Chronic hindbrain administration of oxytocin elicits weight loss in male diet-induced obese mice.

Previous studies indicate that oxytocin (OT) administration reduces body weight in high-fat diet (HFD)-induced obese (DIO) rodents through both reductions in food intake and increases in energy expenditure. We recently demonstrated that chronic hindbrain [fourth ventricular (4V)] infusions of OT evoke weight loss in DIO rats. Based on these findings, we hypothesized that chronic 4V OT would elicit weight loss in DIO mice. We assessed the effects of 4V infusions of OT (16 nmol/day) or vehicle over 28 days on body weight, food intake, and body composition. OT reduced body weight by approximately 4.5% ± 1.4% in DIO mice relative to OT pretreatment body weight (P < 0.05). These effects were associated with reduced adiposity and adipocyte size [inguinal white adipose tissue (IWAT)] (P < 0.05) and attributed, in part, to reduced energy intake (P < 0.05) at a dose that did not increase kaolin intake (P = NS). OT tended to increase uncoupling protein-1 expression in IWAT (0.05 < P < 0.1) suggesting that OT stimulates browning of WAT. To assess OT-elicited changes in brown adipose tissue (BAT) thermogenesis, we examined the effects of 4V OT on interscapular BAT temperature (TIBAT). 4V OT (1 µg) elevated TIBAT at 0.75 (P = 0.08), 1, and 1.25 h (P < 0.05) postinjection; a higher dose (5 µg) elevated TIBAT at 0.75-, 1-, 1.25-, 1.5-, 1.75- (P < 0.05), and 2-h (0.05 < P < 0.1) postinjection. Together, these findings support the hypothesis that chronic hindbrain OT treatment evokes sustained weight loss in DIO mice by reducing energy intake and increasing BAT thermogenesis at a dose that is not associated with evidence of visceral illness.

Use of the PET ligand florbetapir for in vivo imaging of pancreatic islet amyloid deposits in hIAPP transgenic mice.

AIMS/HYPOTHESIS: Islet amyloid deposits contribute to beta cell dysfunction and death in most individuals with type 2 diabetes but non-invasive methods to determine the presence of these pathological protein aggregates are currently not available. Therefore, we examined whether florbetapir, a radiopharmaceutical agent used for detection of amyloid-ß deposits in the brain, also allows identification of islet amyloid in the pancreas. METHODS: Saturation binding assays were used to determine the affinity of florbetapir for human islet amyloid polypeptide (hIAPP) aggregates in vitro. Islet amyloid-prone transgenic mice that express hIAPP in their beta cells and amyloid-free non-transgenic control mice were used to examine the ability of florbetapir to detect islet amyloid deposits in vitro, in vivo and ex vivo. Mice or mouse pancreases were subjected to autoradiographic, histochemical and/or positron emission tomography (PET) analyses to assess the utility of florbetapir in identifying islet amyloid. RESULTS: In vitro, florbetapir bound synthetic hIAPP fibrils with a dissociation constant of 7.9 nmol/l. Additionally, florbetapir bound preferentially to amyloid-containing hIAPP transgenic vs amyloid-free non-transgenic mouse pancreas sections in vitro, as determined by autoradiography (16,475 ± 5581 vs 5762 ± 575 density/unit area, p < 0.05). In hIAPP transgenic and non-transgenic mice fed a high-fat diet for 1 year, intravenous administration of florbetapir followed by PET scanning showed that the florbetapir signal was significantly higher in amyloid-laden hIAPP transgenic vs amyloid-free non-transgenic pancreases in vivo during the first 5 min of the scan (36.83 ± 2.22 vs 29.34 ± 2.03 standardised uptake value × min, p < 0.05). Following PET, pancreases were excised and florbetapir uptake was determined ex vivo by γ counting. Pancreatic uptake of florbetapir was significantly correlated with the degree of islet amyloid deposition, the latter assessed by histochemistry (r = 0.74, p < 0.001). CONCLUSIONS/INTERPRETATION: Florbetapir binds to islet amyloid deposits in a specific and quantitative manner. In the future, florbetapir may be useful as a non-invasive tool to identify islet amyloid deposits in humans.

