrenergic receptors. The mouse medulla expresses a higher density of I(1)-receptors than in the rat, whereas alpha(2)-receptor densities were similar between the two species. In anesthetized, ventilated and paralyzed mice, we tested the hypotensive actions of the I(1)/alpha(2) agonist moxonidine, determined its central site of its actions, and the relative roles of I(1) and alpha(2)-receptors. Experiments were performed in C(57)Bl(6) wild type and alpha(2A)-adrenergic receptor deficient mice. In both types of mice, neuronal activation within the rostral ventrolateral medulla (RVLM) region by glutamate microinjection elicited increases in arterial pressure. Moxonidine (0.5 nmol/site/10 nl) microinjected bilaterally into this vasopressor region decreased arterial pressure by 30% and heart rate by 11% in wild type mice. Efaroxan, the I(1)/alpha(2) antagonist (0.4 nmol) when microinjected into the RVLM elevated blood pressure itself and abolished the action of moxonidine, whereas alpha(2)-blockade with SK&F 86466 had no significant effect on blood pressure and did not attenuate moxonidine's effect. To more definitively test the role of alpha(2)-adrenergic receptors in the action of moxonidine, moxonidine was microinjected into the RVLM of alpha(2A)-adrenergic deficient mice. The decreases in arterial pressure were nearly identical to those of wild type mice, whereas bradycardia was attenuated. Thus, in the mouse moxonidine acts within the RVLM region to lower arterial pressure mainly through the I(1)-imidazoline receptor independent of alpha(2)-adrenergic receptors.

Alterations in respiratory behavior, brain neurochemistry and receptor density induced by pharmacologic suppression of sleep in the neonatal period.

UNLABELLED: The present study examined if drug suppression of active sleep (AS) in the neonate affected the development and expression of respiratory behavior. Secondly, we assessed brain neurochemistry and receptor density in specific supra-medullary brain regions to identify coincident biochemical alterations. Sprague-Dawley newborn rat pups were randomized and divided among six rat mothers (n=10/mother/group), each mother housed separately. Two untreated control (UC) groups received either no interventions or were fed milk vehicle twice daily and were handled similarly to the drug intervention animals. Pharmacological disruption of sleep was achieved by administration (2 groups of each) of either clonidine (CLO) 100 microm/kg, or scopolamine (SCO) 800 microm/kg, given orally twice daily for the first 7 days of life. On postnatal (P) days P10 and P19 of life, pups were assessed for metabolism, minute ventilation (VE), tidal volume (Vt) and frequency (f). On P21 (14 days after the end of drug exposure), pups from each condition were sacrificed and punch biopsies of the frontal cortex, hypothalamus, and hippocampus were examined for hydroxytryptophan (5-HT), and norepinepherine (NE) by HPLC. An equal number of pups were sacrificed and brains examined for muscarinic acetylcholine (mAch), alpha2-adrenergic and I1-imidazoline receptor density. RESULTS: Both CLO and SCO exposed animals had a lower V(t) and respiratory quotient than UC animals (p<0.01). CLO animals exhibited a higher f (p<0.01) and both CLO and SCO exhibited a lower V(t) (p<0.05) than the UC groups; VE was reduced in the SCO groups, compared with CLO and UC groups (p<0.01). Pattern of breathing in response to brief hypoxia exposure was altered for CLO and SCO. The normal decline in VE during sleep was not observed in CLO rats. Both drug exposures resulted in a comparable reduction in hypothalamic NE and 5-HT levels (p<0.05), while in the frontal cortex, and the hippocampus variable changes in NE and 5-HT, occurred. In CLO and SCO rats mAch receptors were increased in cortex, and reduced in hypothalamus; I1-imidazoline receptors were increased in hypothalamus and decreased in hippocampus (p<0.05 for each). In contrast, alpha2-adrenergic receptors were increased in cortex for both CLO and SCO, decreased in hypothalamus for CLO, and decreased in hippocampus for SCO (p<0.05 for each). CONCLUSIONS: these data show that drug-induced neonatal sleep suppression will alter ventilatory pattern, metabolism, and site-specific concentrations of adrenergic neurotransmitters and in receptor density, perhaps as a result of suppression of neonatal AS.

Pharmacology of moxonidine: an I1-imidazoline receptor agonist.

