The Hepatic Vagus Mediates Fat-Induced Inhibition of Diabetic Hyperphagia

Male rats (Sprague-Dawley, B&K International, Gilroy, CA) weighing 250 ± 10 g were individually housed in hanging wire cages in a temperature- (21–23°C) and light-controlled (lights on at 0600–1800) room. Rats were allowed to adapt to their new environment for at least 4 days. All procedures were approved by the University of California San Francisco IACUC. All rats had ad libitum access to pelleted rat chow (Purina Chow #5008; Purina, St. Louis, MO) and tap water except where noted. Food and body weight were measured daily. The lard used in experiments 1, 3, and 4 was purchased locally (Armour lard; 9 kcal/g). The compositions of the equal caloric high-carbohydrate and high-fat diets in experiment 2 were similar to those described (18). High-fat diet contained 63% fat, 13% carbohydrate, and 24% protein, whereas high-carbohydrate diet contained 63% carbohydrate, 13% fat, and 24% protein (Labdiets, Richmond, IN)

Rats were anesthetized with ketamine (75 mg/kg intramuscularly; Abbott Lab, Chicago, IL) and xylazine (10 mg/kg intramuscularly; Butler, Columbus, OH). A midline laparotomy was performed. The common hepatic vagal branch (hepvag) was transected by stretching the fascia containing the common hepatic vagal branch. A myelin-specific dye (Toluidine Blue; Sigma Chemicals, St. Louis, MO) revealed the common hepatic branch as it separates from the left vagal trunk. Stained neural tissue was transected between ventral vagal trunk and liver. Small, blue-stained branches running in the fascia between stomach and liver were also transected. For gastroduodenal vagotomy (duovag), the common hepatic vagus was stretched and followed down to the duodenum. The branches were visualized as above, and all branches on top of and above the duodenum were removed. Sham-operated (sham) rats underwent all procedures except transection of the neural tissue. During all procedures, the abdominal cavity was bathed with saline to prevent drying of the viscera. Ketoprofen (10 mg/kg subcutaneously; Abbott Lab) was given postoperatively.

Seven days later, rats were eating normally and showed normal weight gain, then hepvag, duovag, and sham rats were anesthetized with isoflurane and given either STZ (Sigma Chemicals, St. Louis, MO; 65 mg/kg in citrate buffer pH 4.2, subcutaneously) or the citrate buffer (vehicle, subcutaneously).

Twelve of 20 hepvag rats and 16 of 26 sham rats received STZ; the rest received citrate vehicle (veh). All were fed pellet chow for 6 days until the diabetic rats were hyperphagic. Four rats did not become diabetic (one hepvag-STZ and three sham-STZ rats), and one rat in each diabetic group died of diabetic complications. Five sham-veh, six sham-STZ, four hepvag-veh, and six hepvag-STZ rats received lard in addition to the pellet chow; the remaining rats received pulverized pellets. After 3 days, all rats were decapitated between 0900 and 1000. Blood was collected from the trunk into chilled plastic tubes containing EDTA, kept on ice until centrifuged, and then aliquoted. Brains were immediately removed from the skull and were immersion-fixed in 4% paraformaldehyde in PBS (0.1 mol/l, pH 7.2).

Nine hepvag rats and nine sham rats received STZ. All were given the high-carbohydrate diet for 6 days until all were hyperphagic. One hepvag-STZ and one sham-STZ rat died. Four rats of each group were then switched to the high-fat diet; the others remained on high-carbohydrate diet. After 3 days, rats were decapitated between 0900 and 1000; blood and brains were collected.

Seven duovag rats and five hepvag rats were made diabetic and after 6 days of normal pellet chow were exposed to lard for 3 days. Four sham-veh rats were maintained on normal chow during the course of the experiment. Three of the seven duovag-STZ rats and one hepvag-STZ rat died before the end of the experiment. The remaining procedures were as described in experiment 1.

