Visualization of sympathetic neural innervation in human white adipose tissue

Obesity, defined as an excess of adipose tissue that adversely affects health, is a major cause of morbidity and mortality. However, to date, understanding the structure and function of human adipose tissue has been limited by the inability to visualize cellular components due to the innate structure of adipocytes, which are characterized by large lipid droplets. Combining the iDISCO and the CUBIC protocols for whole tissue staining and optical clearing, we developed a protocol to enable immunostaining and clearing of human subcutaneous white adipose tissue (WAT) obtained from individuals with severe obesity. We were able to perform immunolabelling of sympathetic nerve terminals in whole WAT and subsequent optical clearing by eliminating lipids to render the opaque tissue completely transparent. We then used light sheet confocal microscopy to visualize sympathetic innervation of human WAT from obese individuals in a three-dimensional manner. We demonstrate the visualization of sympathetic nerve terminals in human WAT. This protocol can be modified to visualize other structures such as blood vessels involved in the development, maintenance and function of human adipose tissue in health and disease.     

Electrical activity in white adipose tissue of rat

Intracellular recording of white adipocytes was performed in an in vitro preparation. Resting potential, input resistance and membrane time constant averaged: -34 +/- 9 mV, 295 +/- 161 M omega, and 58 +/- 19 ms respectively (mean +/- SD, n = 32). Intracellular injection of positive and negative square current pulses elicited membrane voltage responses, characterized by a rectification of the voltage change evoked by positive pulses, and a slow return to baseline at the offset of hyperpolarizing pulses. The amplitude and duration of the slow return to resting potential was dependent on membrane potential, pulse duration, and extracellular K+ concentration. This response was depressed when external Ca2+ was replaced by Co2+, and by external application of 4-aminopyridine. These results indicate that white adipocytes can generate membrane voltage responses which may mostly be a consequence of the activity of ionic channels. The properties of the slow return to baseline suggest that it may be due to a transient K+ current.     

Sex differences in sympathetic innervation and browning of white adipose tissue of mice

Background

The higher prevalence of obesity-related metabolic disease in males suggests that female sex hormones provide protective mechanisms against the pathogenesis of metabolic syndrome. Because browning of white adipose tissue (WAT) is protective against obesity-related metabolic disease, we examined sex differences in β3-adrenergic remodeling of WAT in mice.

Methods

Effects of the β3-adrenergic receptor agonist CL316,243 (CL) on browning of white adipose tissue were investigated in male and female C57BL mice. The role of ovarian hormones in female-specific browning was studied in control female C57BL mice and mice with ovarian failure induced by 4-vinylcyclohexene diepoxide treatment for 15 days.

Results

We found that treatment with CL-induced upregulation of brown adipocyte markers and mitochondrial respiratory chain proteins in gonadal WAT (gWAT) of female mice, but was without effect in males. In contrast, CL treatment was equally effective in males and females in inducing brown adipocyte phenotypes in inguinal WAT. The tissue- and sex-specific differences in brown adipocyte recruitment were correlated with differences in sympathetic innervation, as determined by tyrosine hydroxylase immunostaining and western blotting. Levels of the neurotrophins NGF and BDNF were significantly higher in gWAT of female mice. CL treatment significantly increased NGF levels in gWAT of female mice but did not affect BDNF expression. In contrast, estradiol treatment doubled BDNF expression in female adipocytes differentiated in vitro. Ovarian failure induced by 4-vinylcyclohexene diepoxide treatment dramatically reduced BDNF and TH expression in gWAT, eliminated induction of UCP1 by CL, and reduced tissue metabolic rate.

Conclusions

Collectively, these data demonstrate that female mice are more responsive than males to the recruitment of brown adipocytes in gonadal WAT and this difference corresponds to greater levels of estrogen-dependent sympathetic innervation.

     

Regional-dependent increase of sympathetic innervation in rat white adipose tissue during prolonged fasting.

White adipose tissue (WAT) is innervated by the sympathetic nervous system. A role for WAT sympathetic noradrenergic nerves in lipid mobilization has been suggested. To gain insight into the involvement of nerve activity in the delipidation process, WAT nerves were investigated in rat retroperitoneal and epididymal depots after prolonged fasting. A significant increase in tyrosine hydroxylase (TH) content was found in epididymal and, especially, retroperitoneal WAT by Western blotting. Accordingly, an increased immunoreactivity for TH was detected by immunohistochemistry in epididymal and, especially, retroperitoneal vascular and parenchymal noradrenergic nerves. Neuropeptide Y (NPY)-containing nerves were found around arteries and in the parenchyma. Double-staining experiments and confocal microscopy showed that most perivascular and some parenchymal noradrenergic nerves also contained NPY. Detection of protein gene product (PGP) 9.5, a general marker of peripheral nerves, by Western blotting and PGP 9.5-TH by double-staining experiments showed significantly increased noradrenergic nerve density in fasted retroperitoneal, but not epididymal depots, suggesting that formation of new nerves takes place in retroperitoneal WAT in fasting conditions. On the whole, these data confirm the important role of sympathetic noradrenergic nerves in WAT lipid mobilization during fasting but also raise questions about the physiological role of regional-dependent nerve adjustments and their functional significance in relation to white adipocyte secretory products.     