Chronic hindbrain administration of oxytocin is sufficient to elicit weight loss in diet-induced obese rats.

Oxytocin (OT) administration elicits weight loss in diet-induced obese (DIO) rodents, nonhuman primates, and humans by reducing energy intake and increasing energy expenditure. Although the neurocircuitry underlying these effects remains uncertain, OT neurons in the paraventricular nucleus are positioned to control both energy intake and sympathetic nervous system outflow to interscapular brown adipose tissue (BAT) through projections to the hindbrain nucleus of the solitary tract and spinal cord. The current work was undertaken to examine whether central OT increases BAT thermogenesis, whether this effect involves hindbrain OT receptors (OTRs), and whether such effects are associated with sustained weight loss following chronic administration. To assess OT-elicited changes in BAT thermogenesis, we measured the effects of intracerebroventricular administration of OT on interscapular BAT temperature in rats and mice. Because fourth ventricular (4V) infusion targets hindbrain OTRs, whereas third ventricular (3V) administration targets both forebrain and hindbrain OTRs, we compared responses to OT following chronic 3V infusion in DIO rats and mice and chronic 4V infusion in DIO rats. We report that chronic 4V infusion of OT into two distinct rat models recapitulates the effects of 3V OT to ameliorate DIO by reducing fat mass. While reduced food intake contributes to this effect, our finding that 4V OT also increases BAT thermogenesis suggests that increased energy expenditure may contribute as well. Collectively, these findings support the hypothesis that, in DIO rats, OT action in the hindbrain evokes sustained weight loss by reducing energy intake and increasing BAT thermogenesis.

Chronic CNS oxytocin signaling preferentially induces fat loss in high-fat diet-fed rats by enhancing satiety responses and increasing lipid utilization.

Based largely on a number of short-term administration studies, growing evidence suggests that central oxytocin is important in the regulation of energy balance. The goal of the current work is to determine whether long-term third ventricular (3V) infusion of oxytocin into the central nervous system (CNS) is effective for obesity prevention and/or treatment in rat models. We found that chronic 3V oxytocin infusion between 21 and 26 days by osmotic minipumps both reduced weight gain associated with the progression of high-fat diet (HFD)-induced obesity and elicited a sustained reduction of fat mass with no decrease of lean mass in rats with established diet-induced obesity. We further demonstrated that these chronic oxytocin effects result from 1) maintenance of energy expenditure at preintervention levels despite ongoing weight loss, 2) a reduction in respiratory quotient, consistent with increased fat oxidation, and 3) an enhanced satiety response to cholecystokinin-8 and associated decrease of meal size. These weight-reducing effects persisted for approximately 10 days after termination of 3V oxytocin administration and occurred independently of whether sucrose was added to the HFD. We conclude that long-term 3V administration of oxytocin to rats can both prevent and treat diet-induced obesity.

Sympathetic innervation of interscapular brown adipose tissue is not a predominant mediator of oxytocin-elicited reductions of body weight and adiposity in male diet-induced obese mice.

