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1 Department of Physiology, University of California San Francisco, San Francisco, CA, USA
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(Received 7 May 2007;
accepted after revision 19 June 2007;
first published online 21 June 2007)
Corresponding author J. P. Warne: Department of Physiology, Box 0444, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA. Email: james.warne{at}ucsf.edu
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Introduction |
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The vagus nerve represents a two-way highway of communication between the brain and the periphery, as shown by the presence of afferent (sensory) and efferent (motor) fibres of parasympathetic origin (Berthoud, 2004). Afferent nerve fibres from the liver join those originating from the duodenum, pancreas, pylorus and distal gastric antrum (collectively known as the gastroduodenal branch) to form the common hepatic branch of the vagus. The majority of fibres within the common hepatic branch originate from the gastroduodenal branch; however, the liver is represented by fibres originating from the hepatic artery, portal vein and bile ducts, with little or no contribution of fibres from the hepatic parenchyma (Magni & Carobi, 1983; Carobi & Magni, 1985). Fibres at these locations are in prime positions to detect changes in hepatic output as well as sample the hormonal and metabolic milieu entering the liver.
Studies using hepatic branch vagotomy (HV) have shown the common hepatic branch of the vagus nerve to be important in the regulation of food intake. In STZ-diabetic high corticosterone clamped rats, HV promotes voluntary lard intake, suggesting the vagus nerve ordinarily has an inhibitory influence on lard intake, which can be overcome by insulin action (Warne et al. 2007). HV also prevents the lard-induced inhibition of food intake and changes in neuropeptide expression in STZ-diabetic rats (La Fleur et al. 2003, 2005b) and inhibits mercaptoacetate-induced food intake (Langhans, 2000). Hence, signals traversing the common branch of the hepatic vagus clearly regulate, and are responsive to, lard intake. However, it is unclear if the inhibitory signal for lard intake from the common hepatic branch of the vagus is afferent or efferent in nature.
In recent years, understanding of the importance of afferent and efferent signalling through the vagus has become apparent, which has led to the discovery of complex axes that regulate metabolism. For example, in addition to the classical direct actions of insulin on the liver, insulin can act within the brain to suppress liver gluconeogenesis via a vagal signal (Obici et al. 2002; Pocai et al. 2005a,b). Afferent signalling from the hepatic vagus and efferent sympathetic signalling to adipose tissue also regulates energy expenditure, glucose metabolism and fat distribution (Uno et al. 2006). Hence, determining the contribution of afferent and efferent vagal signalling in the regulation of food intake and metabolism is an important therapeutic consideration in pathological conditions where these processes are dysregulated.
Since the common branch of the hepatic vagus exhibits an inhibitory signal for lard intake that can be overcome by insulin action (Warne et al. 2007), we sought to examine if this inihibitory signal is mediated by afferent fibres. The following groups of rats were studied: non-diabetic citrate (the vehicle for STZ) injected and STZ-diabetic with either saline (‘STZ-saline’) or insulin (‘STZ-insulin’) infused into the superior mesenteric vein. These groups were further subdivided into those whose hepatic vagus was treated with the afferent-specific neurotoxin capsaicin and those that were treated with vehicle. All rats received a subcutaneous pellet of corticosterone. After 5 days of recovery, all rats were offered lard in addition to chow for a further 5 days. At the end of the experiment, plasma samples were taken to confirm concentrations of steady-state corticosterone and insulin. Biological actions of both corticosterone and insulin were confirmed by examining adrenal, thymus and spleen weights, liver glycogen content and the plasma levels of leptin, glucagon, glucose, triglycerides, total ketone bodies, free fatty acids (FFAs) and glycerol. White adipose tissue (WAT) depots were excised and weighed to gauge outcomes of lard intake.
