Hindbrain insulin controls feeding behavior

Abstract

Objective: Pancreatic insulin was discovered a century ago, and this discovery led to the first lifesaving treatment for diabetes. While still controversial, nearly one hundred published reports suggest that insulin is also produced in the brain, with most focusing on hypothalamic or cortical insulin-producing cells. However, specific function for insulin produced within the brain remains poorly understood. Here we identify insulin expression in the hindbrain's dorsal vagal complex (DVC), and determine the role of this source of insulin in feeding and metabolism, as well as its response to diet-induced obesity in mice.

Methods: To determine the contribution of Ins2-producing neurons to feeding behavior in mice, we used the cross of transgenic RipHER-cre mouse and channelrhodopsin-2 expressing animals, which allowed us to optogenetically stimulate neurons expressing Ins2 in vivo. To confirm the presence of insulin expression in Rip-labeled DVC cells, in situ hybridization was used. To ascertain the specific role of insulin in effects discovered via optogenetic stimulation a selective, CNS applied, insulin receptor antagonist was used. To understand the physiological contribution of insulin made in the hindbrain a virogenetic knockdown strategy was used.

Results: Insulin gene expression and presence of insulin-promoter driven fluorescence in rat insulin promoter (Rip)-transgenic mice were detected in the hypothalamus, but also in the DVC. Insulin mRNA was present in nearly all fluorescently labeled cells in DVC. Diet-induced obesity in mice altered brain insulin gene expression, in a neuroanatomically divergent manner; while in the hypothalamus the expected obesity-induced reduction was found, in the DVC diet-induced obesity resulted in increased expression of the insulin gene. This led us to hypothesize a potentially divergent energy balance role of insulin in these two brain areas. To determine the acute impact of activating insulin-producing neurons in the DVC, optic stimulation of light-sensitive channelrhodopsin 2 in Rip-transgenic mice was utilized. Optogenetic photoactivation induced hyperphagia after acute activation of the DVC insulin neurons. This hyperphagia was blocked by central application of the insulin receptor antagonist S961, suggesting the feeding response was driven by insulin. To determine whether DVC insulin has a necessary contribution to feeding and metabolism, virogenetic insulin gene knockdown (KD) strategy, which allows for site-specific reduction of insulin gene expression in adult mice, was used. While chow-fed mice failed to reveal any changes of feeding or thermogenesis in response to the KD, mice challenged with a high-fat diet consumed less food. No changes in body weight were identified, possibly resulting from compensatory reduction in thermogenesis.

Conclusions: Together, our data suggest an important role for hindbrain insulin and insulin-producing cells in energy homeostasis.

Keywords: Diet-induced obesity; Dorsal vagal complex; Food intake; Hindbrain; Insulin.

Figure 1

Rat Ins2 promoter cells, found in the hypothalamus and hindbrain, are affected by diet-induced obesity in a neuroanatomically divergent manner. 20x epifluorescent images of A: YFP- expression in the arcuate nucleus (ARC), B: paraventricular nucleus (PVN) and C: the nucleus of the solitary tract (NTS) in Rip-Chr2-YFP mice. Lower magnification image and corresponding mouse brain atlas section of NTS are shown in Figs. S1 and S2. Higher magnification images showcasing individual cells from each area are also shown in Fig. S3. D: Eight weeks of high-fat diet maintenance induced E: weight gain measured at 8wks of diet exposure and F: increased serum insulin levels. G: Expression of Ins2-and H: Pomc-gene expression in the hypothalamus, and I: Ins2-and J: Pomc-gene expression in the brainstem of mice on the HFD for eight weeks. n = 8–10, ∗p < 0.05, ∗∗∗∗p < 0.00001 students t-test. YFP expression is visualized in green, and DAPI (nuclear stain) in blue.

Figure 2

Hyperphagic effect of optogenetic activation of Ins2 cells in the dorsal vagal complex (DVC) of Rip-Chr2-TdTomato mice A: Schematic of optogenetic stimulation target and representative confocal image with cannula tract, cannula tract indicated by white arrows in the enlarged square. B: The 10Hz 10 ms tonic stimulation of the DVC increased cumulative food intake with peak consumption at 60min from stimulation onset. C: Results presented as change in food intake from non-stimulated control. In a separate cohort, animals were additionally cannulated to allow infusion of the insulin receptor antagonist, S961. Optogenetic stimulation in the vehicle infused group increased food intake similarly to the first experiment. D–F: However, infusion of the S961 InsR antagonist into the lateral ventricle blocked the effects of the stimulation. E: Main effect of stimulation compared between the non-stimulated and stimulated vehicle condition. At 1h after reintroduction of food, the stimulated vehicle condition group significantly increased intake of food compared to both stimulated and non-stimulated mice infused with S961. At 2h, S961 infusion alone increased food intake compared to non-stimulated vehicle infused control. The shaded area in the graphs represents the stimulation period for the stimulated mice and for the non-stimulated mice the sham stimulation which involved inserting the optic fiber without turning on the light source. Time point 0 represents presentation of food. Artificial cerebrospinal fluid (ACSF), n = 6, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

Ins2-KD in the DVC alters feeding and thermogenesis selectively during high-fat challenge. A: Isolated mouse pancreatic islets incubated in 20 nmol/L glucose and in vitro treated with Ins2-KD virus released significantly less insulin (in % of total insulin content) than scramble treated control, which released insulin at normal expected level. Virally expressed GFP in the NTS is shown in Fig. S6 and reduction in gene expression achieved by the KD in the DMV is shown in Fig. S7. The Ins2-KD had no impact on B: cumulative food intake, body weight gain at C:16 or D: 32 days on the standard chow diet. There was also no effect of the KD in food intake at E: light switch to dark period or F: after a 16 h h fast. Moreover, G: the 16 h fast did not impact weight lost, H: nor did the treatment impact glucose metabolism. There was no difference in I: spontaneous movement or J: velocity while on the HFD. However, K: cumulative food intake on the HFD was significantly decreased at 100 days. There was no significant difference in the L: weight gain at 50 or 100 days, although there was a tendency towards reduced weight gain in the Ins2-KD group (p = 0.1). Similarly, M: food intake after fasting was reduced in the Ins2-KD group at 100 days on HFD. N: The Ins2-KD group lost less weight than the control during the 16 h fast at 100 days on HFD. The difference in weight loss led us to analyze the surface heat in the tail, the back and between the scapula using a FLIR™ camera at 90–100 days on HFD (O). S: Temperatures on the lower back (core, Q) were lower in the Ins2-KD group at baseline and after fasting. T: tail temperatures (mid-tail, P) were lower only after fasting. U: Intrascapular temperatures representing heat production in intrascapular brown adipose tissue (BAT, R), were similar at baseline and after fasting. V: There were no differences between the groups in glucose homeostasis based on the intraperitoneal glucose tolerance test (ipGTT). n = 15–16, ∗p < 0.05, ∗∗p < 0.01 two-way anova.

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