Previous studies indicate that CNS administration of oxytocin (OT) reduces body weight in high fat diet-induced obese (DIO) rodents by reducing food intake and increasing energy expenditure (EE). We recently demonstrated that hindbrain (fourth ventricular [4V]) administration of OT elicits weight loss and elevates interscapular brown adipose tissue temperature (T IBAT , a surrogate measure of increased EE) in DIO mice. What remains unclear is whether OT-elicited weight loss requires increased sympathetic nervous system (SNS) outflow to IBAT. We hypothesized that OT-induced stimulation of SNS outflow to IBAT contributes to its ability to activate BAT and elicit weight loss in DIO mice. To test this hypothesis, we determined the effect of disrupting SNS activation of IBAT on the ability of 4V OT administration to increase T IBAT and elicit weight loss in DIO mice. We first determined whether bilateral surgical SNS denervation to IBAT was successful as noted by ≥ 60% reduction in IBAT norepinephrine (NE) content in DIO mice. NE content was selectively reduced in IBAT at 1-, 6- and 7-weeks post-denervation by 95.9±2.0, 77.4±12.7 and 93.6±4.6% ( P <0.05), respectively and was unchanged in inguinal white adipose tissue, pancreas or liver. We subsequently measured the effects of acute 4V OT (1, 5 µg ≈ 0.99, 4.96 nmol) on T IBAT in DIO mice following sham or bilateral surgical SNS denervation to IBAT. We found that the high dose of 4V OT (5 µg ≈ 4.96 nmol) elevated T IBAT similarly in sham mice as in denervated mice. We subsequently measured the effects of chronic 4V OT (16 nmol/day over 29 days) or vehicle infusions on body weight, adiposity and food intake in DIO mice following sham or bilateral surgical denervation of IBAT. Chronic 4V OT reduced body weight by 5.7±2.23% and 6.6±1.4% in sham and denervated mice ( P <0.05), respectively, and this effect was similar between groups ( P =NS). OT produced corresponding reductions in whole body fat mass ( P <0.05). Together, these findings support the hypothesis that sympathetic innervation of IBAT is not necessary for OT-elicited increases in BAT thermogenesis and reductions of body weight and adiposity in male DIO mice.

Peripheral oxytocin suppresses food intake and causes weight loss in diet-induced obese rats.

Growing evidence suggests that oxytocin plays an important role in the regulation of energy balance and that central oxytocin administration induces weight loss in diet-induced obese (DIO) animals. To gain a better understanding of how oxytocin mediates these effects, we examined feeding and neuronal responses to oxytocin in animals rendered obese following exposure to either a high-fat (HFD) or low-fat diet (LFD). Our findings demonstrate that peripheral administration of oxytocin dose-dependently reduces food intake and body weight to a similar extent in rats maintained on either diet. Moreover, the effect of oxytocin to induce weight loss remained intact in leptin receptor-deficient Koletsky (fa(k)/fa(k)) rats relative to their lean littermates. To determine whether systemically administered oxytocin activates hindbrain areas that regulate meal size, we measured neuronal c-Fos induction in the nucleus of the solitary tract (NTS) and area postrema (AP). We observed a robust neuronal response to oxytocin in these hindbrain areas that was unexpectedly increased in rats rendered obese on a HFD relative to lean, LFD-fed controls. Finally, we report that repeated daily peripheral administration of oxytocin in DIO animals elicited a sustained reduction of food intake and body weight while preventing the reduction of energy expenditure characteristic of weight-reduced animals. These findings extend recent evidence suggesting that oxytocin circumvents leptin resistance and induces weight-loss in DIO animals through a mechanism involving activation of neurons in the NTS and AP, key hindbrain areas for processing satiety-related inputs.

Alterations in activity and energy expenditure contribute to lean phenotype in Fischer 344 rats lacking the cholecystokinin-1 receptor gene.

CCK is hypothesized to inhibit meal size by acting at CCK1 receptors (CCK1R) on vagal afferent neurons that innervate the gastrointestinal tract and project to the hindbrain. Earlier studies have shown that obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which carry a spontaneous null mutation of the CCK1R, are hyperphagic and obese. Recent findings show that rats with CCK1R-null gene on a Fischer 344 background (Cck1r(-/-)) are lean and normophagic. In this study, the metabolic phenotype of this rat strain was further characterized. As expected, the CCK1R antagonist, devazepide, failed to stimulate food intake in the Cck1r(-/-) rats. Both Cck1r(+/+) and Cck1r(-/-) rats became diet-induced obese (DIO) when maintained on a high-fat diet relative to chow-fed controls. Cck1r(-/-) rats consumed larger meals than controls during the dark cycle and smaller meals during the light cycle. These effects were accompanied by increased food intake, total spontaneous activity, and energy expenditure during the dark cycle and an apparent reduction in respiratory quotient during the light cycle. To assess whether enhanced responsiveness to anorexigenic factors may contribute to the lean phenotype, we examined the effects of melanotan II (MTII) on food intake and body weight. We found an enhanced effect of MTII in Cck1r(-/-) rats to suppress food intake and body weight following both central and peripheral administration. These results suggest that the lean phenotype is potentially driven by increases in total spontaneous activity and energy expenditure.