The I1-imidazoline receptor is a novel neurotransmitter receptor found mainly in the brainstem, adrenal medulla and kidney. The actions of moxonidine are described at the level of individual biomolecules, cells, tissues, organs and finally with integrative functions. The receptor functions at the cellular level works through arachidonic acid and phospholipid signaling cascades in neuronal cells with the net result of inhibiting sympathetic premotor neurons.

Ontogeny of neurokinin-1 receptors in the porcine respiratory system.

We characterized the ontogeny of NK-1 receptor agonist affinity (Kd) and density (Bmax) in membranes from tracheal epithelium, smooth muscle, and lung of pigs aged 1-7 days, 8-21 days, and adult in comparison to contractile responses in vitro. Affinity of [125I] Bolton-Hunter substance P ([125I]BH-SP) in epithelium and smooth muscle was three- to fourfold lower in young piglets than in adults. The Bmax of NK-1 sites in epithelium was elevated by more than twofold at 8-21 days relative to 1-7 days piglets and adults. In the lung, NK-1 density as well as affinity was lower than in trachea, regardless of age. In all three groups, [125I]BH-SP binding was potently inhibited by Gpp(NH)p, in both trachea and lung, implying coupling to G-proteins. Inhibition by Gpp(NH)p was most potent in the adult relative to younger animals, in both tracheal epithelium and smooth muscle. Functional sensitivity to the NK-1 agonists substance P and septide was reduced in neonates, as shown by the higher concentration of agonist required to elicit contractile responses. We conclude that the reduced sensitivity of newborn piglet airways to substance P reflects immaturity of G-protein coupling to NK-1, independent of receptor density.

Imidazoline receptor antisera-selected cDNA clone and mRNA distribution.

A novel cDNA, designated Imidazoline Receptor Antisera-Selected cDNA-1 (iras-1), encodes a 167-kD protein. Two of its predicted peptides (42-43 kD) are immunologically consistent with a previously reported 1(1)-imidazoline binding protein. In the present study, two forms of iras mRNA (6.0 and 9.5 kb) were quantified across fresh rat tissues. Highest levels were found in brain (almost exclusively 6.0 kb in size), followed by liver and lung (9.5 > or = 6.0 kb iras mRNA), kidney (6.0 > 9.5 kb), heart (6.0 kb), spleen (6.0 > or = 9.5 kb), testes (6.0 > 9.5 kb), and skeletal muscle (6.0 > 9.5 kb). A correlation exists (p = 0.71, p = 0.05) between total (6.0 + 9.5 kb) iras mRNA and I1 BMAX values across rat tissues, corrected for housekeeping gene expression. Thus, total iras mRNA appears to be roughly proportional to the density of I1-imidazoline binding sites.

The I1-imidazoline receptor and its cellular signaling pathways.

Two primary questions are addressed. First, do I1-imidazoline binding sites fulfill all the essential criteria for identification as a true receptor? Second, what are the cellular signaling pathways coupled to this novel receptor? I1-imidazoline binding sites show specificity in binding assays, linkage to physiologic functions, appropriate anatomic, and cellular and subcellular localization. Most important, binding affinities correlate with functional drug responses. I1-imidazoline binding sites meet several additional criteria identified with functional receptors: they show physiologic regulation and endogenous ligands and, most crucially, are coupled to cellular signaling events. A series of studies have identified cellular events triggered by I1-imidazoline receptor occupancy. This receptor is not coupled to conventional pathways downstream of heterotrimeric G-proteins, such as activation or inhibition of adenylyl or guanylyl cyclases, stimulation of inositol phospholipid hydrolysis, or induction of rapid calcium fluxes. The I1-imidazoline receptor is coupled to choline phospholipid hydrolysis, leading to the generation of diacylglyceride, arachidonic acid, and eicosanoids. Additional cellular responses include inhibition of Na+/H+ exchange and induction of genes for catecholamine synthetic enzymes. The signaling pathways linked to the I1-imidazoline receptor are similar to those of the interleukin family, implying that I1-receptors may belong to the family of neurocytokine receptors.

Moxonidine acting centrally inhibits airway reflex responses.