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Male rats (Sprague–Dawley, Simonsen, Gilroy, CA, USA) weighing 298 ± 1 g were housed individually in hanging wire cages in a temperature- (22°C) and light- (lights on 06.00–18.00 h) controlled room. Rats were allowed to adapt to their new environment for 4 days before experimentation. All experimental procedures were approved by the University of California San Francisco Institutional Animal Care and Use Committee. The rats had ad libitum access to pelleted rat chow (Purina Chow no. 5008, Purina, St Louis, MO, USA; 3.31 kcal g–1) and water throughout the experiment.
Experimental timeline
All surgical procedures and treatments were performed in one surgery (day 0). The rats (n = 6 per group) were then allowed 5 days to recover, during which incisions, body weight and food and water intake were monitored daily. All rats were also presented with an ad libitum supply of lard (Armour, Omaha, NB, USA; 9 kcal g–1) in a metal cup on day 5. Body weight and solid and liquid intakes were monitored daily for a further 5 days. On day 10, all rats were killed by decapitation and samples were collected.
The model
The rodent model utilized in these studies employs sustained steady-state corticosterone infusion that produces circulating concentrations corresponding to circadian maximum levels (Akana et al. 1985). These levels ensure the widest range of lard intake (la Fleur et al. 2004) such that any effects of our surgical manipulations could be seen. On this background, voluntary lard intake was reduced by inducing insulin-dependent diabetes using STZ. Lard intake can be rescued in STZ-diabetic rodents by steady-state insulin infusions (la Fleur et al. 2004; Warne et al. 2006). Since corticosterone levels are elevated by STZ-diabetes, which produces the characteristics of rodent chronic stress (Scribner et al. 1991; Scribner et al. 1993), implantation of a corticosterone pellet controls for this otherwise confounding variable across all groups by shutting down endogenous corticosterone production (Akana et al. 1992). The site of insulin replacement was chosen based on previous studies. Insulin replaced either subcutaneously using a pellet (la Fleur et al. 2004) or venously through either the superior mesenteric or right external jugular veins (Warne et al. 2006) all promote lard intake in STZ-diabetic rats. However, infusion into the superior mesenteric vein promotes lard intake most reliably and robustly, is strongly affected by the integrity of the common branch of the hepatic vagus (Warne et al. 2007) and infusion into this site most closely mimics the path of insulin from the pancreas, which has the most physiological relevance in terms of tissue exposure to insulin when factoring in liver clearance (Field, 1973).
Surgical procedures and treatments
All rats were anaesthetized using ketamine (75 mg kg–1, I.M.) and xylazine (10 mg kg–1, I.M.). Ketoprofen (10 mg kg–1, S.C.) was provided as an analgesic after surgery but prior to the rat regaining consciousness. After all appropriate surgical manipulations were performed, all incisions were closed using silk suture.
Capsaicin treatment.
The capsaicin treatment protocol was adapted from that of previous studies (Erin et al. 2000; Horn et al. 2001; Foster & Bartness, 2006). Briefly, the common hepatic vagal branch was visualized through the incision into the left side of the body by gently moving aside surrounding tissues, which were held out of the field of view by saline-soaked sterile gauze. The common hepatic vagal branch was located as it separates from the left vagal trunk. One centimetre of the vagus was exposed, gently lifted and a small piece of parafilm was placed underneath. This section of the vagus was then selectively saturated in capsaicin (10 mg ml–1; Sigma-Aldrich, St Louis, MO, USA) in vehicle (10% EtOH, 10% Tween-80, 80% 0.15 M NaCl)) by placing a small capsaicin-saturated cotton bud (
5 mm in diameter) onto the site of interest. Capsaicin was re-applied to the bud at 5 min intervals, such that a total of 1 mg (0.1 ml) of capsaicin was applied to the vagus of each rat. After 30 min the cotton bud was removed, the vagus was cleaned using sterile saline and the parafilm removed. Rats in the vehicle subgroups were treated similarly, with the vagus instead being saturated with vehicle.
STZ-induced diabetes. Diabetes was induced by a subcutaneous injection of STZ (Sigma Chemicals; 65 mg kg–1 in citrate buffer pH 4.2). Rats in the non-diabetic groups were injected with a comparable volume of citrate buffer (2 ml kg–1).