Repetitive Blast Promotes Chronic Aversion to Neutral Cues Encountered in the Peri-Blast Environment.

Repetitive mild traumatic brain injury (mTBI) has been called the "signature injury" of military service members in the Iraq and Afghanistan wars and is highly comorbid with post-traumatic stress disorder (PTSD). Correct attribution of adverse blast-induced mTBI and/or PTSD remains challenging. Pre-clinical research using animal models can provide important insight into the mechanisms by which blast produces injury and dysfunction-but only to the degree by which such models reflect the human experience. Avoidance of trauma reminders is a hallmark of PTSD. Here, we sought to understand whether a mouse model of blast reproduces this phenomenon, in addition to blast-induced physical injuries. Drawing on well-established work from the chronic stress and Pavlovian conditioning literature, we hypothesized that even while one is anesthetized during blast exposure, environmental cues encountered in the peri-blast environment could be conditioned to evoke aversion/dysphoria and re-experiencing of traumatic stress. Using a pneumatic shock tube that recapitulates battlefield-relevant open-field blast forces, we provide direct evidence that stress is inherent to repetitive blast exposure, resulting in chronic aversive/dysphoric-like responses to previous blast-paired cues. The results in this report demonstrate that, although both single and repetitive blast exposures produce acute stress responses (weight loss, corticosterone increase), only repetitive blast exposure also results in co-occurring aversive/dysphoric-like stress responses. These results extend appreciation of the highly complex nature of repetitive blast exposure; and lend further support for the potential translational relevance of animal modeling approaches currently used by multiple laboratories aimed at elucidating the mechanisms (both molecular and behavioral) of repetitive blast exposure.

Effects of Combined Oxytocin and Beta-3 Receptor Agonist (CL 316243) Treatment on Body Weight and Adiposity in Male Diet-Induced Obese Rats.

Previous studies have indicated that oxytocin (OT) reduces body weight in diet-induced obese (DIO) rodents through reductions in energy intake and increases in energy expenditure. We recently demonstrated that hindbrain [fourth ventricular (4V)] administration of OT evokes weight loss and elevates interscapular brown adipose tissue temperature (T IBAT ) in DIO rats. What remains unclear is whether OT can be used as an adjunct with other drugs that directly target beta-3 receptors in IBAT to promote BAT thermogenesis and reduce body weight in DIO rats. We hypothesized that the combined treatment of OT and the beta-3 agonist, CL 316243, would produce an additive effect to decrease body weight and adiposity in DIO rats by reducing energy intake and increasing BAT thermogenesis. We assessed the effects of 4V infusions of OT (16 nmol/day) or vehicle (VEH) in combination with daily intraperitoneal injections of CL 316243 (0.5 mg/kg) or VEH on food intake, T IBAT , body weight and body composition. OT and CL 316243 alone reduced body weight by 7.8 ± 1.3% (P < 0.05) and 9.1 ± 2.1% (P < 0.05), respectively, but the combined treatment produced more pronounced weight loss (15.5 ± 1.2%; P < 0.05) than either treatment alone. These effects were associated with decreased adiposity, adipocyte size, energy intake and increased uncoupling protein 1 (UCP-1) content in epididymal white adipose tissue (EWAT) (P < 0.05). In addition, CL 316243 alone (P < 0.05) and in combination with OT (P < 0.05) elevated T IBAT and IBAT UCP-1 content and IBAT thermogenic gene expression. These findings are consistent with the hypothesis that the combined treatment of OT and the beta-3 agonist, CL 316243, produces an additive effect to decrease body weight. The findings from the current study suggest that the effects of the combined treatment on energy intake, fat mass, adipocyte size and browning of EWAT were not additive and appear to be driven, in part, by transient changes in energy intake in response to OT or CL 316243 alone as well as CL 316243-elicited reduction of fat mass and adipocyte size and induction of browning of EWAT.

