pubmed.ncbi.nlm.nih.gov

Parabrachial Interleukin-6 Reduces Body Weight and Food Intake and Increases Thermogenesis to Regulate Energy Metabolism - PubMed

  • ️Tue Jan 01 2019

Parabrachial Interleukin-6 Reduces Body Weight and Food Intake and Increases Thermogenesis to Regulate Energy Metabolism

Devesh Mishra et al. Cell Rep. 2019.

Abstract

Chronic low-grade inflammation and increased serum levels of the cytokine IL-6 accompany obesity. For brain-produced IL-6, the mechanisms by which it controls energy balance and its role in obesity remain unclear. Here, we show that brain-produced IL-6 is decreased in obese mice and rats in a neuroanatomically and sex-specific manner. Reduced IL-6 mRNA localized to lateral parabrachial nucleus (lPBN) astrocytes, microglia, and neurons, including paraventricular hypothalamus-innervating lPBN neurons. IL-6 microinjection into lPBN reduced food intake and increased brown adipose tissue (BAT) thermogenesis in male lean and obese rats by increasing thyroid and sympathetic outflow to BAT. Parabrachial IL-6 interacted with leptin to reduce feeding. siRNA-mediated reduction of lPBN IL-6 leads to increased weight gain and adiposity, reduced BAT thermogenesis, and increased food intake. Ambient cold exposure partly normalizes the obesity-induced suppression of lPBN IL-6. These results indicate that lPBN-produced IL-6 regulates feeding and metabolism and pinpoints (patho)physiological contexts interacting with lPBN IL-6.

Keywords: PVN; brown adipose tissue; cold exposure; diet-induced obesity; energy balance; hindbrain; lateral PBN; obesity; sex differences; thyroid.

Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1

Interaction of IL-6 Gene Expression with Sex and Diet in the Parabrachial Nucleus (A–F) Mice, 5 weeks old at the start of the experiment, were fed a normal chow or a high-fat diet for 8 weeks. Measurements shown were taken at 8 weeks on the respective diet. (A) Body weight of male mice at 13 weeks of age (n = 10, for all groups). (B) IL-6 gene expression in male mice in the parabrachial nucleus as detected by qPCR. (C) Expression of other inflammation-associated genes (n = 8–9) in male mice in the parabrachial nucleus as detected by qPCR. (D) qPCR of IL-6 expression in other food intake-associated brain regions in male mice, hypothalamus (HYP), amygdala (AMYG), and hippocampus (HIPP) (n = 6–10). (E) Body weight of female mice at 13 weeks old (n = 10, for all groups). (F) qPCR of IL-6 and IL-1 gene expression in the parabrachial nucleus of female mice. IL-1 was below the detection threshold. (G) Body weight of male rats on a high-fat/high-sugar diet (n = 5). (H) White adipose tissue mass in male rats on a chow or a high fat/high-sugar diet. (I) IL-6 gene expression, as detected by qPCR, in male rats maintained on a chow or a high-fat/high-sugar diet, 14 weeks on the tissue collection day. (J and K) Body weight (J) and IL-6 expression (K) in female rats maintained on a chow or a high-fat/high-sugar diet for 14 weeks. (L–S) IL-6 mRNA is displayed in green, and cell nuclei is displayed in blue (DAPI). (L) Lateral parabrachial nucleus IL-6 mRNA was detected using fluorescent in situ hybridization (RNAScope). (M–P) DAPI (M), IL-6 (N), DAPI with IL-6 (O), and a high-resolution image of single cells in the lPBN showing IL-6 and DAPI (P). (Q–S) To understand the cellular origin of IL-6 in the lPBN, we used RNAScope to co-localize IL-6 mRNA with neuronal (Rbfox3; red; Q), glial (GFAP; orange; R), or microglial (AIF1; gray; S) mRNA markers. Gene expression data were normalized to the housekeeping gene Ppia and are presented as mean ± SEM. PBN, parabrachial nucleus; Il-6, interleukin-6; Il-1, interleukin-1; Il-6r, IL-6 receptor; Tnf-α, tumor necrosis factor alpha; GWAT, gonadal white adipose tissue; IWAT, inguinal white adipose tissue; X, cycle threshold values in real-time PCR were out of the defined threshold point of 40 cycles, indicating that IL-1 levels were too low to be evaluated and compared. For comparison of male and female IL-6 gene expression, see Figure S1. For results of approximate quantification of co-expression of each of the cellular markers with IL-6 mRNA, see Figure S2. All gene expression presented here as bar graphs was detected by real-time qPCR. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.001; compared with respective controls, unpaired Student’s t test comparisons.