Insulin replacement. Steady-state insulin (3 U day–1; Humulin R U500, Eli Lilly and Company, Indianapolis, IL, USA) or saline was infused into the superior mesenteric vein of STZ-treated rats. Catheters (PE5 tubing, 1.5 cm, fused to PE60 tubing, 1.5 cm) attached to osmotic minipumps (Alzet, model 2002, Alza, Palo Alto, CA, USA) were inserted into the vein and sealed in place using sterile glue (Vetabond, 3M Animal Care Products, St Paul, MN, USA), as previously described (Warne et al. 2006).
Corticosterone treatment. Steady-state circulating corticosterone levels were achieved by placing a 100 mg pellet of corticosterone (100%; Steraloids Inc., Newport, RI, USA) subcutaneously through a small incision in the back.
Sample collection
After the rats were killed, trunk blood was collected into chilled tubes containing 100 µl EDTA (65 mg ml–1). Tubes were centrifuged at 4°C, plasma collected and stored at –80°C. The hepatic branch of the vagus was dissected out, placed in a mould containing Tissue-Tek (Sakura Finetek USA Inc., Torrance, CA, USA) and immediately frozen. Liver biopsies (150 mg) were quickly collected from the lobus sinister lateralis, snap frozen and stored at –80°C. The rest of the body was put onto ice for subsequent dissection and weighing of the WAT fat depots (subcutaneous (scWAT), epididymal (eWAT), perirenal (pWAT) and mesenteric (mWAT)), the thymus, adrenals and spleen. At this time, position of the catheters and osmotic mini-pumps was verified. In all cases, one end of the catheter was attached to the mini-pump, and the other end securely inserted into the superior mesenteric vein.
Immunocytochemistry
Calretinin- and tyrosine hydroxylase (TH)-immuno-reactive nerve fibres of the hepatic branch of the vagus were detected by immunocytochemistry as previously described (Foster & Bartness, 2006). Sections of frozen vagus (10 µm thick) were cut, mounted onto glass slides and kept frozen at –80°C until processed. The sections were defrosted, washed in distilled water and fixed in 4% paraformaldehyde for 15 min. The sections were then washed (2 x 5 min) in distilled water and incubated in 0.3% H2O2 for 30 min at room temperature to block endogenous peroxidase activities. Following three washes (2 x 0.015 M phosphate-buffered saline (PBS), 1 x 0.15 M PBS, each wash 10 min), the sections were blocked in 0.15 M PBS containing 2% normal goat serum (NGS) and 0.4% Triton X-100 for 20 min. Sections were then incubated overnight with the primary antibodies (in 0.15 M PBS containing 2% NGS and 0.4% Triton X-100), mouse anti-calretinin (1: 500; Chemicon International, CA, USA) or mouse anti-TH (1: 500; Chemicon International) in a humid chamber at 4°C. Antibody specificity was demonstrated by incubating sections without the primary antibody; each procedure resulted in no immunoreactivity. After incubation with the primary antibody, the slides were rinsed in 0.015 M PBS (3 x 5 min) and incubated with horse anti-mouse biotinylated IgG (1: 200 in 0.015 M PBS + 2% NGS + 0.4% Triton X-100) for 20 min at room temperature. The slides were washed (0.015 M PBS, 3 x 5 min) and incubated in a solution containing the avidin–biotin horseradish peroxidase complex (Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Immunoreactivity was visualized using 0.075% diaminobenzidine and 0.02% H2O2 in 0.05 M Tris buffer (pH 7.6) as the chromagen for 3 min. The slides were washed with tap water, counterstained with cresyl violet, dehydrated through a graded series of ethanol concentrations, immersed in xylene (2 x 5 min) and coverslipped in permount. Sections were then visualized by light microscopy. Positive fibres, including all within nerve bundles, were counted manually and numbers were standardized per square millimetre of the vagus section.