Hindbrain Administration of Oxytocin Reduces Food Intake, Weight Gain and Activates Catecholamine Neurons in the Hindbrain Nucleus of the Solitary Tract in Rats.

Existing studies show that CNS oxytocin (OT) signaling is important in the control of energy balance, but it is unclear which neurons may contribute to these effects. Our goals were to examine (1) the dose-response effects of acute OT administration into the third (3V; forebrain) and fourth (4V; hindbrain) ventricles to assess sensitivity to OT in forebrain and hindbrain sites, (2) the extent to which chronic 4V administration of OT reduces weight gain associated with the progression of diet-induced obesity, and (3) whether nucleus tractus solitarius (NTS) catecholamine neurons are downstream targets of 4V OT. Initially, we examined the dose-response effects of 3V and 4V OT (0.04, 0.2, 1, or 5 µg). 3V and 4V OT (5 µg) suppressed 0.5-h food intake by 71.7 ± 6.0% and 60 ± 12.9%, respectively. 4V OT (0.04, 0.2, 1 µg) reduced food intake by 30.9 ± 12.9, 42.1 ± 9.4, and 56.4 ± 9.0%, respectively, whereas 3V administration of OT (1 µg) was only effective at reducing 0.5-h food intake by 38.3 ± 10.9%. We subsequently found that chronic 4V OT infusion, as with chronic 3V infusion, reduced body weight gain (specific to fat mass) and tended to reduce plasma leptin in high-fat diet (HFD)-fed rats, in part, through a reduction in energy intake. Lastly, we determined that 4V OT increased the number of hindbrain caudal NTS Fos (+) neurons (156 ± 25) relative to vehicle (12 ± 3). The 4V OT also induced Fos in tyrosine hydroxylase (TH; marker of catecholamine neurons) (+) neurons (25 ± 7%) relative to vehicle (0.8 ± 0.3%). Collectively, these findings support the hypothesis that OT within the hindbrain is effective at reducing food intake, weight gain, and adiposity and that NTS catecholamine neurons in addition to non-catecholaminergic neurons are downstream targets of CNS OT.

Intranasal oxytocin reduces weight gain in diet-induced obese prairie voles.

Oxytocin (OT) elicits weight loss in diet-induced obese (DIO) rodents, nonhuman primates and humans by reducing food intake and increasing energy expenditure. In addition to being important in the regulation of energy balance, OT is involved in social behaviors including parent-infant bonds, friendships, and pair bonds. However, the impact of social context on susceptibility to diet-induced obesity (DIO) and feeding behavior (including food sharing) has not been investigated in a rodent model that forms strong social bonds (i.e. prairie vole). Our goals were to determine in Prairie voles (Microtus ochrogaster) whether i) social context impacts susceptibility to DIO and ii) chronic intranasal OT reverses DIO. Voles were housed in divided cages with holes in the divider and paired with a same-sex animal with either the same food [high fat diet (HFD)/HFD, [low fat diet (LFD; chow)/chow], or the opposite food (HFD/chow or chow/HFD) for 19 weeks. HFD-fed voles pair-housed with voles maintained on the HFD demonstrated increased weight relative to pair-housed voles that were both maintained on chow. The study was repeated to determine the impact of social context on DIO susceptibility and body composition when animals are maintained on purified sugar-sweetened HFD and LFD to enhance palatability. As before, we found that voles demonstrated higher weight gain on the HFD/HFD housing paradigm, in part, through increased energy intake and the weight gain was a consequence of an increase in fat mass. However, HFD-fed animals housed with LFD-fed animals (and vice versa) showed intermediate patterns of weight gain and evidence of food sharing. Of translational importance is the finding that chronic intranasal OT appeared to reduce weight gain in DIO voles through a decrease in fat mass with no reduction in lean body mass. These effects were associated with transient reductions in food intake and increased food sharing. These findings identify a role of social context in the pathogenesis of DIO and indicate that chronic intranasal OT treatment reduces weight gain and body fat mass in DIO prairie voles, in part, by reducing food intake.

Mechanisms of the anorexia of aging in the Brown Norway rat.