Fourteen hepvag rats and 14 sham rats received STZ, and 7 sham rats received vehicle. Two sham-STZ rats died of diabetic complications. All rats were fed normal chow for 5 days until diabetic rats were hyperphagic. Three days after STZ injection, a tail nick was performed (2 h after lights on) to withdraw 0.5 ml of blood for hormone measurements. One hour before lights off, seven hepvag-STZ and seven sham-STZ rats received lard in addition to the pelleted chow, whereas others received pulverized pellets. In the morning of the second day (41 h later), all rats were anesthetized as described in liver energy status.

Eighteen hepvag rats and 23 sham rats received STZ; 4 nonoperated rats received vehicle. One hepvag-STZ and two sham-STZ rats died. STZ rats were hyperphagic on normal chow after 5 days. Groups of hepvag-STZ and sham-STZ rats were anesthetized, and the livers were freeze-clamped at 6, 13, and 30 h after presentation of lard. Five sham-STZ rats and four veh rats received pulverized pellets and were anesthetized as described above.

Glucose was measured using the glucose oxidase assay on a plate reader (Sigma Diagnostics, St. Louis, MO), and corticosterone, insulin, and leptin were determined with radioimmunoassays (ICN Biochemicals, Orangeburg, NY, and Linco Research, St. Charles, MO, respectively).

Brains were sliced at 30 μm from the optic chiasm to the ventromedial nuclei, and 1:6 sections at the appropriate level were stained for CRF (anti-rabbit CRF [lot c-70], diluted 1:10,000, gift from Dr. W Vale, Salk Institute, San Diego, CA), NPY, and galanin (anti-rabbit NPY or anti-rabbit galanin, diluted 1:6,000; gifts from Dr. R.M. Buijs, NIBR, the Netherlands). Immunostaining was performed as described previously (27). For ensuring that the exposure to DAB was similar between groups, sections were stained in 24-well plates; all sections were stained with the same solution. DAB exposure was limited so that we did not measure maximum DAB staining. Semiquantitative densitometric analysis was performed on relative levels of NPY-peptide (Arc [medial part]), CRF peptide (median eminence outer layer [bregma, −2.8 mm]), and the galanin peptide (anterior parvocellular PVN [bregma, −1.4 mm]). An illumination objective of 10× was used, and digital images were made with a digital camera connected to a computer with NIH Image software (ImageJ-1.29×). Optical density readings through any given cell group, corrected for background, were taken at regularly spaced (120 μm) intervals. Mean group values were determined from averaged readings of multiple sections (at least three) from individual rats (four per group). The number of slices analyzed for each rat varied depending on the region and distribution of the peptide (three of four sections per rat) (28,29).

Rats were anesthetized (ketamine/acepromazine [10:1; 1 ml/kg intramuscularly]), and a piece of the median lobe (∼3 g) was freeze-clamped, homogenized, and stored at −70°C and analyzed later as described elsewhere (30). The phosphorylation potential was calculated as [ATP]/([ADP]) × [Pi].

Data were analyzed by two-way ANOVA using two factors: group (sham-veh, sham-diab, vag-veh, vag-diab) and treatment (no lard, lard or high-carbohydrate diet, high-fat diet). Significant interaction (P < 0.05) was followed by post hoc tests of individual group differences (Tukey-Kramer). In the text, tables, and figures, all data are presented as means ± SE.

Caloric intake, body weight gain, and corticosterone concentrations were significantly increased after common branch hepatic vagotomy (Figs. 1 and 2A and B, Table 1), and there was a trend toward increased insulin concentrations (P = 0.09; Table 1). Both sham-veh and hepvag-veh rats increased total caloric intake when lard was present (effect of lard, F[1,14] = 31, P < 0.001), eating similar amounts of lard and chow over 3 days (effect of hepvag, F[1,7] = 1; P = 0.35). It is interesting that lard ingestion increased leptin and insulin significantly only in hepvag-veh rats (leptin:interaction effect, F[3,32] = 5.2; P < 0.004; insulin interaction, F[3,32] = 3.0; P < 0.05; Fig. 2B, Table 1).

