The DMH plays an important role in maintaining energy homeostasis. Lesions of the DMH resulted in hypophagia and reduced body weight (Bellinger and Bernardis, 2002
). Disinhibition of neurons in the DMH provoked nonshivering thermogenesis and elevated core body temperature (Zaretskaia et al., 2002
). Despite these observations, the neural mechanisms underlying these actions of the DMH remain undetermined. Here we establish a critical role for NPY in the DMH in regulating energy homeostasis by using AAV-mediated RNAi to knock down NPY expression in the DMH of intact rats.
We first assessed the effect of DMH NPY knockdown on regulation of body weight. Consistent with the orexigenic effect of DMH NPY (Yang et al., 2009
), we found that DMH NPY knockdown significantly decreased diet-induced hyperphagia, resulted in slower weight gain on both RC and HF diets and reduced body fat mass. In addition, we noted selective effects of DMH NPY on inguinal adiposity and BAT thermogenesis. DMH NPY knockdown resulted in development of brown adipocytes in inguinal WAT, increased UCP1 expression in the inguinal and interscapular BAT and increased energy expenditure and cold-induced thermogenesis. DMH NPY knockdown promoted inguinal lipid mobilization and decreased diet-induced fat accumulation. DMH NPY knockdown also resulted in increased locomotor activity. Together, our results demonstrated that DMH NPY affects multiple aspects of energy homeostasis including food intake, body adiposity, thermogenesis, energy expenditure and physical activity.
Two types of fat, WAT and BAT, exist in mammals including in adult humans (Cypess et al., 2009
; van Marken Lichtenbelt et al., 2009
; Virtanen et al., 2009
). While WAT stores excess calories, BAT burns fat to produce heat via nonshivering thermogenesis as a defense against cold. Both types of fat are innervated by the SNS (Bartness and Bamshad, 1998
; Cannon and Nedergaard, 2004
). Activation of the sympathetic innervation induces lipolysis in WAT (Fredholm and Karlsson, 1970
; Weiss and Maickel, 1968
), and produces thermogenesis through mitochondrial UCP1 in BAT (Cannon and Nedergaard, 2004
). Sympathetic activation via treatment of β-adrenergic agonist or cold stress has also been demonstrated to cause development of brown adipocytes in white fat pads (Himms-Hagen et al., 1994
; Jimenez et al., 2003
; Nagase et al., 1996
). In contrast, intracerebroventricular administration of NPY increases WAT lipoprotein lipase activity (suggesting increased lipid storage) and decreases BAT GDP binding activity (indicating decreased thermogenic activity) in addition to its orexigenic effect (Billington et al., 1991
) and central administration of NPY also suppresses sympathetic activity in interscapular BAT in rats (Egawa et al., 1991
). These observations imply that central NPY may serve as a neuromodulator of the SNS controlling both WAT lipogenesis and BAT thermogenesis. Our current findings provide support for this view and further identify DMH NPY as an important contributing factor to these effects. We found that DMH NPY knockdown resulted in development of brown adipocytes (or white into brown adipocyte transformation) in inguinal WAT and reduced inguinal fat accumulation and that sympathetic denervation prevented this brown adipocyte formation. DMH NPY knockdown also resulted in increased UCP1 expression in the interscapular BAT. These results indicate that DMH NPY normally modulates SNS signaling to influence adiposity and energy homeostasis and that knockdown of NPY expression in the DMH results in increases in peripheral sympathetic tone selectively in the inguinal fat and interscapular brown fat. As a result, Ucp1
gene expression was up-regulated in the inguinal fat and interscapular BAT of NPY knockdown rats, leading to increased thermogenesis and overall increased energy expenditure; and increases in Cpt1a
gene expression with a trend for a decrease in Fas
gene expression in the inguinal fat of NPY knockdown rats appear to cause increased fatty acid oxidation in adipose tissue (increased lipid mobilization), and overall reduce body adiposity.
Bamshad and colleagues (1998)
have investigated the central nervous system origins of SNS outflow to WAT. By using viral transsynaptic retrograde tracer, they found that viral tracer was less detected in the DMH in animals receiving epididymal viral injection than those receiving inguinal injection (Bamshad et al., 1998
), implying that the central nervous control of inguinal WAT is more DMH-related than that of epididymal WAT. In support of this view, we found that DMH NPY knockdown specifically affected lipid mobilization and brown adipocyte formation in inguinal WAT through the SNS. This suggests that DMH NPY is an important factor influencing sympathetic innervation in inguinal WAT, but not epididymal WAT. Overall, in combination with the evidence that the DMH is involved in thermoregulation (Dimicco and Zaretsky, 2007
), our results suggest that NPY in the DMH may serve to modulate actions of both inguinal WAT and interscapular BAT in maintaining energy homeostasis.