Figure 2
Figure 2

IL-6 Acts in the lPBN to Reduce Food Intake and Increase Thermogenesis (A) Representative confocal image of the injection site pasted into the corresponding rat brain atlas section is displayed. (B–M) IL-6 or vehicle control was microinjected in the lPBN of male rats. (B) Chow intake was measured 1 h and 6 h post-injection. (C) Core temperature was measured every minute for 5 h post-injection. Injections were performed at 10.00. (D) Core temperature averaged over 4 h starting immediately post-injection. (E) Spontaneous activity was measured for 5 h post-injection. Injections were performed at 10.00. (F) Cumulative spontaneous locomotor activity over 4 h. (G and H) Representative images of rat infrared thermographic pictures used to assess BAT temperature after vehicle (G) and IL-6 injection (H). (I) BAT temperature was measured 4 h–5 h post-IL-6 microinjections into the lPBN in another cohort of rats and compared to controls. (J–M) Intra-lPBN IL-6 was injected in another cohort of rats fed a high-fat/high-sugar-fed diet for 3 weeks at the time of testing and food intake expressed as total calories consumed (J), chow intake (K), sucrose solution intake (L), and lard intake (M) were measured 6 h post-IL-6 microinjections. BAT, brown adipose tissue; lPBN, lateral parabrachial nucleus; IL-6, interleukin-6; VEH, artificial cerebrospinal fluid. Data represent mean ± SEM. ∗p < 0.05, paired comparisons Student’s t test. n = 5–9.

Figure 3
Figure 3

Cooperative Interaction of Leptin and IL-6 at the Level of the lPBN (A–I) Leptin was microinjected into the lPBN of 11-week-old male rats. (A) Chow intake was continuously measured (minute by minute) immediately starting after injections as displayed on the line graph (n = 6). (B–D) Cumulative chow intake at the following time points: 6 h (B), 12 h (C), and 24 h (D), as indicated with black arrows on the line graph. (E) Body weight 24 h post-lPBN leptin microinjections. (F) Core body temperature was telemetrically collected every 5 min in another group of rats (n = 18) microinjected with leptin into the lPBN. (G) Core temperature is averaged over the 6 h post-injection time period. (H) Spontaneous home-cage locomotor activity was telemetrically collected (n = 18) in rats microinjected with leptin into the lPBN. (I) Sum of all post-injection locomotor counts accumulated for 6 h post-injections. (J and K) In a new cohort of 11-week-old male rats (n = 12) chow intake and BAT temperature were measured after intra-lPBN delivery of sub-threshold doses of IL-6 (0.1 μg) or leptin (0.1 μg) alone, or delivery of the two substances together into the lPBN. (J) Cumulative chow intake collected over 6 h post-injections. (K) BAT temperature measured with infrared thermography displayed as temperature change from pre-injection baseline to 4–5 h after injections (n = 12). (L) Representative confocal image of a cannula tract in lPBN. lPBN, lateral parabrachial nucleus; IL-6, interleukin-6; BAT, brown adipose tissue. For intra-lPBN effect of leptin on heart rate, see Figure S3, and for effect of the combination of leptin and IL-6 on 24-h chow intake, see Figure S4, and on tail temperature, see Figure S5. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; two-way ANOVA (J and K) and one-way ANOVA (B–E, G, and I) were used for comparisons with Sidak’s post hoc test.

Figure 4
Figure 4

Disrupted Feeding and Body Weight Control in lPBN IL-6 Knockdown (A) Representative image of rat coronal brain atlas drawing and male rat tissue section showing lPBN-targeted AAV infusions. (B–E) GFP expression (B), DAPI (C), and the two signals together showing AAV infection (D) in the lPBN cells. High-resolution DAPI (in cyan: A; in blue: C–E). (F) IL-6 gene expression as detected by qPCR in control rats and rats with siRNA-mediated knockdown of IL-6 gene (n = 9–12). (G) Cumulative body weight gain of the IL-6 KD group compared to controls (n = 17 for all groups). Day 0 represents the day of siRNA introduction. (H) Cumulative chow intake in the same rats. (I–M) Acute light cycle measurements over a period of 6 h of total caloric intake (I), intake of lard (J), intake of chow (K), intake of sucrose (L), and intake of water (M) in male rats maintained on a high-fat/high-sugar diet for 3 weeks. (N) Inguinal white adipose tissue of IL-6 KD (n = 17) in comparison to controls (n = 17). (O) Gonadal white adipose tissue mass in the same rats. For daily chronic intake on two different obesogenic diets, see Figures S6 and S7. The n = 17 reflects data combined from two independent experiments/cohorts of rats. IL-6 KD, interleukin-6 knockdown; lPBN, lateral parabrachial nucleus; scp, superior cerebellar peduncle; IWAT, inguinal white adipose tissue; GWAT, gonadal white adipose tissue. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; #p = 0.07. Two-way ANOVA, followed by Sidak’s post hoc test, or unpaired Student’s t test when appropriate.