We examined the role of I1-imidazoline (I1-IR) receptors in control of airway function, by testing the effects of systemic administration of the I1-IR agonist moxonidine on reflex responses of tracheal smooth muscle (TSM) tone to either lung deflation or mechanical stimulation of intrapulmonary rapidly adapting receptors. Experiments were performed in either alpha-chloralose anesthetized or decorticate, paralyzed, and mechanically ventilated beagle dogs. Moxonidine (10-100 micrograms/kg) administered via three different routes (femoral vein, muscular branch of superior thyroid artery, and vertebral artery) attenuated TSM responses to stimulation of airway sensory nerve fibers by two different ways and caused a decrease in arterial pressure and heart rate. These effects were dose dependent and were significantly reversed by efaroxan (an I1-IR and alpha 2-adrenergic blocker) administered via the vertebral artery. Intravertebral efaroxan abolished the hemodynamic effects of moxonidine. Intravenous moxonidine (10-100 micrograms/kg) did not alter airway smooth muscle responses to electrical stimulation of the peripheral vagus nerve. In addition, in vitro moxonidine (1-100 micrograms/ml) had no effect on contractile responses to increasing doses of acetylcholine. These findings indicate that moxonidine may act at a central site to suppress reflex airway constriction, even when given into the systemic circulation. Given the presence of I1-IR sites and alpha 2-adrenergic receptors in brain regions participating in airway reflexes, these receptor classes may be involved in brainstem control of the cholinergic outflow to the airways.

Molecular pathology in the obese spontaneous hypertensive Koletsky rat: a model of syndrome X.

The SHROB rat is a unique strain with genetic obesity, hypertriglyceridemia, hyperinsulinemia, renal disease with proteinuria, and genetically determined hypertension, characteristics paralleling human Syndrome X. The obese phenotype results from a single homozygous recessive trait, designated faK, and is allelic with the Zucker fatty trait (fa), but of distinct origin. The faK mutation is a premature stop codon in the extracellular domain of the leptin receptor, resulting in a natural receptor knockout. The SHROB are glucose intolerant compared to heterozygous or wild-type SHR, but retain fasting euglycemia even on a high sucrose diet, suggesting that diabetes requires polygenic interaction with additional modifier genes. Insulin-stimulated phosphorylation of tyrosine residues on the insulin receptor and on the associated docking protein IRS-1 are reduced in skeletal muscle and liver compared to SHR, due mainly to diminished expression of insulin receptor and IRS-1 proteins. Despite multiple metabolic derangements and severe insulin resistance, hypertension is not exacerbated in SHROB compared to SHR. Thus, insulin resistance and hypertension are independent in this model. Increased activity of the sympathetic nervous system may be a common factor leading by separate pathways to hypertension and to insulin resistance. We studied the chronic effects of sympathetic inhibition with moxonidine on glucose metabolism in SHROB. Moxonidine (8 mg/kg/day), a selective I1-imidazoline receptor agonist, not only reduced blood pressure but also ameliorated glucose intolerance. Moxonidine reduced fasting insulin by 47% and plasma free fatty acids by 30%. Moxonidine enhanced expression and insulin-stimulated phosphorylation of IRS-1 in skeletal muscle by 74 and 27%, respectively. Thus, central sympatholytic therapy not only counters hypertension but also insulin resistance, glucose tolerance, and hyperlipidemia in the SHROB model of Syndrome X.

Mechanisms of antihyperglycemic effects of moxonidine in the obese spontaneously hypertensive Koletsky rat (SHROB).

Increased activity of the sympathetic nervous system may be a critical factor in the development of impaired insulin secretion and insulin resistance. We studied the chronic effects of sympathetic inhibition with moxonidine on glucose metabolism in the spontaneously hypertensive genetically obese rat (SHROB). This unique animal model closely resembles human syndrome X, expressing insulin resistance, genetic obesity, spontaneous hypertension, and hyperlipoproteinemia. Moxonidine, a selective imidazoline receptor agonist, was administered to lean spontaneous hypertensive rats (SHR) and SHROBs for 90 days in food at 8 mg/kg/day and significantly reduced mean blood pressure. Moxonidine treatment reduced fasting insulin levels by 71% in SHROB and lowered plasma free fatty acids by 25%. In SHR, moxonidine treatment decreased free fatty acids by 17% compared with controls. During an oral glucose tolerance test, blood glucose levels in moxonidine-treated SHROB were reduced relative to untreated controls from 60 min onwards. Insulin secretion was facilitated at 30 min (83% greater) and 60 min (67% greater) postchallenge compared with control SHROB. In skeletal muscle, moxonidine treatment increased the expression of the insulin receptor beta subunit by 19% in SHROB but was without effect in SHR. The level of insulin receptor substrate-1 (IRS-1) protein was decreased by 60% in control SHROB compared with lean SHR. Moxonidine treatment enhanced the expression and insulin-stimulated phosphorylation of IRS-1 protein in skeletal muscle in SHROB by 74 and 27%, respectively, and in SHR by 40 and 56%, respectively. Moxonidine increased the levels of expression of IRS-1 protein in liver in SHR by 275% and in SHROB by 260%. These findings indicate that chronic inhibition of sympathetic activity with moxonidine therapy can lower free fatty acids and significantly improve insulin secretion, glucose disposal, and expression of key insulin signaling intermediates in an animal model of obese hypertension.

