The role of α1-adrenergic receptors in regulating metabolism: increased glucose tolerance,leptin secretion and lipid oxidation

The role of α₁-adrenergic receptors (α₁-ARs) and their subtypes in metabolism is not well known. Most previous studies were performed before the advent of transgenic mouse models and utilized transformed cell lines and poorly selective antagonists. We have now studied the metabolic regulation of the α_1A- and α_1B-AR subtypes in vivo using knock-out (KO) and transgenic mice that express a constitutively active mutant (CAM) form of the receptor, assessing subtype-selective functions. CAM mice increased glucose tolerance while KO mice display impaired glucose tolerance. CAM mice increased while KO decreased glucose uptake into white fat tissue and skeletal muscle with the CAM α_1A-AR showing selective glucose uptake into the heart. Using indirect calorimetry, both CAM mice demonstrated increased whole body fatty acid oxidation, while KO mice preferentially oxidized carbohydrate. CAM α_1A-AR mice displayed significantly decreased fasting plasma triglycerides and glucose levels while α_1A-AR KO displayed increased levels of triglycerides and glucose. Both CAM mice displayed increased plasma levels of leptin while KO mice decreased leptin levels. Most metabolic effects were more efficacious with the α_1A-AR subtype. Our results suggest that stimulation of α₁-ARs results in a favorable metabolic profile of increased glucose tolerance, cardiac glucose uptake, leptin secretion and increased whole body lipid metabolism that may contribute to its previously recognized cardioprotective and neuroprotective benefits.     

Chronic stress aggravates glucose intolerance in leptin receptor-deficient (db/db) mice

Genetic predisposition and environmental challenges interact to determine individual vulnerability to obesity and type 2 diabetes. We previously established a mouse model of chronic subordination stress-induced hyperphagia, obesity, metabolic like-syndrome and insulin resistance in the presence of a high-fat diet. However, it remains to be established if social stress could also aggravate glucose intolerance in subjects genetically predisposed to develop obesity and type 2 diabetes. To answer this question, we subjected genetically obese mice due to deficiency of the leptin receptor (db/db strain) to chronic subordination stress. Over five weeks, subordination stress in db/db mice led to persistent hyperphagia, hyperglycemia and exacerbated glucose intolerance altogether suggestive of an aggravated disorder when compared to controls. On the contrary, body weight and fat mass were similarly affected in stressed and control mice likely due to the hyperactivity shown by subordinate mice. Stressed db/db mice also showed increased plasma inflammatory markers. Altogether our results suggest that chronic stress can aggravate glucose intolerance but not obesity in genetically predisposed subjects on the basis of a disrupted leptin circuitry.     

Partial leptin receptor gene deletion in transgenic mice prevents expression of the membrane-bound isoforms except for Ob-Rc

Examination of random insertional mutations in transgenic animals harbouring an abnormal phenotype contributes to the discovery of new genes and/or the understanding of already known genes. Here we describe a transgenic mouse line showing early-onset obesity as consequence of the transgene insertion. Molecular genetic analysis revealed a partial deletion of the leptin receptor (Lepr, Ob-R) gene including the coding sequences downstream of exon 17'. This defect prevents the expression of all described membrane-bound isoforms of Ob-R except for isoform Ob-Rc in the homozygous transgenic animals. Thus, this mouse model might be useful for the investigation of the function of the short Ob-R isoforms.     

Norepinephrine Controls Both Torpor Initiation and Emergence via Distinct Mechanisms in the Mouse

Some mammals, including laboratory mice, enter torpor in response to food deprivation, and leptin can attenuate these bouts of torpor. We previously showed that dopamine β-hydroxylase knockout (Dbh −/−) mice, which lack norepinephrine (NE), do not reduce circulating leptin upon fasting nor do they enter torpor. To test whether the onset of torpor in mice during a fast requires a NE-mediated reduction in circulating leptin, double mutant mice deficient in both leptin (ob/ob) and DBH (DBL MUT) were generated. Upon fasting, control and ob/ob mice entered torpor as assessed by telemetric core T_b acquisition. While fasting failed to induce torpor in Dbh −/− mice, leptin deficiency bypassed the requirement for NE, as DBL MUT mice readily entered torpor upon fasting. These data indicate that sympathetic activation of white fat and suppression of leptin is required for the onset of torpor in the mouse. Emergence from torpor was severely retarded in DBL MUT mice, revealing a novel, leptin-independent role for NE in torpor recovery. This phenotype was mimicked by administration of a β₃ adrenergic receptor antagonist to control mice during a torpor bout. Hence, NE signaling via β₃ adrenergic receptors presumably in brown fat is the first neurotransmitter-receptor system identified that is required for normal recovery from torpor.     