Plasma and liver assays
Plasma corticosterone, insulin, glucagon and leptin concentrations were assessed by radioimmunoassays at half volumes (MP Biomedicals, Orangeburg, NY, USA; Linco Research Inc., St Charles, MO, USA). Plasma glucose, triglycerides, glycerol, total ketone bodies and FFAs were measured colourimetrically using commercially available kits (Mega Diagnostics, Los Angeles, CA, USA; Sigma-Aldrich; Wako Chemicals, Neuss, Germany). Liver glycogen was extracted in 30% potassium hydroxide (30 min, 100°C), precipitated using ethanol (95%, equal volume), pelleted by centrifugation (800 g, 30 min, 4°C), reconstituted in distilled water and measured using a colourimetric assay outlined previously (Lo et al. 1970). Glycogen values were standardized to milligrams wet liver weight. All assays were as previously described (la Fleur et al. 2004; Warne et al. 2006, 2007).
Statistical analyses
All data are presented as the mean ± standard error of the mean (S.E.M.). Data were analysed by 2-way ANOVA (factors being diabetic status and capsaicin versus vehicle treatment of the nerves). Significant (P < 0.05) effects were followed by post hoc tests of individual group differences (Tukey's test). Where appropriate, repeated measures ANOVA was employed to monitor changes with time.
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Food intake is presented in Fig. 2. As shown in Fig. 2A, after surgery, chow intake rose in all groups until day 3; thereafter the STZ-treated groups continued to increase chow intake with time, whereas the citrate-treated (hence non-diabetic) control groups stabilized chow intake. Presentation of lard on day 5 caused a reduction in chow intake in all groups (day 6 measures). Thereafter, the citrate-treated groups exhibited reduced chow intake compared with previous, no-choice intake. The STZ-treated groups, however, diverged from day 7 onwards; this depended on the infusion into the superior mesenteric vein. STZ-treated rats infused with saline progressively increased chow intake back to pre-choice levels, whereas those rats infused with insulin ate less chow than these groups. Total chow intake for the 5 days of choice illustrated a significant (P < 0.001) difference between the insulin-manipulated groups, but no effect of vagal manipulation. Specifically, the STZ-treated groups ate significantly (P < 0.001) more than the citrate-treated groups. Insulin infusion into the STZ-treated rats reduced chow intake, compared with saline infusion, but hyperphagia remained evident.
Lard intake is shown in Fig. 2C and D. As illustrated in Fig. 2C, presentation of lard on day 5 caused all rats to consume the lard equally during the first day of availability (day 6 data). Thereafter, the insulin- and vagus-associated patterns were evident. Total 5 day intake of lard is presented in Fig. 2D. In the citrate-injected groups, capsaicin treatment of the vagus did not modify lard intake. STZ injection significantly (P < 0.05) reduced lard intake, which was prevented by capsaicin treatment, but not to the level of the non-diabetic controls. Examination of the pattern of lard intake of the STZ-injected, capsaicin-treated group showed that lard intake was comparable on the second, forth and fifth days of lard presentation (days 7, 9 and 10; Fig. 2C), at levels slightly lower than the insulin-replaced counterparts. Lard intake on the third day of lard availability (day 8) was low, and comparable to the STZ-injected, vehicle-treated group. Insulin infusion into the superior mesenteric vein elevated lard intake to levels of non-diabetic controls. Capsaicin treatment did not modify lard intake in the insulin-infused groups.
Total caloric intake is presented in Fig. 2E and F. Similar to chow intake, STZ induced a significant (P < 0.01) increase in total caloric intake. As revealed by 2-way ANOVA, insulin replacement significantly (P < 0.05) reduced total intake (Fig. 2F). Post hoc analysis showed significant (P < 0.05) differences between the saline- and insulin-infused groups that received capsaicin treatment, but not those treated with vehicle on the common branch of the hepatic vagus.