Five days after STZ injection, hepvag-STZ and sham-STZ rats were hyperphagic (0 h; Fig. 1A and B; effect of STZ, F[1,36] = 76; P < 0.001). During the first 24 h of lard, sham-STZ and hepvag-STZ rats maintained total caloric intake that was similar to the preceding day, but they substituted 30–40% of the total calories with calories from lard. A similar percentage was seen in the vehicle-treated rats (24 h; Fig. 1C and D). During the subsequent 24 h, sham-STZ rats decreased caloric intake to nondiabetic levels, whereas hepvag-STZ rats did not decrease intake, eating the same number of calories as on the previous day (48 h; Fig. 1B; interaction effect, F[1,18] = 12.5; P < 0.003). This pattern continued in the hepvag-STZ rats for the third 24-h period, whereas sham-STZ rats increased their total caloric intake slightly, predominantly from high carbohydrate chow (72 h; Fig. 1). Both sham-veh and hepvag-veh groups increased total caloric intake with 40% calories from lard over the total 72 h that lard was available (Fig. 1C), whereas the sham-STZ and hepvag-STZ rats decreased lard intake after the first 24 h (effect of lard, F[2,34] = 7.5; P < 0.002; Fig. 1D).

During lard access, hepvag-STZ rats (as with sham-STZ and hepvag-STZ groups without lard) did not gain body weight, despite the higher caloric intake; the sham-STZ group tended to lose weight, probably because of lower caloric intake (Fig. 2A and B). At the end of the experiment, STZ decreased insulin and leptin and increased glucose and corticosterone, independent of hepatic vagotomy (Table 1). Furthermore, lard ingestion in diabetic rats did not alter plasma concentrations of glucose, insulin, leptin, or corticosterone.

In vehicle-treated rats, common branch hepatic vagotomy and/or lard ingestion did not have clear effects on NPY and CRF peptide levels. Lard ingestion tended to decrease CRF peptide in hepvag-veh rats (Fig. 2C). STZ-induced diabetes decreased CRF peptide in the median eminence (ME; Fig. 2C) and increased NPY-icc in the Arc (Fig. 2D). Lard ingestion decreased caloric intake in sham-STZ, decreased NPY to nondiabetic levels (interaction effect (F[1,12] = 13; P < 0.004), and tended to increase CRF (interaction effect, F[1,12] = 4.9; P = 0.051); in hepvag-STZ rats lard ingestion had no effect on CRF- and NPY peptide levels (Fig. 1C–E).

Because diabetic rats in experiment 1 refused to eat lard after the first day of exposure, we used a forced high-fat diet to test whether the hepatic vagal control is general to high fat in the diet. Only hepvag-STZ and sham-STZ groups were used, and the rats were given a high-fat and high-carbohydrate diet (both 3.3 kcal/g) similar to that described before (18). Five days after inducement of diabetes with a single injection of STZ, rats were hyperphagic on the high-carbohydrate diet (hepvag-STZ from 33 ± 1.0 to 48 ± 2.2 and sham-STZ from 31 ± 0.5 to 51 ± 2.9 cal/100 g body wt/day). Again we saw a clear decrease in caloric intake to nondiabetic levels in sham-STZ rats eating fat (caloric intake in sham-STZ rats on the high-fat diet was not significantly different from that before diabetes was induced; P = 0.2). In contrast, caloric intake in the hepvag-STZ rats eating a high-fat diet was similar to that of hepvag-STZ rats eating the high-carbohydrate diet (Fig. 3A). Changes in body weight over the period of the experiment were not different among the four groups (data not shown), and there were no differences in plasma concentrations of glucose, insulin, or leptin (Table 2). Plasma corticosterone concentrations all were elevated for the time of the day (>10 μg/dl), although they decreased significantly in sham-STZ rats that were switched to a high-fat diet (Table 2).

The decrease in caloric intake in sham-STZ rats that were switched to a high-fat diet was accompanied by significant decreases in NPY peptide in Arc and increases in CRF peptide in ME. Hepatic vagotomy completely blocked these fat-induced changes (Fig. 3B, C, and E). Like NPY peptide, galanin peptide was decreased in sham-STZ rats that were switched to a high-fat diet, and this was blocked by hepatic vagotomy (Fig. 3D and E).