Reduced Body Weight and Increased Energy Expenditure in Transgenic Mice Over-Expressing Soluble Leptin Receptor

Background

Soluble leptin receptor (OBRe), one of several leptin receptor isoforms, is the only bona fide leptin binding protein in plasma. Our earlier studies demonstrated that OBRe modulates leptin levels in circulation. Both clinical and in vitro studies have shown that OBRe expression is inversely correlated to body weight and leptin levels. However, it is not clear whether OBRe plays an active role, either in collaboration with leptin or independently, in the maintenance of body weight.

Methodology/Principal Findings

To investigate the function of OBRe in the regulation of energy homeostasis, we generated transgenic mice that express OBRe under the control of human serum amyloid P (hSAP) component gene promoter. The transgene led to approximately doubling of OBRe in circulation in the transgenic mice than in wild type control mice. Transgenic mice exhibited lower body weight at 4 weeks of age, and slower rate of weight gain when compared with control mice. Furthermore, transgenic mice had lower body fat content. Indirect calorimetry revealed that transgenic mice had reduced food intake, increased basal metabolic rate, and increased lipid oxidation, which could account for the differences in body weight and body fat content. Transgenic mice also showed higher total circulating leptin, with the majority of it being in the bound form, while the amount of free leptin is comparable between transgenic and control mice.

Conclusions

These results are consistent with the role of OBRe as a leptin binding protein in regulating leptin''s bioavailability and activity.     

Effects of Intracerebroventricular Infusion of Leptin in Obese Zucker Rats

AL-BARAZANJI, KAMAL A, ROBIN E BUCKINGHAM, JONATHAN RS ARCH, ANDREA HAYNES, DANUTA E MOSSAKOWSKA, DIANE L McBAY, STEPHEN D HOLMES, MARK T McHALE, XIN-MIN WANG, ISRAEL S GLOGER. Effects of intracerebro-ventricular infusion of leptin in obese Zucker rats. The obese Zucker rat (OZR) exhibits a missense mutation in the cDNA for the leptin receptor, producing a single amino acid substitution in the extracellular domain of the receptor. A mutation in the leptin receptor gene of the db/db mouse prevents the synthesis of the long splice variant of the receptor. The possibility that the OZR, like the db/db mouse, is refractory to the actions of murine leptin was tested by infusing the protein intracerebroventricularly via a minipump for 7 days. Lean Zucker rats (LZR) infused with leptin acted as positive controls, and other groups of OZR and LZR were infused with vehicle. In LZR, leptin reduced body-weight and food intake and increased brown adipose tissue (BAT) temperature. Plasma corticosterone increased (61%) in these rats, and plasma triglycerides fell (78%). Leptin treatment improved tolerance to an oral glucose load (16% reduction in the area under the blood glucose curve) while lowering plasma insulin. In OZR, the actions of leptin were blunted. Food intake was slightly, but not significantly, reduced. Although there was a reduction in the rate of increase in body mass, the effect of leptin was about half that seen in LZR. BAT temperature and glucose tolerance were unchanged. In contrast to the elevated plasma corticosterone seen in LZR, leptin reduced the level of this hormone (27%) in OZR. In OZR and LZR treated with leptin, the plasma leptin levels were increased 24-fold and 47-fold, respectively. The results suggest that leptin retains some efficacy in OZR, although these rats are less responsive than LZR.     