All rats lost body weight throughout the study (Fig. 3), in keeping with previous observations (Warne et al. 2006). STZ injection further exacerbated the weight loss, compared with citrate-injected (non-diabetic) controls. The STZ-induced weight loss was partially prevented by insulin infusion, an action that was curtailed by capsaicin treatment of the common branch of the hepatic vagus. Body weight loss was halted and reversed by provision of lard on day 5.
WAT weight showed effects of STZ, insulin replacement and capsaicin treatment, as shown in Fig. 4. Capsaicin treatment of the vagi of the citrate-injected controls had no effect on the weight of any WAT depot examined. STZ treatment (saline infused) served to significantly (P < 0.05) reduce the weight of all fat pads examined. Insulin infusion into STZ-treated rats served to significantly (P < 0.05) elevate mWAT weight above non-diabetic levels (Fig. 4A), restore eWAT weight (Fig. 4C) and partially restore pWAT weight (Fig. 4D) to that of the citrate-injected controls, without affecting scWAT weight (Fig. 4B), in keeping with previous observations (Warne et al. 2006, 2007). Capsaicin treatment prevented the effects of STZ (saline-infused group) on mWAT weight (Fig. 4A).
Figure 5 shows plasma hormone levels and hormone-sensitive tissue weights. Plasma insulin (Fig. 5A) and leptin (Fig. 5B) levels were significantly (P < 0.001) reduced by STZ treatment with saline infusion, compared with citrate-injected controls. Insulin infusion into the STZ-injected rats elevated circulating insulin and leptin levels. Capsaicin treatment of the hepatic vagus had no effect on these hormones. Corticosterone levels (Fig. 5C) were unaffected by any insulin or hepatic vagal experimental manipulation, showing the efficacy of the corticosterone pellet.
The adrenal weights (Fig. 5D) were typically low, showing the efficacy of the corticosterone treatment; however, significant differences (P < 0.05) were observed between STZ-insulin capsaicin-treated group and the citrate and the STZ-saline vehicle-treated groups. No other differences were observed. Spleen weight (Fig. 5E) was significantly (P < 0.05) elevated by STZ injection and unaffected by insulin infusion or capsaicin treatment. In contrast, thymic weight (Fig. 5F) generally showed no differences among groups. However, the citrate-injected, capsaicin-treated group was significantly (P < 0.05) heavier than the citrate-injected vehicle-treated, STZ-saline vehicle-treated and STZ-insulin capsaicin-treated groups. Collectively, the organ weights presented in Fig. 5, together with the total caloric intake presented in Fig. 2 and body weight changes presented in Fig. 3, show the long-term efficacy of corticosterone treatment, STZ-induced diabetes and insulin replacement throughout the experiment and are validated by the terminal plasma measures of corticosterone and insulin (Fig. 5).
Plasma metabolites were affected by STZ treatment, insulin replacement and capsaicin treatment (Fig. 6). Capsaicin treatment did not affect any of the plasma metabolites or liver glycogen in the citrate-injected rats. STZ injection with saline infusion resulted in significant (P < 0.05) elevations in triglycerides (Fig. 6A), FFA (Fig. 6C) and total ketone bodies (Fig. 6D). Capsaicin treatment of the STZ-saline group further elevated triglycerides compared with STZ-saline, vagal vehicle-treated rats. In the vehicle-treated STZ-diabetic rats, insulin infusion reduced plasma triglycerides, FFA and total ketone bodies. Capsaicin treatment significantly (P < 0.05) attenuated the actions of insulin on FFA and total ketone bodies and partially attenuated the effects on plasma triglycerides.
Plasma glucagon (Fig. 7A), liver glycogen (Fig. 7B) and plasma glucose (Fig. 7C) showed interrelated patterns of change with STZ, insulin and capsaicin treatments. Both plasma glucagon and glucose were significantly (P < 0.01) elevated and liver glycogen content was significantly (P < 0.01) reduced by STZ treatment. Capsaicin treatment of the common hepatic vagus further significantly (P < 0.05) increased plasma glucose and glucagon levels, whilst further reducing liver glycogen content. Insulin replacement partially reduced glucose levels and fully reduced glucagon levels to non-diabetic, citrate-injected, levels whilst significantly (P < 0.05) elevating liver glycogen content. Capsaicin treatment in this instance partially attenuated (P < 0.05) the actions of insulin on liver glycogen and glucagon and completely attenuated the actions of insulin on plasma glucose levels.