Effects of fasting and food restriction on sympathetic activity in brown adipose tissue in mice

Summary The activity of the sympathetic nervous system in mice that were either fed ad libitum, food restricted or fasted was estimated by measuring the accumulation of dopamine following the inhibition of dopamine -hydroxylase activity. Mice in each group were injected with the dopamine -hydroxylase inhibitor 1-cyclohexyl-2-mercaptoimidazole and were exposed to either 30°C (warm) or 4°C (cold). Mice were killed 1 h after the injection. Both heart and brown adipose tissue were then quickly removed and homogenized in ice-cold perchloric acid. Dopamine and noradrenaline were determined using high performance liquid chromatography. Regardless of whether mice were warm or cold exposed, both content and concentration of brown adipose tissue and dopamine were predictably higher in 1-cyclohexyl-2-mercaptoimidazole-injected mice than in non-injected animals. In mice fed ad libitum, post-injection content and concentration of dopamine in both brown adipose tissue and heart were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice, post-injection concentrations of dopamine in brown adipose tissue were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice there was no difference between warm- and cold-exposed animals with respect to post-injection contents and concentrations of dopamine in heart tissue. In fasted mice there was no difference between warm- and cold-exposed animals in post-injection content of dopamine in brown adipose tissue. This study provides further evidence that fasting, in contrast to food restriction, may blunt the tissue sympathetic nervous system response in brown adipose tissue of cold-exposed mice.Abbreviations BAT brown adipose tissue - CHMI 1-cyclohexyl-2-mercaptoimidazole - DA dopamine - DHBA dihydroxybenzylamine - EDTA ethylenediaminetetra-acetic acid - HPLC high performance liquid chromatography - NA noradrenaline - PCA perchloric acid - SNS sympathetic nervous system     

The stimulation of lipogenesis in white adipose tissue from fed rats by corticosterone

The direct effects of a physiological concentration of corticosterone (50 ng ml-1) in presence of insulin (200 microU ml-1) on lipid synthesis and CO2 production from glucose and glycerol release were evaluated in vitro in white adipose tissue after pre-incubation with the hormones. Lipid synthesis was 27% higher after 24 h and 66% higher after 48h pre-incubation with corticosterone and insulin compared with insulin alone. Basal and adrenaline-stimulated glycerol release and CO2 production were unchanged after pre-incubation with both hormones compared with insulin alone. We propose that corticosterone acts as a pro-lipogenic hormone on adipose tissue in the fed rat, in contrast to its glucose sparing effects in the fasted animal.     

Fish oil and corn oil induced differential effect on beiging of visceral and subcutaneous white adipose tissue in high-fat-diet-induced obesity

Obesity is characterised by excessive accumulation of fat in white adipose tissue (WAT) which is compartmentalised into two anatomically and functionally diverse depots - visceral and subcutaneous. Advice to substitute essential polyunsaturated fatty acids (PUFAs) for saturated fatty acids is a cornerstone of various obesity management strategies. Despite an array of reports on the role of essential PUFAs on obesity, there still exists a lacuna on their mode of action in distinct depots i.e. visceral (VWAT) and subcutaneous (SWAT). The present study aimed to evaluate the effect of fish oil and corn oil on VWAT and SWAT in high-fat-diet-induced rodent model of obesity. Fish oil (FO) supplementation positively ameliorated the effects of HFD by regulating the anthropometrical and serum lipid parameters. FO led to an overall reduction in fat mass in both depots while specifically inducing beiging of adipocytes in SWAT as indicated by increased UCP1 and PGC1α. We also observed an upregulation of AMPKα and ACC1/2 phosphorylation on FO supplementation in SWAT suggesting a role of AMPK-PGC1α-UCP1 axis in beiging of adipose tissue. On the other hand, corn oil supplementation did not show any improvements in adipose tissue metabolism in both the depots of adipose tissue. The results were analysed using one-way ANOVA followed by Tukey's test in Graphpad Prism 5.0. Combined together our results suggest that n-3 PUFAs exert their anti-obesity effect by regulating adipokine secretion and inducing beiging of SWAT, hence increasing energy expenditure via thermogenic upregulation.     

Effects of noradrenaline and insulin on electrical activity in white adipose tissue of rat.

An active change in membrane voltage responses to hyperpolarizing pulses has been identified by intracellular recording on an in vitro preparation of white adipose tissue. This change was characterized by a slow return to baseline at the offset of the pulses. Amplitude and duration of the slow return to baseline were dependent on extracellular K+ concentration, and were diminished by external application of Ba2+. Such properties suggest that this electrical response can be mainly due to activation of transient K+ conductances. The effects that noradrenaline and insulin have over the slow return to baseline have been also studied. While external addition of noradrenaline decreased amplitude and duration of this electrical response, insulin produced the opposite effect. These results suggest that noradrenaline and insulin could modulate K+ conductances in white adipocytes.     