Food intake and body weight gain were not different between duovag and hepvag rats. Six days after the administration of STZ, both groups were hyperphagic compared with the vehicle group (Fig. 4A, 0 h). During the first 24 h of lard presence, both STZ groups maintained total caloric intake similar to that of the preceding day (Fig. 1A, 24 h), but they substituted 30–40% of the total with calories from lard. During the subsequent 24 h, duovag-STZ rats decreased caloric intake to nondiabetic levels, whereas hepvag-STZ rats did not decrease intake, eating the same amount of calories as the previous day (48 h; Fig. 4A and B). This feeding pattern continued in the hepvag-STZ rats for the third 24-h period, whereas caloric intake in duovag-STZ rats remained decreased (72 h; Fig. 4A and B). Body weight loss after STZ was comparable between both groups. Lard ingestion and subsequent inhibition of food intake in duovag-STZ rats decreased body weight further, whereas hepvag-STZ rats gained weight, although still significantly less than sham-veh rats (Fig. 4C). As in experiment 1, Arc NPY peptide levels in hepvag-STZ rats with lard were significantly higher than in sham rats, whereas duovag-STZ rats with lard had similar NPY peptide levels as sham-veh rats that were not given lard (Fig. 4D). STZ in both hepvag and duovag rats increased plasma concentrations of glucose and corticosterone and decreased plasma concentrations of insulin and leptin. There were no differences between these values in the STZ groups (Table 3).

Five days after STZ, hepvag-STZ and sham-STZ rats were hyperphagic compared with the sham-veh group (47.5 ± 1.0, 44.5 ± 1.7, and 29.1 ± 0.8 respectively; effect of STZ, F[2,29] = 44; P < 0.001). Caloric intake between the two STZ groups was not different (P = 0.2). After lard, total caloric intake in sham-STZ and hepvag-STZ rats showed the usual patterns (Fig. 5A, left). Sham-STZ rats decreased caloric intake after 24 h of lard, whereas hepvag-STZ rats remained hyperphagic. Without lard, STZ decreased hepatic energy status in both hepvag-STZ and sham-STZ rats: hepatic ATP, the ATP/ADP ratio, and the phosphorylation potential all were decreased. There were no significant differences in hepatic energy status between the STZ groups, and lard ingestion did not change hepatic energy status (Fig. 5B–D, left).

We next measured hepatic energy status from initiation of lard inhibition in caloric intake occurred. Hepvag-STZ and sham-STZ rats again ate similar calories on the day lard was first given and significantly more calories than sham-veh rats (Fig. 5E, right). STZ again decreased hepatic energy status (Fig. 5F–H). After 5 h of lard (lard was given at lights off), hepvag-STZ and sham-STZ rats consumed similar amounts of calories (±30 cal/100 g body wt; Fig. 5E), with lard representing 57.3% ± 3.3 and 58.7% ± 3.8 of total intake, respectively. Between 5 and 13 h after lard access, hep-STZ and sham-STZ rats ate only chow and no lard, resulting in a cumulative intake at 13 h of ±40 cal/100 g body wt (Fig. 5E). Thus, over the first 24-h period of lard access, rats consumed lard during the first 5 h of the dark period. Between 13 and 30 h of lard access, inhibition of caloric intake occurred in sham-STZ rats; hep-STZ rats maintained caloric intake, although they did not eat more lard (Fig. 5E).

Hepatic energy status in hepvag-STZ and sham-STZ rats during 30 h of lard access did show significant differences (effect of time, ATP F[2,24] = 6.4, P < 0.006; ATP/ADP ratio F[2,24] = 3.5, P < 0.05); phosphorylation ratio F[2,24] = 4.6; P < 0.03). No significant differences emerged between hepvag-STZ and sham-STZ rats in any measures of hepatic energy status. All measures in hepvag-STZ and sham-STZ rats at all times were significantly lower than in sham-veh rats (F[2,34] = 3.5; P < 0.05), and post hoc analysis revealed significant differences for sham-veh versus hepvag-STZ and sham-veh versus sham-STZ at all times (P < 0.05).