Plasma proteomic profiles of bovine growth hormone transgenic mice as they age

Attenuation of the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis results in extended lifespan in many organisms including mice. Conversely, GH transgenic mice have excess GH action and die prematurely. We have studied bovine (b) GH transgenic mice (n = 9) and their wild type (WT) littermates (n = 8) longitudinally and have determined several age-related changes. Compared to WT mice, bGH mice lost fat mass, became hypoglycemic and had lower insulin levels at older ages despite being hyperinsulinemic when young. To examine plasma protein differences in bGH mice relative to controls, samples at 2, 4, 8, 12 and 16 months of age were analyzed by two-dimensional gel electrophoresis followed by identification using mass spectrometry. We found several differences in plasma proteins of bGH mice compared to controls, including increased apolipoprotein E (five isoforms), haptoglobin (four isoforms) and mannose-binding protein-C (one out of three isoforms), and decreased transthyretin (six isoforms). In addition, clusterin (two out of six isoforms) and haptoglobin (four isoforms) were up-regulated in bGH mice as a function of age. Finally, alpha-2 macroglobulin (seven isoforms) was altered in an isoform-specific manner with two isoforms increased and two decreased in bGH mouse plasma compared to controls. In conclusion, identification of these proteins suggests that bGH mice exhibit an increased inflammatory state with an adverse lipid profile, possibly contributing to their diminished life expectancy. Also, these newly discovered plasma proteins may be indicative or ‘biomarkers’ of a shortened lifespan.     

Temperature dependence of O2 consumption; opposite effects of leptin and etomoxir on respiratory quotient in mice

Objectives : The aims were to compare the temperature dependence of the metabolic rate in young ob/ob mice with that in mature ob/ob and db/db mice and to examine the effect on the metabolic substrate preference of leptin and etomoxir in ob/ob, C57BL/6J (wild‐type), and db/db mice. Research Methods and Procedures : In vivo oxygen consumption and carbon dioxide production were continuously measured by indirect calorimetry, and body temperature and total locomotor activity were measured by an implanted transponder. Leptin, etomoxir, or vehicle was administered intraperitoneally. Results : The temperature dependence of the metabolic rate of mature ob/ob and db/db mice were similar to that in wild‐type mice. In young 6‐week‐old ob/ob mice, the metabolic rate was almost doubled at 15 °C. Leptin (2 × 3 mg/kg) decreased the respiratory quotient (RQ) and carbon dioxide production but did not alter oxygen consumption, body temperature, or locomotor activity in ob/ob and C57BL/6J mice and had no effect in the db/db mice. Etomoxir (2 × 30 mg/kg) enhanced RQ and decreased oxygen consumption, carbon dioxide production, and body temperature in ob/ob, C57BL/6J, and db/db mice. Total locomotor activity was reduced in ob/ob and C57BL/6J mice. Discussion : In young ob/ob mice, the temperature sensitivity was enhanced compared with mature mice. Leptin and etomoxir had opposite effects on metabolic substrate preference. Leptin and lowered environmental temperature increased the relative fat oxidation as indicated by decreased RQ, possibly through activation of the sympathetic nervous system.     

Dietary flavonoids suppress azoxymethane-induced colonic preneoplastic lesions in male C57BL/KsJ-db/db mice

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/db mice with obesity and diabetic phenotypes. Male db/db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68–91% and BCAC by 64–71%, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans.     

Leptin-Dependent Control of Glucose Balance and Locomotor Activity by POMC Neurons

Leptin plays a pivotal role in regulation of energy balance. Via unknown central pathways, leptin also affects peripheral glucose homeostasis and locomotor activity. We hypothesized that, specifically, pro-opiomelanocortin (POMC) neurons mediate those actions. To examine this possibility, we applied Cre-Lox technology to express leptin receptors (ObRb) exclusively in POMC neurons of the morbidly obese, profoundly diabetic, and severely hypoactive leptin receptor-deficient Lepr^db/db mice. Here, we show that expression of ObRb only in POMC neurons leads to a marked decrease in energy intake and a modest reduction in body weight in Lepr^db/db mice. Remarkably, blood glucose levels are entirely normalized. This normalization occurs independently of changes in food intake and body weight. In addition, physical activity is greatly increased despite profound obesity. Our results suggest that leptin signaling exclusively in POMC neurons is sufficient to stimulate locomotion and prevent diabetes in the severely hypoactive and hyperglycemic obese Lepr^db/db mice.     