Lipoprotein lipase activity in white adipose tissue of rats subjected to exercise--rest cycles

This study was conducted to determine serum lipid levels and the activity of lipoprotein lipase in epididymal white adipose tissue of rats undergoing exercise training. During the 8-week period of treatment, one group of rats was kept sedentary and the remaining animals were exercise trained either continually (1 h of daily treadmill running) or intermittently (alternate weeks of daily running and inactivity). Exercise training, either continual or intermittent, decreased postprandial serum total and high-density lipoprotein cholesterol concentrations, which returned to sedentary levels in the intermittently trained animals following a week of rest. Lipoprotein lipase activity in whole epididymal adipose pad was lower in rats trained continually than in the sedentary group at the end of the treatment. The intermittent training program elicited large fluctuations in both the specific (per milligram of protein) and total (per tissue) activity of lipoprotein lipase in white adipose tissue. During rest periods, enzyme activity rose to levels that were higher than those of sedentary rats, whereas lipase activity was below that of sedentary animals following a week of running. In the last exercise--rest cycle, body weight gain of the intermittently trained rats was nearly abolished during the week of running, but it increased above that of sedentary animals during weeks of rest. The present results suggest that the modulation of lipoprotein lipase activity in white adipose tissue is one of the adaptations that take place to accommodate the fluctuations in the rate of energy deposition that occur in the rat during an intermittent training program.     

Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.     

Involvement of the sympathetic nervous system in lipolytic activity in brown adipose tissue of cold acclimated rats

In cold acclimated rats, in vitro, NE led to a significant increase in release of FFA and glycerol in denervated IBAT. In vivo, study of arteriovenous differences showed that the denervated BAT loses its full capacity to utilize FFA and glycerol released by NE. After denervation an increase of blood flow in Sulzer's vein was observed. This effect appeared immediately after intervention whereas the effect on fat metabolism appeared later. In cold acclimated rats, the sympathetic nervous system appears to be an important regulator of fatty acid metabolism in BAT.     

Relationship between hypothalamic noradrenergic activity and the sympathetic activity in interscapular brown adipose tissue after cold-swim stress in rats.

Noradrenaline (NA) activities in both hypothalamus and interscapular brown adipose tissue (IBAT) were simultaneously assessed before and after cold-swim stress in rats. The technique of gas chromatography-mass spectrometry was employed for the analysis of NA and its primary neuronal metabolite, 3,4-dehydroxy-phenylethylenglycol (DHPG), and the ratio of DHPG to NA was used as an index of NA activity. The ratios of DHPG/NA in both hypothalamus and IBAT were significantly elevated 5 and 20 min after cold-swim stress. Moreover, we found that there is a highly significant positive relationship between the hypothalamic DHPG/NA ratio and the ratio of DHPG/NA in IBAT (r = 0.872, p less than 0.0001). This observation strongly supports the concept in which hypothalamic NA neurons play an important role in modulating the sympathetic outflow.     

Intracerebroventricular administration of urotensin II regulates food intake and sympathetic nerve activity in brown adipose tissue

To clarify the functional roles of urotensin II in regulating energy balance, we investigated the effects of a central infusion of urotensin II on food intake, uncoupling protein (UCP) 1 mRNA expression, temperature, and sympathetic nervous system activity in brown adipose tissue (BAT), a site that regulates energy expenditure in rodents. A bolus central infusion of urotensin II at a dose of 1 nmol/rat into the third cerebral ventricle decreased food intake (p<0.05). Additionally, urotensin II induced c-Fos-like-immunoreactivity (c-FLI) in the paraventricular nucleus (PVN) as compared with that in the control (phosphate buffered saline [PBS]-treated) group. Furthermore, urotensin II increased BAT UCP 1 mRNA expression (p<0.05). Finally, central infusion of urotensin II significantly increased BAT sympathetic nerve activity, which was accompanied by a significant elevation in BAT temperature (p<0.05) in rats. Taken together, central infusion of urotensin II regulates food intake and BAT sympathetic nerve activity in rats.     

Sexual dimorphism in white and brown adipose tissue with obesity and inflammation

This article is part of a Special Issue “Energy Balance”.     

Enzyme histochemical differentiation of white adipose tissue in the rat

Subcutaneous adipose tissues from fetal and young rats were studied with enzyme histochemical techniques. Lipid staining and histological evaluation were also utilized to compare the development of a wide variety of enzyme activities to cytoplasmic lipid deposition and morphological differentiation of adipocytes. Three distinct stages of adipose-tissue differentiation were postulated. In stage III, adipocytes were morphologically differentiated (rounded, basal-lamina positive) and enzyme reactive for many enzymes. In stage II, however, adipocytes were reactive for some enzymes but were not morphologically differentiated. Stage I adipose tissue was histologically distinct from connective tissue but did not contain lipid-laden cells or enzyme-reactive cells. Stages I and II (95%) were predominant in fetuses, whereas stage III (90%) was predominant in young animals. Histochemical analysis of adipocytes in newborn rats established the metabolic competence of these cells despite their small size. These studies indicate that enzymatic differentiation of adipocytes clearly precedes morphological differentiation.