Consistent with several other studies, we show that a high-fat diet normalizes food intake in STZ-diabetic rats (17,18). Furthermore, hypothalamic NPY peptide decreases and CRF peptide increases, supporting the data of Chavez et al. (18), who found that the mRNAs of these peptides were normalized in diabetic rats consuming a high-fat diet. Because presenting a high-fat diet forces intake and does not represent daily life, we also studied rats with the choice of consuming fat. Fat consumed by choice also results in fat-induced inhibition of caloric intake and normalization of CRF and NPY peptide-ir to nondiabetic levels. Furthermore, lard ingestion by diabetic rats either by choice or forced with a high-fat diet does not change plasma concentrations of glucose, insulin, leptin, or corticosterone, consistent with findings of others (18).

Striking, common branch hepatic vagotomy completely blocks both the fat-induced decrease in caloric intake in diabetic rats and normalization of hypothalamic peptides. Moreover, unlike common branch hepatic vagotomy, gastroduodenal vagotomy does not prevent the fat-induced inhibition of caloric intake and normalization of NPY peptide levels in STZ-induced diabetes. Our findings, therefore, strongly suggest that in diabetic rats, the ingested fat results in a signal to the CNS that is mediated by the hepatic vagal nerve innervating the liver and not the proximal gastroduodenal region.

It has been assumed that the actions of insulin and/or leptin in the brain are important for the increased food intake that occurs in diabetes (6,31). Our data show that without changes in plasma concentrations of insulin, leptin, or corticosterone, the hepatic vagus mediates changes in feeding and hypothalamic neuropeptides in diabetic rats. In rats on the forced high-fat diet, all plasma corticosterone concentrations are elevated for the time of the day (>10 mg/dl) but decrease significantly in sham-operated diabetic rats that were switched to the high-fat diet compared with those maintained on the high-carbohydrate diet. Because corticosterone concentrations in sham-STZ rats on high-fat diet are not significantly different from those in hepvag-STZ groups on high-carbohydrate and high-fat diets, it is unlikely that corticosterone mediates the fat-induced inhibition of caloric intake. Furthermore, this corticosterone concentration in diabetic rats is sufficient to promote hyperphagia (15). Thus, the decrease in corticosterone concentrations observed in sham-STZ rats on high-fat diet are not likely to mediate the decrease in food intake seen after high-fat diet. Changes in NPY and CRF peptides are associated with diabetic hyperphagia (6–9). In addition, mRNAs for other (an)orexogenic peptides in the hypothalamus, proopiomelanocortin (POMC), agouti-related peptide (AGRP), and galanin are altered by diabetes (14,32). It is interesting that the changes in POMCmRNA, AGRPmRNA, and body weight gain induced by diabetes were partially reversed after systemic insulin treatment, whereas NPYmRNA and CRFmRNA and food intake were totally reversed (14,33). It could well be that these peptides are more related to changes in energy expenditure or body weight than to food intake. Thus, although NPY and AGRP are synthesized in the same arcuate neurons, their expression may change separately. This suggests that (an)orexogenic neuropeptides may serve different roles in feeding and body weight regulation in rats with uncontrolled diabetes. Further experiments are necessary to determine whether POMCmRNA and AGRPmRNA are also associated with fat-induced changes in diabetes. Another peptide associated with food intake and fat preference in general is galanin. Because both hyperphagia in diabetes (32) and fat preference (34) are correlated with increases in the hypothalamic peptide galanin, we measured galanin peptide in the anterior portion of the PVN in both sham-STZ and hepvag-STZ rats on high-carbohydrate and high-fat diets. As with NPY, galanin was decreased significantly in sham-STZ rats that were switched to a high-fat diet, and this was blocked by common branch hepatic vagotomy. Whether the fat-induced decrease in caloric intake in sham-STZ rats correlates with galanin and fat preference or with food ingestion in general is not clear but will be the subject of further study.