Aging is associated with a loss of the ability to maintain homeostasis in response to physiologic and environmental disturbances. Age-related dysregulation of food intake and energy balance appears to be the result of impaired responsiveness of hypothalamic integrative circuitry to metabolic cues, which can lead to lack of appropriate food intake (the anorexia of aging) and thus to inappropriate weight loss in response to acute or chronic illness or other stressors. Using the Brown Norway (BN) male rat model, we have shown that old animals fail to appropriately increase food intake after the metabolic challenge of a 72 h fast, resulting in the failure to re-gain lost body weight upon refeeding. Leptin levels increase with adiposity and age, and remain elevated above levels of young animals even after a 72 h fast, suggesting that hyperleptinemia may be influencing the energy balance dysregulation. It is unclear whether this age-related response is due to a failure of the network of hypothalamic neurons to appropriately integrate hormonal and neural inputs, or due to a failure of the neurons to produce the appropriate neuropeptides. We hypothesize that sequential, age-related alterations in the expression patterns of neuropeptides that maintain melanocortinergic tone, and in the hormone mediators that inform the system of the state of energy balance, result in a diminished ability to maintain energy homeostasis with increasing age. We have undertaken a number of interventional approaches to test this hypothesis, including manipulations of the hormones ghrelin, insulin and testosterone, and direct application of neuropeptides to the central nervous system in these animals.

Circulating irisin levels and muscle FNDC5 mRNA expression are independent of IL-15 levels in mice.

Interleukin-15 (IL-15) and irisin are exercise-induced myokines that exert favorable effects on energy expenditure and metabolism. IL-15 can induce PGC-1α expression, which in turn induces expression of irisin and its precursor, FNDC5. Therefore, the present study tested the hypothesis that increases in circulating irisin levels and muscle FNDC5 mRNA expression are dependent on IL-15. Circulating irisin levels and gastrocnemius muscle FNDC5 mRNA expression were examined following acute exercise in control and IL-15-deleted (IL-15 KO) mice, following injection of IL-15 into IL-15 KO mice, and in transgenic mice with elevated circulating IL-15 levels (IL-15 Tg mice). Circulating IL-15 levels and muscle PGC-1α and PPARδ mRNA expressions were determined as positive controls. No effect of IL-15 deletion on post-exercise serum irisin levels or muscle FNDC5 mRNA expression was detected. While serum IL-15 levels and muscle PGC-1α expression were elevated post-exercise in control mice, both serum irisin levels and muscle FNDC5 expression decreased shortly after exercise in both control and IL-15 KO mice. A single injection of recombinant IL-15 into IL-15 KO mice that significantly increased muscle PPARδ and PGC-1α mRNA expressions had no effect on circulating irisin release, but modestly induced muscle FNDC5 expression. Additionally, serum irisin and gastrocnemius muscle FNDC5 expression in IL-15 Tg mice were similar to those of control mice. Muscle FNDC5 mRNA expression and irisin release are not IL-15-dependent in mice.

Central Nervous System Delivery of Intranasal Insulin: Mechanisms of Uptake and Effects on Cognition.

Intranasal insulin has shown efficacy in patients with Alzheimer's disease (AD), but there are no preclinical studies determining whether or how it reaches the brain. Here, we showed that insulin applied at the level of the cribriform plate via the nasal route quickly distributed throughout the brain and reversed learning and memory deficits in an AD mouse model. Intranasal insulin entered the blood stream poorly and had no peripheral metabolic effects. Uptake into the brain from the cribriform plate was saturable, stimulated by PKC inhibition, and responded differently to cellular pathway inhibitors than did insulin transport at the blood-brain barrier. In summary, these results show intranasal delivery to be an effective way to deliver insulin to the brain.

Testosterone (T)-induced changes in arcuate nucleus cocaine-amphetamine-regulated transcript and NPY mRNA are attenuated in old compared to young male brown Norway rats: contribution of T to age-related changes in cocaine-amphetamine-regulated transcript and NPY gene expression.