Table 1 compares the results obtained in this study with that of a previous study that utilized a similar model, in which the common hepatic branch of the vagus was cut (HV) as opposed to treated with capsaicin. This enables dissection of the effects of afferent and efferent fibres in the regulation of food intake, fat deposition and plasma variables, as outlined in the discussion.
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For the majority of variables examined, non-diabetic rats showed no effect of capsaicin treatment. However, these rats had high, steady-state corticosterone levels which, consequently, elevated circulating insulin levels in comparison with those expected for intact, naïve rats with freely secreted corticosterone of a similar age and at a similar time of day (typically around 2 ng ml–1, e.g. La Fleur et al. 2005a). Tipping the important corticosterone: insulin balance (Strack et al. 1995) by manipulating insulin levels in groups of rats that otherwise share similar, steady-state corticosterone concentrations is probably the key factor in revealing the role of the hepatic vagus in the maintenance of metabolic homeostasis.
In addition to our findings, other studies have shown that afferent signalling is important in regulating fat intake. For example, rats with general autonomic afferent denervation by capsaicin over-eat high fat on at least the first test (Chavez et al. 1997). Furthermore, jejunal and portal infusions of lipids increase the activity of hepatic vagal afferents (Randich et al. 2001). This has been suggested to complement, or be an alternative to, celiac vagal afferent-mediated, lipid-induced inhibition of food intake (Cox et al. 2000, 2004; Randich et al. 2000). In this and our previous study (Warne et al. 2007; Table 1), chow intake balance changes with lard intake such that total intake is unaffected by any manipulations of the hepatic vagus. This supports other studies that have shown that the hepatic vagus does not influence total caloric intake (e.g. Louis-Sylvestre et al. 1980; Bellinger et al. 1984; Bellinger & Williams, 1988). Hence, the hepatic vagal afferents are responsive to dietary lipids and can signal to modify composition of the diet, but not total caloric intake.
Since capsaicin treatment of the common hepatic vagus does not modify lard intake in either non-diabetic or STZ-diabetic, insulin-replaced rats, this suggests insulin is acting via a mechanism independent of the hepatic vagus. A direct, central site of insulin action is a possible candidate, since there is widespread presence of the insulin receptors throughout the brain (Havrankova et al. 1978). Notably, two important sites involved in reward, namely the ventral tegmental area and the substantia nigra pars compacta, express insulin receptors (Figlewicz et al. 2003). Hence, there is capacity for central insulin to affect rewarding behaviour, such as choosing palatable food intake especially under the motivational influence of high corticosterone levels (Pecoraro et al. 2006). Indeed, choice is important considering that intracerebroventricular administration of high insulin concentrations can reduce palatable food intake (Figlewicz et al. 2004), which is not evident after a choice diet of saturated macronutrients (Van Dijk et al. 1997). In contrast, the arcuate nucleus is clearly the epicentre for the inhibitory actions of insulin, as well as leptin, on food intake (Niswender & Schwartz, 2003). Insulin and leptin actions at this site probably account for the reduction in total caloric intake of STZ-diabetic rats by insulin replacement, which also accounts for why total intake was unaffected by capsaicin treatment of the hepatic vagus.
Chronic stress and glucocorticoids result in elevated pleasurable food intake in both rodents (la Fleur et al. 2004; Pecoraro et al. 2004) and humans (Epel et al. 2001), although total caloric intake might be unaffected or even reduced (Gibson, 2006). In our model, high steady-state corticosterone levels were utilized in this study that mimic those observed with chronic stress. STZ-diabetes itself is a stressor, as evidenced by similar behavioural, autonomic, endocrine and neuroendocrine characteristics to chronic stress in rodents (Scribner et al. 1993); therefore it was essential to equalize corticosterone concentrations in all groups. In the presence of high corticosterone levels, but defective insulin production due to STZ treatment, pleasurable food intake is not stimulated. Hence, elevated insulin levels in concert with elevated corticosterone are probably responsible for the increased pleasurable food intake associated with chronic stress.