The thermoregulatory and metabolic responses to photoperiod manipulations of the Macedonian mouse (Mus macedonicus), a post-fire invader

1. The Macedonian mouse (Mus macedonicus), a small (15 g) mesic rodent distributed in the Mediterranean ecosystem, is a species which invades post fire habitats in the first stages of habitat recovery. 2. In order to assess the seasonal acclimatization of thermoregulatory and metabolic mechanisms, the response of several physiological variables to photoperiod manipulations under a constant T_a were studied. 3. Our results show that mice acclimated to a short photoperiod increased their resistance to cold, while acclimation to a long photoperiod increased their resistance to high T_as and the effectiveness of their thermoregulatory mechanisms. Body mass increased in mice acclimated to a short photoperiod. We conclude that photoperiod is an important environmental cue for seasonal acclimatization of thermoregulatory and metabolic mechanisms in this species.     

Humanized TLR7/8 Expression Drives Proliferative Multisystemic Histiocytosis in C57BL/6 Mice

A humanized TLR7/TLR8 transgenic mouse line was engineered for studies using TLR7/8 ligands as vaccine adjuvants. The mice developed a spontaneous immune-mediated phenotype prior to six months of age characterized by runting, lethargy, blepharitis, and corneal ulceration. Histological examination revealed a marked, multisystemic histiocytic infiltrate that effaced normal architecture. The histological changes were distinct from those previously reported in mouse models of systemic lupus erythematosus. When the mice were crossed with MyD88^−/− mice, which prevented toll-like receptor signaling, the inflammatory phenotype resolved. Illness may be caused by constitutive activation of human TLR7 or TLR8 in the bacterial artificial chromosome positive mice as increased TLR7 and TLR8 expression or activation has previously been implicated in autoimmune disease.     

The effects of age on uptake, serum half-life and bioavailability of mouse [D-Leu-4]OB3, a synthetic peptide amide with leptin-like activity, in male C57BL/6J mice

We have previously shown that the activity of a synthetic peptide amide corresponding to amino acid residues 116–130 of mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116 and 122 (Ser-Cys-Ser-Leu-Pro-Glu-Thr). This peptide was named mouse OB3. The potency of OB3 was improved by replacing the l-leucine residue at position four with its d-isomer. Intraperitoneal administration (ip) of mouse OB3 or [D-Leu-4]OB3 to ob/ob and db/db mice reduces food intake, body weight gain and serum glucose levels, and enhances insulin sensitivity. These effects of OB3 and [D-Leu-4]OB3 are very pronounced in young mice and diminish with age. In the present study, we measured uptake, serum half-life, and bioavailability of mouse [D-Leu-4]OB3 in mice of different ages. Groups of male C57BL/6J mice, six and 25 weeks of age, were given a single ip injection of 1 mg mouse [D-Leu-4]OB3 in PBS. Five, 10, 20, 40, 60, 120, or 180 min after injection, the mice were anesthetized and exsanguinated. Serum samples were prepared and assayed for mouse [D-Leu-4]OB3 content by competitive ELISA. In six week-old mice, the maximum concentration of mouse [D-Leu-4]OB3 was reached in 10 min, and the serum half-life was approximately 52.5 min. In 25 week-old mice, however, mouse [D-Leu-4]OB3 peaked in 5 min, and the serum half-life was approximately 30.6 min. The relative bioavailability of mouse [D-Leu-4]OB3 in six and 25 week-old mice was determined by measuring the area under the uptake curves. Bioavailability of mouse [D-Leu-4]OB3 was approximately 20% greater in six week-old mice than in 25 week-old mice. The results of this study indicate that at least some pharmacokinetic parameters of peptide uptake change as mice age. They also suggest that differences in uptake, serum half-life, and relative bioavailability of mouse [D-Leu-4]OB3 may contribute, at least in part, to the reduced efficacy of bioactive leptin-related peptides we have consistently observed in ob/ob and db/db mice as they age.     