Decreased transport of leptin across the blood-brain barrier in rats lacking the short form of the leptin receptor.

Leptin is produced in adipose tissue in the periphery, but its satiety effect is exerted in the CNS that it reaches by a saturable transport system across the blood-brain barrier (BBB). The short form of the leptin receptor has been hypothesized to be the transporter, with impaired transport of leptin being implicated in obesity. In Koletsky rats, the splice variant that gives rise to the short form of the leptin receptor contains a point mutation that results in marked obesity. We studied the transport of leptin across the BBB in Koletsky rats and found it to be significantly less than in their lean littermates. By contrast, Sprague-Dawley rats matched in weight to each of these two groups showed no difference in the blood-to-brain influx of leptin. HPLC showed that most of the leptin crossing the BBB in rats remained intact and capillary depletion showed that most of the leptin reached the parenchyma of the brain. The results indicate that the short form of the leptin receptor is involved in the transport of leptin across the BBB.

Arachidonic acid release from PC12 pheochromocytoma cells is regulated by I1-imidazoline receptors.

Rat PC 12 pheochromocytoma cells lack alpha2-adrenergic receptors but express plasma membrane I1-imidazoline receptors. In response to the I1-agonist moxonidine, diglycerides are generated via phosphatidylcholine-selective phospholipase C, and prostaglandin E2 is released. This report characterizes I-receptor-mediated release of arachidonic acid, the precursor to the prostaglandins. PC12 cells were incubated with [3H]arachidonic acid for 24 h and superfused with 0.01% bovine serum albumin in Krebs' physiological buffer at 1 ml/min. Calcium ionophore increased arachidonic acid release only marginally, implying that in PC12 cells arachidonic acid release is not driven by calcium. The I1-agonist moxonidine at concentrations between 10 nM and 1.0 microM rapidly elicited up to two-fold increases in [3H]arachidonic acid release. Guanabenz, a potent alpha2-agonist and I2-ligand, had no effect. The selective I1-antagonist efaroxan blocked the action of moxonidine. The phospholipase A2 inhibitor aristolochic acid had no effect, suggesting that arachidonic acid release may be through an indirect pathway, possibly involving diglycerides. Thus, I1-imidazoline receptors in PC12 cells are coupled to arachidonic acid release through an as yet unknown pathway.

Phenotypic consequences of a nonsense mutation in the leptin receptor gene (fak) in obese spontaneously hypertensive Koletsky rats (SHROB).

The genetically obese Koletsky rat (SHROB, fak) has a novel point mutation of the leptin receptor at amino acid +763, resulting in a premature stop codon in the leptin receptor extracellular domain. This implies that all leptin receptor isoforms should be absent in this model. We examined the phenotypic consequences of this mutation on leptin and leptin receptor mRNA in hypothalamus and peripheral tissues from SHROB and their lean SHR littermates. Despite the mutation, mRNA for both the long (ObRa) and the short (ObRb) form were expressed at comparable levels in SHROB and SHR in brain and throughout peripheral tissues. Adipose tissue mRNA for leptin was two to three times greater in SHROB compared to SHR (P < 0.01), while circulating leptin concentration was 170 times greater than SHR littermates (P < 0.01), suggesting extreme leptin resistance in SHROB. Leptin was also detected in the cerebrospinal fluid (CSF) of SHR and SHROB (13.8 and 27.2 pmol/L, respectively); however, the CSF/plasma ratio for leptin was 32-fold greater in SHR than in SHROB. To assess the putative action of leptin and leptin receptors on insulin-mediated glucose transport, muscles from SHR and SHROB were incubated in vitro with recombinant human leptin. Leptin directly suppressed insulin-mediated glucose transport by 50% in skeletal muscle from SHR but not in obese SHROB rats lacking all forms of the leptin receptor. These results suggest that the natural leptin receptor knockout in the SHROB represents a unique rat model to define the functional role(s) of leptin in central and peripheral energy metabolism.