Maria J. Barnes

HF diets increase hypothalamic PTP1B and induce leptin resistance through both leptin-dependent and -independent mechanisms

Protein tyrosine phosphatase 1B (PTP1B) contributes to leptin resistance by inhibiting intracellular leptin receptor signaling. Mice with whole body or neuron-specific deletion of PTP1B are hypersensitive to leptin and resistant to diet-induced obesity. Here we report a significant increase in PTP1B protein levels in the mediobasal hypothalamus (P = 0.003) and a concomitant reduction in leptin sensitivity following 28 days of high-fat (HF) feeding in rats. A significant increase in PTP1B mRNA levels was also observed in rats chronically infused with leptin (3 microg/day icv) for 14 days (P = 0.01) and in leptin-deficient ob/ob mice infused with leptin (5 microg/day sc for 14 days; P = 0.003). When saline-infused ob/ob mice were placed on a HF diet for 14 days, an increase in hypothalamic PTP1B mRNA expression was detected (P = 0.001) despite the absence of circulating leptin. In addition, although ob/ob mice were much more sensitive to leptin on a low-fat (LF) diet, a reduction in this sensitivity was still observed following exposure to a HF diet. Taken together, these data indicate that hypothalamic PTP1B is specifically increased during HF diet-induced leptin resistance. This increase in PTP1B is due in part to chronic hyperleptinemia, suggesting that hyperleptinemia is one mechanism contributing to the development of leptin resistance. However, these data also indicate that leptin is not required for the increase in hypothalamic PTP1B or the development of leptin resistance. Therefore, additional, leptin-independent mechanisms must exist that increase hypothalamic PTP1B and contribute to leptin resistance.

Leptin receptor mRNA in rat brain astrocytes.

We recently reported that mouse astrocytes express leptin receptors (ObR), and that obesity induces upregulation of astrocytic ObR. To provide further evidence of the importance of astrocytic ObR expression, we performed double-labeling fluorescent in situ hybridization (FISH) and immunohistochemistry in the rat hypothalamus. Laser confocal microscopic image analysis showed that ObR mRNA was present in glial fibrillary acidic protein (+) cells that show distinctive astrocytic morphology as well as in neurons. In addition to the presence of ObR mRNA, ObR protein was shown in both astrocytes and neurons in the rat hypothalamus by double-labeling immunohistochemistry. In cultured rat C6 astrocytoma cells treated with different doses of lipopolysaccharide for 6h, the mRNA for ObRa or ObRb did not show significant changes, as measured by quantitative RT-PCR. However, the protein expression of both ObRa and ObRb, determined by Western blotting, was increased after the C6 cells were treated with either lipopolysaccharide or tumor necrosis factor-alpha. The results indicate that astrocytic ObR expression is present in rats as well as mice, and that it probably plays a role in the neuroinflammatory response.

Increased expression of mu opioid receptors in animals susceptible to diet-induced obesity.

Stimulation of mu opioid receptors preferentially increases the intake of a high fat diet. In this paper we investigated whether there was a difference in the expression of mu opioid receptors between animals susceptible (Osborne-Mendel) or resistant (S5B/Pl) to obesity induced by eating a high fat diet. Immunohistochemical studies demonstrated that Osborne-Mendel rats eating a chow diet had an increased number of mu opioid receptors in the arcuate nucleus when compared to S5B/Pl rats. These immunohistochemical findings were supported by Real Time-PCR which demonstrated that the mRNA level of mu opioid receptors was also increased in the hypothalamus of Osborne-Mendel rats compared to S5B/Pl rats. Low doses of the mu opioid receptor agonist DAMGO [d-Ala(2)-N-Me-Phe(4)-Glycol(5)]-enkephalin administered to Osborne-Mendel rats caused a significant increase in the preference for a diet high in fat. The same doses of DAMGO switched the diet preference of S5B/Pl rats to high fat but did not significantly increase food intake. The combination of these findings suggests that the increased levels of hypothalamic mu opioid receptors in Osborne-Mendel rats may contribute to their preference for a diet high in fat and increase their susceptibility to becoming obese.

Central administration of the RFamide peptides, QRFP-26 and QRFP-43, increases high fat food intake in rats

Pyrogultamylated arginine-phenylalanineamide peptide (QRFP) is strongly conserved across species and is a member of the family of RFamide-related peptides, with the motif Arg-Phe-NH(2) at the C-terminal end. The precursor peptide for QRFP generates a 26-amino acid peptide (QRFP-26) and a 43-amino acid peptide (QRFP-43), both of which bind to the G protein-coupled receptor, GPR103. Recently, QRFP has been characterized in rats, mice and humans and has been reported to have orexigenic properties. In rodents, prepro-QRFP mRNA is expressed in localized regions of the mediobasal hypothalamus, a region implicated in feeding behavior. Increased intake of a high fat diet contributes to increased weight gain and obesity. Therefore, the current experiments investigated the effects of QRFP administration in rats and the effects of a high fat diet on prepro-QRFP mRNA and GPR103 receptor mRNA levels. Intracerebroventricular administration of QRFP-26 (3.0nM, 5.0nM) and QRFP-43 (1.0nM, 3.0nM) dose-dependently increased 1h, 2h, and 4h cumulative intake of high fat (55% fat), but not low fat (10% fat) diet. In Experiment 2, hypothalamic prepro-QRFP mRNA levels and GPR103 receptor mRNA levels were measured in rats fed a high fat or a low fat diet for 21 days. Prepro-QRFP mRNA was significantly increased in the ventromedial nucleus/arcuate nucleus of the hypothalamus of rats fed a high fat diet compared to those fed a low fat diet, while GPR103 mRNA levels were unchanged. These findings suggest that QRFP is a regulator of dietary fat intake and is influenced by the intake of a high fat diet.