The age-related decrease in serum T levels is associated with impairments in food intake and weight regulation and alterations in brain peptides that regulate energy balance. To test the hypothesis that reduced T levels contribute to altered hypothalamic cocaine-amphetamine-regulated transcript (CART) and NPY gene expression, the mRNA content of these neuropeptides was measured by in situ hybridization in sham-operated (intact), castrated, and T-replaced castrated young and old male Brown Norway rats. T levels in T-replaced young and old rats were similar to those in intact young animals. Compared with castrated rats, arcuate nucleus CART mRNA was lower and NPY mRNA was higher in both young and old T-replaced castrated animals, suggesting reciprocal regulation of these peptides by T; these T-induced changes were localized primarily in the rostral arcuate and were markedly attenuated in old animals. Compared with intact animals, paraventricular nucleus CART mRNA was lower in castrated animals and similar in T-replaced young and old rats. We conclude that hypothalamic CART and NPY neurons remain responsive to T regulation in old rats, albeit less so than in young animals, suggesting that the age-related reduction of T contributes in part to altered brain neuropeptide gene expression favoring anorexia and wasting with aging.

Troglitazone treatment of aging Brown Norway rats improves food intake and weight gain after fasting without increasing hypothalamic NPY gene expression.

Compared to younger animals, aged male Brown Norway (BN) rats demonstrate increased body fat and serum insulin, and lower prepro-neuropeptide Y (ppNPY) mRNA content in the arcuate nucleus (ARC), and blunted food intake (FI) and body weight (BW) gain in response to a 72 h fast. Since centrally administered insulin decreases FI and weight of young rats and inhibits fasting-induced increases of NPY gene expression, we hypothesized that hyperinsulinemia in old rats contributes to an age-related central dysregulation of energy balance. Young, middle-aged and old BN rats were fed chow with troglitazone (Trog; 200 mg/kg BW/d) or without drug for 75 d (Experiment 1) or 66 d (Experiment 2). Rats were then fasted for 72 h, refed for 2 weeks and sacrificed after an overnight fast (Experiment 1) or fasted for 72 h and sacrificed (Experiment 2). Serum insulin and leptin were measured from trunk blood and brains were analyzed for ppNPY mRNA by in situ hybridization. In Experiment 1, troglitazone treatment resulted in increased post-fast weight gain, rate of gain and FI in old rats. Troglitazone decreased serum insulin by 50% in old rats, while leptin levels decreased 20-30% in all age groups in Experiment 1. No differences in serum insulin or leptin were detectable with troglitazone treatment in Experiment 2, due to the extreme suppression caused by the 72 h fast. Troglitazone treatment did not increase ARC NPY gene expression either after a 72 h fast and re-feeding for 2 weeks (Experiment 1) or immediately after a 72 h fast (Experiment 2). These findings suggest that increased insulin levels may contribute to age-related impairments of FI and BW regulation. However, improvements in these defects in energy regulation induced by troglitazone do not appear to result from changes in NPY gene expression, and may be due to alterations in other hypothalamic neuropeptides that regulate energy balance.

Assessment of murine exercise endurance without the use of a shock grid: an alternative to forced exercise.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in naïve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

IL-15 is required for postexercise induction of the pro-oxidative mediators PPARδ and SIRT1 in male mice.