WAT contains mature adipocytes for storage of lipids and other types of cells including preadipocytes, fibroblasts, pericytes, endothelial cells, and various blood cells in the stromal-vascular fraction (SVF) (Ailhaud et al., 1992
). Although white fat progenitor cells have been demonstrated to reside in the adipose SVF (Tang et al., 2008
), types of brown fat precursor cells in WAT or whether precursor cells in the SVF of WAT possess the ability to develop into both white and brown adipocytes is unclear. Reversible physiological transdifferentiation between WAT and BAT implies that white and brown adipocytes are mixed in most fat dopets in rodents (including inguinal WAT, Cinti, 2009
). Barbatelli et al. (2010)
further reported that the emergence of cold-induced brown adipocytes in mouse white fat depots (including inguinal WAT) is determined predominantly by white to brown adipocyte transdifferentiation. This trandifferentiation is thought to be directly derived from mature white adipocytes as determined by adipocytes with intermediate features between white and brown adipocytes (referred as transdifferentiating paucilocular adipocytes, Barbatelli et al., 2010
). The present study did not find clear UCP1 immunoreactive paucilocular cells in inguinal fat of NPY knockdown rats as proposed above. In fact, we found numerous clusters of brown-like adipocytes surround by white adipocytes as well as various UCP1 immunoreactive unilocular adipocytes in inguinal adipose tissue of NPY knockdown rats. We further found a significant elevation of Ppar-γ
expression, an essential factor for adipogenesis, in this inguinal fat tissue. Therefore, although there is still the possibility of white into brown adipocyte transdifferentiation in this rat model, our results imply that development of brown adipocytes in inguinal WAT resulting from DMH NPY knockdown may be directly derived from brown fat-like precursor cells in the SVF. To this end, the identification of such brown fat-like precursor cells or determination of this develomental origin merits further investigation.
In addition, we demonstrated a role for DMH NPY in regulation of spontaneous physical activity. We found that knockdown of NPY expression in the DMH resulted in increased locomotor activity. Based on the evidence that nonexercise activity thermogenesis from spontaneous physical activity may play a pivotal role in protection against fat gain (Levine et al., 1999
), our results suggest that the effect of DMH NPY on physical activity may also contribute to its influence on body weight control. Moreover, consistent with the previous report of the nocturnal effect of DMH NPY on feeding behavior (Yang et al., 2009
), DMH NPY knockdown produced a dark phase-specific effect on locomotor activity. These results provide additional evidence indicating a potential role for DMH NPY in regulation of day-night rhythms. Previous studies have suggested a role for the DMH in food-entrainable circadian behavior. Although there is some controversy over the effect of DMH lesions on food anticipatory activity (Gooley et al., 2006
; Landry et al., 2006
), a robust oscillation of Per1
expression has been found in the DMH under restricted feeding (Mieda et al., 2006
). Whether NPY in the DMH is involved in this circadian regulation remains to be determined.
The finding of a role for DMH NPY in glucose homeostasis is also intriguing. Previous studies have shown that the DMH contains both glucoreceptive and glucose-sensitive neurons and lesions of the DMH alter feeding response to exogenous glucose and insulin (Bellinger and Bernardis, 2002
), implicating this region in the regulation of glucose homeostasis. We found that DMH NPY knockdown enhanced insulin sensitivity, improved glucose tolerance and prevented diet-induced hyperglycemia and hyperinsulinemia. These results indicate an important role of NPY in the DMH in the regulation of glucose homeostasis. Whether this is a direct result of reduced NPY expression in the DMH or a consequence of the demonstrated brown adipocytes in inguinal fat, activation of interscapular BAT, and resulting in increased thermogenesis and subsequent lean phenotypes is unclear. Nevertheless, we demonstrate that alterations in DMH NPY signaling influence insulin sensitivity and glucose homeostasis, but the mechanisms through which DMH NPY acts to affect insulin action and regulate glucose levels merit further investigation.
In summary, we demonstrate the physiological importance of DMH NPY in energy homeostasis. DMH NPY affects food intake, body adiposity, thermogenesis, energy expenditure and physical activity to regulate body weight. These results indicate that orexigenic NPY in the DMH normally serves as a key factor in maintaining energy homeostasis and also point to the DMH as a potential target site for therapies aimed at combating obesity and/or diabetes.