This and our previous studies have shown that STZ treatment reduces the weight of all WAT depots and insulin replacement into the superior mesenteric vein prevented, partially or totally, the decrease in mWAT, pWAT and eWAT weight, compared with saline-infused counterparts (Warne et al. 2006). The finding that insulin replacement increases mWAT weight to above that of non-diabetic levels suggests that there is an increased propensity towards deposition of fat into this depot after lard intake. A combination of local insulin action and corticosterone are probably responsible for this. Corticosterone redistributes fat from subcutaneous to omental (preferentially mWAT) depots in rats and humans (Thakore et al. 1998; Bell et al. 2000; Dallman et al. 2004, 2007). Our findings that scWAT was unaffected by insulin replacement support this notion. Since superior mesenteric-infused insulin traverses through mWAT, local action could prime this depot for receipt of lipids above the other omental depots. In contrast, the exclusive elevation in mWAT after capsaicin-induced lard intake suggests that this is the first site for fat deposition, probably due to its proximity to the sites of absorption. The lack of insulin ‘priming’ of other WAT depots might account for their lack of change in weight with lard intake under those conditions.
Capsaicin treatment of STZ-treated rats, irrespective of saline or insulin infusion, elevated plasma glucose, triglyceride and glucagon levels and reduced liver glycogen content. The overall levels were influenced, however, by prevailing insulin levels that regulate glucose and triglyceride uptake. Glucagon reduces liver glycogen content, consequently elevating glucose output (Greenberg et al. 2006), and stimulating adipocyte lipolysis (Schade et al. 1979), hence triglyceride release. Since neural innervation of the pancreas can regulate 
-cell glucagon output (Havel & Taborsky, 1989), and the common branch of the hepatic vagus receives fibres from the pancreas (Berthoud, 2004), afferent signalling through the common hepatic branch of the vagus could signal to the brain for consequent manipulation of glucagon levels. This might complete a homeostatic feedback loop that limits extreme glucose and triglyceride output. An intact efferent signalling pathway from the brain through the hepatic vagus is clearly important since HV, which transects both afferent and efferent fibres, does not result in significant changes in plasma glucose or liver glycogen content (Warne et al. 2007; Table 1).
As shown in this study and others (e.g. Bartness & Rowland, 1983), insulin acts to prevent the elevation of circulating levels of FFA and total ketone bodies evident in STZ-diabetes. Capsaicin treatment negated the actions of insulin on these metabolites, which suggests that a component of afferent hepatic vagal signalling regulates these processes, corroborated by similar findings after HV for FFA (Warne et al. 2007; Table 1). Both release of FFA and inhibition of ketogenesis can be achieved by stimulation of sympathetic nerves (Beuers et al. 1986; Bartness et al. 2005). Insulin, acting via an afferent signal through the hepatic vagus, could signal the brain to modify these efferent sympathetic signals. Hence, the loss of afferent, parasympathetic signalling might eliminate a feedback loop by which insulin reduces FFA release and ketogenesis when these glucose alternative energy supplies are not needed.
Collectively, these data suggest that afferent signalling through the common hepatic branch of the vagus inhibits lard, but not chow, intake and regulates plasma metabolite levels depending on the insulin: corticosterone balance of the rat. Considering that chronic stressors are an omnipresent part of our current life and work styles (Wardle & Gibson, 2002; Kunz-Ebrecht et al. 2003) and might contribute to the obesity epidemic (Dallman et al. 2006), vagal manipulation could therefore represent a novel therapeutic avenue to regulate dysregulated or unhealthy (but palatable) food intake.
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