Olfactory Bulbectomy Increases Food Intake and Hypothalamic Neuropeptide Y in Obesity-Prone but not Obesity-Resistant Rats

Obese individuals often suffer from depression. The olfactory bulbectomy (OBX) model is an animal model of depression that produces behavioral, physiological, and neurochemical alterations resembling clinical depression. The OBX model was employed to assess depression-related changes in food intake in obesity-prone, Osborne-Mendel (OM) rats and obesity-resistant, S5B/Pl rats. OBX increased food intake in OM rats beginning 7 days following surgery, however, OBX did not alter food intake in S5B/Pl rats at any time point. Fourteen days following surgery, OBX significantly increased locomotor activity (total lines crossed and rears) in the openfield test in OM and S5B/Pl rats. Fifteen days following surgery, prepro-neuropeptide Y (NPY) mRNA levels were significantly increased in the hypothalamus of bulbectomized OM rats and in the medial nucleus of the amygdala of bulbectomized OM and S5B/Pl rats. OBX decreased NPY Y2 receptor mRNA levels in the hypothalamus and medial nucleus of the amygdala in OM rats, while increasing NPY Y2 receptor mRNA levels in the medial nucleus of the amygdala of S5B/Pl rats. These data indicate that though both obesity-prone and obesity-resistant strains were susceptible to the locomotor effects of OBX, food intake and hypothalamic prepro-NPY mRNA were only increased in OM rats. Therefore, strain specific alterations in hypothalamic NPY may account for increased food intake in the obesity-prone rats following OBX, and suggests a potential mechanism to explain the comorbidity of obesity and depression.

Co-localization of TRHR1 and LepRb receptors on neurons in the hindbrain of the rat

We have reported a highly cooperative interaction between leptin and thyrotropin releasing hormone (TRH) in the hindbrain to generate thermogenic responses (Hermann et al., 2006) (Rogers et al., 2009). Identifying the locus in the hindbrain where leptin and TRH act synergistically to increase thermogenesis will be necessary before we can determine the mechanism(s) by which this interaction occurs. Here, we performed heat-induced epitope recovery techniques and in situ hybridization to determine if neurons or afferent fibers in the hindbrain possess both TRH type 1 receptor and long-form leptin receptor [TRHR1; LepRb, respectively]. LepRb receptors were highly expressed in the solitary nucleus [NST], dorsal motor nucleus of the vagus [DMN] and catecholaminergic neurons of the ventrolateral medulla [VLM]. All neurons that contained LepRb also contained TRHR1. Fibers in the NST and the raphe pallidus [RP] and obscurrus [RO] that possess LepRb receptors were phenotypically identified as glutamatergic type 2 fibers (vglut2). Fibers in the NST and RP that possess TRHR1 receptors were phenotypically identified as serotonergic [i.e., immunopositive for the serotonin transporter; SERT]. Co-localization of LepRb and TRHR1 was not observed on individual fibers in the hindbrain but these two fiber types co-mingle in these nuclei. These anatomical arrangements may provide a basis for the synergy between leptin and TRH to increase thermogenesis.

Leptin and thyrotropin-releasing hormone: Cooperative action in the hindbrain to activate brown adipose thermogenesis

Explanations of leptin induction of thermogenesis typically involve primary detection elements in the hypothalamus. In turn, these circuits control medullary raphe neurons that regulate spinal efferent sympathetic projections to heat-producing brown adipose tissue (BAT). The hindbrain may be capable of considerable thermoregulatory capacity independent of the hypothalamus, though little is known about the site(s), mechanism(s) of action, or the physiological consequences of leptin action in the hindbrain. Several reports describe the presence of leptin receptor in the solitary nucleus, and there is functional evidence that leptin can act in the dorsal medulla to suppress feeding. We examined the effects of leptin, applied to the dorsal medulla, on BAT thermogenesis. Leptin alone (< or =25 microg) had no independent effect on BAT thermogenesis. We hypothesized that, while leptin may not be capable of activating thermocontrol mechanisms in the hindbrain directly, it may modulate the efficacy of other neural signals involved in the control of thermogenesis such as thyrotropin-releasing hormone (TRH). We tested the hypothesis that leptin and TRH, acting in the hindbrain, co-regulate thermogenesis. As expected, TRH (0.1 microg), alone, produces a small increase (+0.75 degrees C) in BAT temperature. Co-application of leptin (5 mug) and TRH (0.1 microg) to the dorsal medulla produces an increase in BAT and core temperature more than 300% greater than TRH alone (+3.5 degrees C). This effect is undiminished in the acute decerebrate rat, suggesting that the effect is mediated entirely by the hindbrain.

