Proteomic analysis of rat hypothalamus revealed the role of ubiquitin-proteasome system in the genesis of DR or DIO.

Obesity has become a global epidemic, contributing to the increasing burdens of cardiovascular disease and type 2 diabetes. However, the precise molecular mechanisms of obesity remain poorly elucidated. The hypothalamus plays a major part in regulating energy homeostasis by integrating all kinds of nutritional signals. This study investigated the hypothalamus protein profile in diet-induced obese (DIO) and diet-resistant (DR) rats using two dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF-MS analysis. Twenty-two proteins were identified in the hypothalamus of DIO or DR rats. These include metabolic enzymes, antioxidant proteins, proteasome related proteins, and signaling proteins, some of which are related to AMP-activated protein kinase (AMPK) signaling or mitochondrial respiration. Among these proteins, in comparison with the normal-diet group, Ubiquitin was significantly decreased in DR rats but not changed in DIO rats, while Ubiquitin carboxyl-terminal esterase L1 (UCHL-1) was decreased in DIO rats but not changed in DR rats. The expression level of Ubiquitin and UCHL-1 were further validated using Western blot analysis. Our study reveals that Ubiquitin and UCHL-1 are obesity-related factors in the hypothalamus that may play an important role in the genesis of DR or DIO by interfering with the integrated signaling network that control energy balance and feeding.

Peroxiredoxin1: a potential obesity-related factor in the hypothalamus.

BACKGROUND: Obesity has become a global epidemic and contributes to the increasing burden of type 2 diabetes, cardiovascular disease and some cancers worldwide. However, the precise molecular mechanisms of obesity remain poorly elucidated. Recent data indicate monitoring of brain energy status especially in the hypothalamus, is crucial for energy homeostasis. Anti-oxidant defenses are suggested to play an important role in production of a well-balanced signal, which is necessary for adjusted neuronal activities. MATERIAL/METHODS: Hypothalamus proteomes of the diet-induced obesity (DIO) and diet-resistant (DR) rats were analyzed using 2-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF-MS analysis. RESULTS: The 2-DE maps showed a resolution of 1280±15, 1250±10, 1260±12 protein spots in normal, DIO and DR groups, respectively. Twenty-two proteins were identified in the hypothalamus of DIO or DR rats. These proteins are involved in metabolism enzymes, antioxidant proteins, proteasome-related proteins and signaling proteins. In comparison with normal-diet group, peroxiredoxin1 (Prx1) was significantly decreased in DR group and it was increased in DIO group. The expression of Prx1 was further validated by Western blot analysis. CONCLUSIONS: The differential expression of Prx1 in DR and DIO groups indicated that the antioxidant protein may play an important role in the genesis of DR or DIO. This study provides new clues to illuminate the mechanisms involved in the progress of DIO and DR in the hypothalamus.

Electroacupuncture in the treatment of obesity.

Obesity is becoming one of the most common health problems in the world. Many other disorders, such as hypertension and diabetes are considered as the consequences of obesity. Since effective remedies are rare (only two drugs, Orlistat and Sibutramine, were officially approved by the US Food and Drug Administration for long-term obesity treatment so far), researchers are trying to discover new therapies for obesity, and acupuncture is among the most popular alternative approaches. To facilitate weight reduction, one can use manual acupuncture, electroacupuncture (EA) or transcutaneous electrical acupoint stimulation (TEAS). As the parameters of the EA or TEAS can be precisely characterized and the results are more or less reproducible, this review will focus on EA as a treatment modality for obesity. Results obtained in this laboratory in recent five years will be summarized in some detail.

Electroacupuncture suppresses expression of gastric ghrelin and hypothalamic NPY in chronic food restricted rats.

Electroacupuncture (EA) has been reported to reduce body weight in overweight subjects in clinical practice, as well as in rats and mice with diet-induced obesity. In the present study, this effect of EA was tested in lean rats subjected to long-term food restriction (FR, food was offered only 1 h/day). Two hertz EA administered once every other day produced a further reduction in body weight in FR rats. Exploration of the mechanism involved revealed significant downregulation of the orexigenic peptides: ghrelin in the stomach, and neuropeptide Y (NPY) but not Agouti-related peptide (AgRP) in the hypothalamus, which was in line with the reduction in food intake in rats receiving EA stimulation as compared with those receiving restraint only. Uncoupling protein 3 (UCP3), involved in accelerating energy expenditure, was not significantly altered. These results suggest that the EA-induced body weight reduction was due mainly to a decrease in food intake rather than an increase in energy expenditure. A reduction in the orexigenic peptides ghrelin and NPY may be involved in the underlying mechanism.

Distribution of beacon immunoreactivity in the rat brain.

Beacon is a novel peptide isolated from the hypothalamus of Israeli sand rat. In the present study, we determined the distribution of beacon in the rat brain using immunohistochemical approach with a polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73). The hypothalamus represented the major site of beacon-immunoreactive (IR) cell bodies that were concentrated in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Additional immunostained cells were found in the septum, bed nucleus of the stria terminalis, subfornical organ and subcommissural organ. Beacon-IR fibers were seen with high density in the internal layer of the median eminence and low to moderate density in the external layer. Significant beacon-IR fibers were also seen in the nucleus of the solitary tract and lateral reticular formation. The beacon neurons found in the PVN were further characterized by double label immunohistochemistry. Several beacon-IR neurons that resided in the medial PVN were shown to coexpress corticotrophin-releasing hormone (CRH) and most labeled beacon fibers in the external layer of median eminence coexist with CRH. The topographical distribution of beacon-IR in the brain suggests multiple biological activities for beacon in addition to its proposed roles in modulating feeding behaviors and pituitary hormone release.