Exogenous leptin promotes the recovery of regressed ovary in fasted ducks

The present study was undertaken to examine the effect of administered recombinant mouse leptin on the recovery of regressed ovary in fasted ducks. Twenty-eight ducks were divided into five groups: fed ad libitum (control; n = 5), fasted control (FC; n = 5), fasted + low dose of leptin (F + L; n = 5), fasted + medium dose of leptin (F + M; n = 5) and fasted + high dose of leptin (F + H; n = 3). All four fasted groups were fasted for 2 days and then ad libitum and the ducks were treated with leptin at doses of 0 (control and FC), 50 (F + L), 250 (F + M) and 1000 (F + H) μg/kg body weight/day on day 3–5. Results showed that a moderate dose of leptin (250 μg/kg body weight/day) injected during the re-feeding period: (i) promoted the recovery of the regressed ovary as evidenced by an increase in ovary weight and recovery of yellow hierarchical follicles; (ii) elevated the plasma 17β-estradiol (E₂) level; (iii) increased the mRNA levels of ovary follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and estrogen receptor-β (ER-β). Furthermore, the results also showed that a high dose of leptin (1000 μg/kg body weight/day) may have a negative effect on the recovery of the regressed ovary. In conclusion, this study indicates that, in ducks, leptin may be involved in the recovery of the regressed ovary caused by 2 days of fasting. This effect may be related to increased plasma E₂ levels and stimulation of the mRNA levels of ovarian FSHR, LHR and especially ER-β.     

Interplay between obesity and aging on myocardial geometry and function: Role of leptin-STAT3-stress signaling

BackgroundUncorrected obesity facilitates premature aging and cardiovascular anomalies. This study examined the interaction between obesity and aging on cardiac remodeling and contractile function. Methods: Cardiac echocardiographic geometry, function, morphology, intracellular Ca²⁺ handling, oxidative stress (DHE fluorescence), STAT3 and stress signaling were evaluated in young (3-mo) and old (12- and 18-mo) lean and leptin deficient ob/ob obese mice. Cardiomyocytes from young and old lean and ob/ob mice were treated with leptin (1 nM) for 4 h in vitro prior to assessment of mechanical and biochemical properties. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obese mice at young and old age were evaluated for comparison. Results: Our results displayed reduced survival in ob/ob mice. Obesity but less likely older age dampened echocardiographic, geometric, cardiomyocyte function and intracellular Ca²⁺ properties, elevated O₂^? and p^47phox NADPH oxidase levels with a more pronounced geometric change at older age. Immunoblot analysis revealed elevated p^47phox NADPH oxidase and dampened phosphorylation of STAT3, with a more pronounced response in old ob/ob mice, the effects were restored by leptin. Obesity and aging inhibited phosphorylation of Akt, eNOS, AMPK, and p38 while promoting phosphorylation of JNK and IκB. Leptin reconciled cardiomyocyte dysfunction, O₂^? yield, p^47phox upregulation, STAT3 dephosphorylation and stress signaling in ob/ob mice although its action on stress signaling cascades were lost at old age. High fat diet-induced and db/db obesity displayed aging-associated cardiomyocyte anomalies reminiscent of ob/ob model albeit lost leptin response.ConclusionsOur data suggest disparate age-associated obesity response in cardiac remodeling and contractile dysfunction due to phosphorylation of Akt, eNOS and stress signaling-related oxidative stress.     

Generation of Leptin Receptor Bone Marrow Chimeras: Recovery From Irradiation,Immune Cellularity,Cytokine Expression,and Metabolic Parameters

Leptin regulates appetite and metabolism but also immunity and inflammation. Although functional leptin receptors (LepR) are expressed on hematopoietic cells, the role of these receptors in regulating immune function in vivo remains controversial. To clarify this issue, we performed bone marrow (BM) transplantation between obese db/db mice, lacking LepR, and wild‐type (WT) mice. Results indicate that expression of LepR on BM‐derived cells directly, though partially, regulates spleen and thymus cellularity, although the environment of db mice contributes to maintaining reduced cellularity of these organs. Selective expression of LepR on BM‐derived cells also modulates leptin and adiponectin levels, with induction of a more favorable adipokine environment in the WT→db/db group. However, LepR signaling in BM‐derived cells is not involved in regulation of body weight (BW) and composition, glycemia, hepatosteatosis or adipose tissue inflammation, although it modulates expression of interleukin (IL)‐1β in the brain. Finally, data indicate that db mice have an increased susceptibility to irradiation compared to WT mice in terms of BW loss and recovery of leukocyte counts in peripheral blood. Therefore, interpretation of results obtained using BM chimeras between WT and db mice should take into account the difference in radiation sensitivity between the two types of animals.