Physical exercise induces transient upregulation of the pro-oxidative mediators peroxisome proliferator-activated receptor-δ (PPARδ), silent information regulator of transcription (sirtuin)-1 (SIRT1), PPARγ coactivator 1α (PGC-1α), and PGC-1ß in skeletal muscle. To determine the role of the cytokine IL-15 in acute postexercise induction of these molecules, expression of these factors after a bout of exhaustive treadmill running was examined in the gastrocnemius muscle of untrained control and IL-15-knockout (KO) mice. Circulating IL-15 levels increased transiently in control mice after exercise. Control mice, but not IL-15-KO mice, upregulated muscle PPARδ and SIRT1 protein after exercise, accompanied by a complex pattern of mRNA expression for these factors. However, in exhaustive exercise, control mice ran significantly longer than IL-15-KO mice. Therefore, in a second experiment, mice were limited to a 20-minute run, after which a similar pattern of induction of muscle PPARδ and SIRT1 protein by control mice only was observed. In a separate experiment, IL-15-KO mice injected systemically with recombinant IL-15 upregulated muscle PPARδ and SIRT1 mRNA within 30 minutes and also exhibited increased muscle PPARδ protein levels by 3 hours. After exercise, both control and IL-15-KO mice downregulated IL-15 receptor-α (IL-15Rα) mRNA, whereas IL-15Rα-deficient mice exhibited constitutively elevated circulating IL-15 levels. These observations indicate IL-15 release after exercise is necessary for induction of PPARδ and SIRT1 at the protein level in muscle tissue and suggest that exercise releases IL-15 normally sequestered by the IL-15Rα in the resting state. These findings could be used to develop an IL-15-based strategy to induce many of the metabolic benefits of physical exercise.

IL-15 overexpression promotes endurance, oxidative energy metabolism, and muscle PPARδ, SIRT1, PGC-1α, and PGC-1ß expression in male mice.

Endurance exercise initiates a pattern of gene expression that promotes fat oxidation, which in turn improves endurance, body composition, and insulin sensitivity. The signals from exercise that initiate these pathways have not been completely characterized. IL-15 is a cytokine that is up-regulated in skeletal muscle after exercise and correlates with leanness and insulin sensitivity. To determine whether IL-15 can induce any of the metabolic adaptations associated with exercise, substrate metabolism, endurance, and molecular expression patterns were examined in male transgenic mice with constitutively elevated muscle and circulating IL-15 levels. IL-15 transgenic mice ran twice as long as littermate control mice in a run-to-exhaustion trial and preferentially used fat for energy metabolism. Fast muscles in IL-15 transgenic mice exhibited high expression of intracellular mediators of oxidative metabolism that are induced by exercise, including sirtuin 1, peroxisome proliferator-activated receptor (PPAR)-δ, PPAR-γ coactivator-1α, and PPAR-γ coactivator-1ß. Muscle tissue in IL-15 transgenic mice exhibited myosin heavy chain and troponin I mRNA isoform expression patterns indicative of a more oxidative phenotype than controls. These findings support a role for IL-15 in induction of exercise endurance, oxidative metabolism, and skeletal muscle molecular adaptations induced by physical training.

Overexpression of interleukin-15 in mice promotes resistance to diet-induced obesity, increased insulin sensitivity, and markers of oxidative skeletal muscle metabolism.

Interleukin-15 (IL-15) is a cytokine that is highly expressed in skeletal muscle. In addition to its well-characterized effects on innate immunity, IL-15 has been proposed to modulate skeletal muscle and adipose tissue mass, as well as insulin sensitivity. In the present study, an IL-15 gain-of-function model, transgenic mice with skeletal muscle-specific oversecretion of IL-15 (IL-15 Tg mice), was utilized to test the hypotheses that IL-15 promotes insulin sensitivity and resistance to diet-induced obesity (DIO) by increasing circulating adiponectin levels, and that IL-15 regulates skeletal muscle metabolism without inducing overt muscle hypertrophy. Compared to closely related control mice, IL-15 Tg mice exhibited lower total body fat following high-fat feeding, lower intra-abdominal fat following both low- and high-fat feeding, and greater insulin sensitivity. However, this was not accompanied by increased total or high molecular weight serum adiponectin levels in IL-15 Tg mice. While overall lean body mass did not differ, IL-15 Tg mice exhibited increased mass of the oxidative soleus muscle, and increased expression of mRNA encoding the slow isoform of troponin I (TnnI 1) in the predominately glycolytic extensor digitorum longus muscle. Skeletal muscle tissue from IL-15 Tg mice also exhibited alterations in the expression of several genes associated with fatty acid metabolism, such as SIRT1, SIRT4, and uncoupling protein 2 (UCP2). These findings suggest changes in oxidative metabolism, rather than induction of adiponectin expression, appear to be responsible for the DIO-resistant and more insulin-sensitive phenotype of IL-15 Tg mice.