Up-regulation of the expression of cocaine and amphetamine-regulated transcript peptide by electroacupuncture in the arcuate nucleus of diet-induced obese rats.

It was reported that acupuncture or electro-acupuncture (EA) is effective in reducing the body weight for obese patients, although the mechanisms remain obscure. In a previous study, we have found that rats fed with high-fat (HIF) diet developed diet-induced obesity (DIO) with a concomitant decrease in the hypothalamic content of the cocaine and amphetamine-regulated transcript (CART) peptide, a peptide with anorexiogenic effect. To assess the central effect of EA on DIO rat, we revealed that EA up-regulated the expression of CART peptide in the arcuate nucleus (ARC) of the DIO rats. After feeding with HIF diet for 14 weeks, the DIO rats received EA stimulation three times per week for 4 weeks. The expression of CART peptide in ARC was measured using immunohistochemistry. The plasma ACTH was measured with ELISA. EA caused a reduction of both body weight and energy intake in DIO rats and increased the expression of CART peptide in ARC. The plasma ACTH was increased in response to restraint stress, but EA produced no further increase in ACTH levels. The results suggest that EA can up-regulate the expression of CART peptide to approach normal level, resulting in an inhibition of food intake and a reduction of body weight in DIO rats.

Changes of hypothalamic alpha-MSH and CART peptide expression in diet-induced obese rats.

Two hypothalamic peptides, cocaine and amphetamine-regulated transcript (CART) and alpha-melanocyte-stimulating hormone (alpha-MSH), recognized as anorexigenic neuropeptides to suppress the feeding behavior, were monitored in rats fed with a high-fat (HIF) diet for 14 weeks. While half of the rats developed obesity (diet-induced obese, DIO), some did not (diet resistant, DR). Compared to the DR rats and the control rats (fed with standard chow), DIO rats were accompanied by a markedly higher energy intake and a decrease in the number of neurons carrying alpha-MSH and CART peptide in the arcuate nucleus of the hypothalamus. Failure of hypothalamic anorexigenic peptides CART and alpha-MSH to increase their content in response to HIF diet may play a key role for overly high energy consumption, resulting in obesity.

Diet-induced obese rats exhibit impaired LKB1-AMPK signaling in hypothalamus and adipose tissue.

AMPK not only acts as a sensor of cellular energy status but also plays a critical role in the energy balance of the body. In this study, LKB1-AMPK signaling was investigated in diet-induced obese (DIO) and diet resistant (DR) rats. In hypothalamus, DIO rats had lower level of LKB1, AMPKα and pAMPKα than chow-fed or DR rats. Both orexigenic peptide NPY and anorexigenic peptide POMC expression were reduced in hypothalamus of DIO rats. i.c.v. injection of AICAR, an activator of AMPK, increased NPY expression but did not alter POMC expression in DIO rats. In periphery, LKB1 protein content and pAMPKα level were lower in the adipose tissue of DIO rats compared to chow-fed and DR rats. Moreover, pAMPKα and LKB1 protein levels obtained from epididymal fat pad were inversely correlated with epididymal fat mass. LKB1 protein content and pAMPKα in skeletal muscle of DIO rats were not different from those in the muscles of chow-fed and DR rats. In summary, DIO rats, but not DR rats, have impaired LKB1-AMPK signaling in hypothalamus and adipose tissue, suggesting the disturbed energy balance observed in DIO rats is related with abnormalities of AMPK signaling in a tissue specific manner.

Arcuate nucleus of hypothalamus is involved in mediating the satiety effect of electroacupuncture in obese rats.

Obesity is a major health problem in the world. Since effective remedies are rare, researchers are trying to discover new therapies for obesity, and acupuncture is among the most popular alternative approaches. This study investigated the anti-obesity mechanisms of EA, using a rat model of diet-induced obesity. After feeding with a high-fat diet for 9 weeks, a number of rats who gained weight that surpassed the maximal body weight of rats in the chow-fed group were considered obese and employed in the study. A 2 Hz EA treatment at the acupoints ST36/SP6 with the intensity increasing stepwise from 0.5-1-1.5 mA was given once a day for 30 min. Rats treated with EA showed significantly decreased food intake and reduced body weight compared with the rats in DIO and restraint group. EA treatment increased peptide levels of α-MSH and mRNA levels of its precursor POMC in the arcuate nuclear of hypothalamus (ARH) neurons. In addition, the cerebral spinal fluid (CSF) content of α-MSH was elevated by EA application. ARH lesions by monosodium glutamate abolished the inhibition effect of EA on food intake and body weight. A non-acupoint stimulation did not show the benefit effect on food intake inhibition and body weight reduction compared with restraint and ST36/SP6 EA treatment. We concluded that EA treatment at ST36/SP6 acted through ARH to significantly inhibit food intake and body weight gain when fed a high-fat diet and that the stimulation of α-MSH expression and release might be involved in the mechanism.