Although several studies suggest that fat ingestion in diabetic rats is not aversive (35–37), we cannot rule out the possibility that diabetic rats, eating lard or a high-fat diet, inhibit their food intake because lard and fat consumed during the first day cause malaise. However, the fat-induced inhibition of food intake is almost certainly not a consequence only of malaise. The absolute quantity of lard eaten by the diabetic rats was similar to that eaten by vehicle-treated rats, and vehicle-treated rats did not decrease food intake after eating lard, and diabetic rats did not increase their total caloric intake in the first 24 h that lard was available. Furthermore, adrenalectomized-diabetic rats, which show moderate diabetic signs with normal food intake and no body weight loss (38,39), show a similar inhibition in food intake after lard consumption while eating 75% less lard than diabetic rats with intact adrenals (S.E. la Fleur and M.F. Dallman, unpublished data). Further studies are necessary to determine whether malaise plays a role in the fat-induced inhibition in food intake.

Experiments on neural feedback in the fat-induced feeding signal and hepatic energy status (19–22,24,25) led us to hypothesize that hepatic energy status might signal fat-induced changes observed in STA-diabetic rats. Consistent with earlier reports, hepatic energy status was decreased in STZ-diabetic rats (26). Fat ingestion by choice, however, did not change hepatic energy status at any of the times studied. The effects of lard on caloric intake in diabetic rats occurred between 6 and 30 h, and during these times, we still observed significantly decreased hepatic energy status in both sham-STZ and hepvag-STZ rats, whereas at the 30-h time point, the sham-STZ rats clearly started to inhibit their feeding (Fig. 5). This suggests that a shift in hepatic energy status does not underlie the fat-induced changes in caloric intake and hypothalamic peptide expression when rats have the choice of eating lard. However, because hepatic vagotomy in STZ-induced diabetes did not alter hepatic energy status compared with nonvagotomized diabetic rats, this suggests that the surgical procedure does not interfere with hepatic energy balance. This supports our hypothesis that a neural signal afferent to the CNS is involved in the fat-induced inhibition of food intake rather than a denervation-induced change in the liver, per se. Because afferent fibers of the hepatic vagus are intermingled with efferent fibers, the influence of hepatic vagal efferent regulation on feeding regulation in diabetes cannot be excluded.

In addition to the role of the hepatic vagus in diabetic hyperphagia, the changes after common hepatic branch vagotomy in nondiabetic rats were interesting. Common hepatic branch vagotomy results in a small increase in food intake, body weight gain, and corticosterone concentrations and tends to increase insulin concentrations. Increased food intake, weight gain, and insulin after hepatic vagotomy have been reported by some (40,41); others have not found this (42–44). Lack of hepatic vagotomy effects may well be a consequence of a postsurgical loss of body weight (40). Our experience shows that preventing weight loss (through prophylactic use of ketoprofen at surgery [45]) is essential for eliciting the effects of hepatic vagotomy on features of obesity. It is interesting that short-term lard consumption in hepvag-veh rats increases body weight and leptin significantly and tends to increase insulin and decrease CRF peptide levels in the median eminence. These changes are not observed in sham-veh rats, which consume similar amounts of calories with a similar percentage of calories from lard. To our knowledge, only one report has studied the first days of fat consumption in a mouse strain that is very sensitive to diet-induced obesity. Increased body weight occurred after 1 week, although increased leptin was already observed on days 1 and 2 (46). Other studies in rats show changes in body weight and leptin levels after 2 weeks of eating a high-fat diet (47,48). The metabolic changes that occur after 72 h of lard intake in hepvag-veh rats are similar to those observed after 72 h of central infusion of dexamethasone, which also leads to increased body weight gain and food intake (49). Taken together, we speculate that features of obesity are accelerated by removing the common hepatic vagal branch and that the common hepatic vagal branch plays a role in the onset of diet-induced obesity. A series of long-term studies with high-fat diet, however, will be necessary to clarify the role of the hepatic vagus in (diet-induced) obesity.

We have revealed a hepatic vagal afferent pathway to the brain that is important in feeding and regulating neuropeptide responses in type 1 diabetes; delineation of this pathway and its controls may provide new insights into a potentially important diabetic neuropathy. With time, diabetic neuropathy may blunt signals that normally ensure maintenance of energy balance.

This work was supported by grants from the Dutch Diabetes Research Foundation and NIH (DK-28172).