Leptin into the rostral ventral lateral medulla (RVLM) augments renal sympathetic nerve activity and blood pressure.

Leptin is a hormone released from adipose tissue. While this hormone normally acts to reduce feeding behavior and increase energy expenditure, in obesity, resistance to these effects occurs even though the hormone is released in large amounts. Although leptin no longer works to suppress feeding in the obese, leptin retains its potent effects on other autonomic functions such as blood pressure regulation. Leptin has been associated with hypertension and increased sympathetic autonomic activity. Therefore, leptin is emerging as a major contributor to the hypertensive state observed in obesity. Sympathetic control of blood pressure is maintained principally by autonomic reflex control circuits in the caudal brainstem. The rostral ventral-lateral medulla (RVLM) is the primary regulator of the sympathetic nervous system, sending excitatory fibers to sympathetic preganglionic neurons to regulate sympathetic control over resistance vessels and blood pressure. Previous studies from our laboratory have shown that neurons in the ventral lateral medulla express leptin receptors (ObRb). Our present study using pseudo-rabies multi-synaptic retrograde tract tracing and immunohistochemical methods revealed that neurons within the RVLM that send sympathetic projections to the kidney express leptin receptors. Acute microinjection of leptin (1 and 3 μg; 40 nL) into the RVLM evoked a significant increase in Mean Arterial Pressure (MAP) and renal sympathetic nerve activity (RSNA). When the 3 μg dose of leptin was preceded with a leptin antagonist, (SLAN-4; 1 ng), it attenuated the cardiovascular response of leptin. Taken together, these data suggest that leptins actions within the RVLM may influence blood pressure and renal sympathetic nerve activity.

Sensitivity to the satiating effects of Exendin 4 is decreased in obesity-prone Osborne-Mendel rats compared to obesity-resistant S5B/Pl rats

Background:Osborne–Mendel (OM) rats are prone to obesity when fed a high-fat diet, whereas S5B/Pl (S5B) rats are resistant to diet-induced obesity when fed the same diet. OM rats have a decreased satiation response to fatty acids infused in the gastrointestinal tract, compared to S5B rats. One possible explanation is that OM rats are less sensitive to the satiating hormone, glucagon-like peptide 1 (GLP-1). GLP-1 is produced in the small intestine and is released in response to a meal. The current experiments examined the role of GLP-1 in OM and S5B rats.Methods:Experiment 1 examined preproglucagon mRNA expression in the ileum of OM and S5B rats fed a high-fat (55% kcal) or low-fat (10% kcal) diet. Experiment 2 investigated the effects of a 2 h high-fat meal after a 24 h fast in OM and S5B rats on circulating GLP-1 (active) levels. Experiment 3 examined the effects of exendin-4 (GLP-1 receptor agonist) administration on the intake of a high-fat or a low-fat diet in OM and S5B rats.Results:Preproglucagon mRNA levels were increased in the ileum of OM rats compared to S5B rats and were increased by high-fat diet in OM and S5B rats. OM and S5B rats exhibited a similar meal-initiated increase in circulating GLP-1 (active) levels. Exendin-4 dose dependently decreased food intake to a greater extent in S5B rats compared to OM rats. The intake of low-fat diet, compared to the intake of high-fat diet, was more sensitive to the effects of exendin-4 in these strains.Conclusions:These results suggest that though OM and S5B rats have similar preproglucagon mRNA expression in the ileum and circulating GLP-1 levels, OM rats are less sensitive to the satiating effects of GLP-1. Therefore, dysregulation of the GLP-1 system may be a mechanism through which OM rats overeat and gain weight.

Food deprivation increases the mRNA expression of μ-opioid receptors in the ventral medial hypothalamus and arcuate nucleus

Activation of micro-opioid receptors makes animals hyperphagic and increases their preference for a high-fat diet. Previous studies have suggested that this receptor population plays a role in mediating the hyperphagia that is associated with food deprivation. In this paper, we tested the hypothesis that food deprivation will increase the expression of micro-opioid receptors in the ventral medial hypothalamus and arcuate nucleus (VMH/ARC). Food deprivation resulted in a significant increase in the mRNA expression of micro-opioid receptors in the VMH/ARC and the lateral hypothalamus (LH) after 48 h of fasting but not after 24 or 12 h of fasting in either the light or dark. We did not observe a change in the mRNA expression of kappa- or delta-opioid receptors after food deprivation. When food-deprived animals were given a choice between a low-fat diet and a high-fat diet, they were hyperphagic and consumed significantly more of the high-fat diet. When the micro-opioid receptors were blocked with beta-funaltrexamine (selective mu-opioid receptor antagonist), prior to giving food-deprived animals access to both a low-fat and high-fat diet, it significantly decreased the percentage of high-fat diet consumed. These data demonstrate that hypothalamic micro-opioid receptors may contribute to the hyperphagia and increased preference for a high-fat diet that is associated with food deprivation.