Figure 5
Figure 5

Mechanisms Underlying Disrupted Thermogenesis in lPBN IL-6 Knockdown (A–S) Virally introduced siRNA, designed to knockdown IL-6 gene expression, was delivered into the lPBN, as shown in Figure 4, and all thermogenic and locomotor testing was conducted in male rats 5–6 weeks after siRNA introduction. All tissue and plasma samples were collected from the same rats 6 weeks later. (A) Core body temperature was telemetrically collected every minute for 48 h (n = 8 each in control and IL-6 KD group). (B) 48-h average of all collected core temperature values. (C) Average core temperature of two consecutive dark phases. (D) Average core temperature from two consecutive light phases. (E) Spontaneous activity collected every minute for 48 h. (F) 48-h sum of all collected spontaneous activity values. (G) Sum of activity from two consecutive dark phases. (H) Sum of activity from two consecutive light phases. (I) Representative images of infrared thermography of BAT region in control and IL-6 KD. (J) BAT temperature as detected by infrared thermography during the light phase (n = 8 and 9, control and IL-6 KD, respectively). (K) BAT gene expression, as detected by qPCR, of the following genes: UCP1, thermogenesis permissive mitochondrial protein; PRDM16, a transcription factor required for suppression of white-fat-selective genes in BAT and DIO2, an enzyme responsible for conversion of the low-activity form thyroxine (T4) to the active form 3, 3′, 5-triiodothryonine (T3). Ucp1, uncoupling protein 1; Prdm16, PR domain zinc-finger protein 16; Dio2, type II deiodinase. (L) UCP-1 protein levels in BAT in the IL-6 KD rats compared to control rats (n = 8 each). Image above the graph shows the representative western blot bands for each treatment group. (M) Plasma T3 levels in IL-6 KD rats and controls (n = 8, each). (N) Plasma T4 levels. (O) Plasma TSH levels. (P) TRH gene expression in the hypothalamus as detected by qPCR (n = 5, each). (Q) TSHb expression in the pituitary (n = 5, each) as detected by qPCR. (R) Quantification of TH protein expression in BAT tissue (n = 12). (S) Representative images of TH expression (shown in brown) in BAT of controls and IL-6 KD, along with a negative control. TSHb, thyroid-stimulating hormone beta subunit; TRH, thyroxine-releasing hormone; BAT, brown adipose tissue; TH, tyrosine hydroxylase. The results presented here were derived from tissues of rats with physiology and metabolism assessed and displayed in Figure 4. All gene expression presented here as bar graphs was detected by real-time qPCR. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; unpaired Student’s t test comparisons.

Figure 6
Figure 6

Physiological Contexts Interacting with lPBN IL-6 (A–F) Virally introduced siRNA, designed to knockdown IL-6 gene expression, was delivered into the lPBN, as shown in Figure 4, and anxiety-like behavior and concurrent locomotor activity testing was conducted in male rats 7 weeks after siRNA introduction. (A) Time spent in the center of an open-field arena (n = 7–8). (B) Time spent in the periphery of the open-field arena. (C) Total locomotor activity of IL-6 KD rats in comparison to controls. (D) Time in the open arms of the elevated plus maze (EPM) of IL-6 KD rats in comparison to control rats. (E) Total locomotor activity of IL-6 KD and controls. (F) Expression of Crh in the hypothalamus, as detected by qPCR, in IL-6 KD and control rats. (G) Gene expression levels of IL-6, as detected by qPCR, in PBN of 11-week-old chow-fed rats challenged with a 30-min acute-restraint stress (n = 7–8). (H and I) PBN IL-6 mRNA expression after 48-h exposure to ambient cold in 11-week-old chow-fed male (n = 6 per treatment group) (H) and female (n = 7 per treatment group) rats (all from another new cohort) (I). (J–L) A new cohort of male mice, fed a high-fat diet for 8 weeks prior to testing, and 13 weeks old at the time of testing, was exposed to cold for 48 h, and brain tissues were immediately collected after cold exposure for testing IL-6 mRNA levels in the PBN of (J) chow- and (K) HFD-fed mice (n = 6–10). Comparison of IL-6 levels in the PBN of cold-exposed chow- and HFD-fed mice (L). All gene expression presented here as bar graphs was detected by real-time qPCR. Data represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗∗p < 0.00001; unpaired Student’s t test comparisons. OF, open field.

Figure 7
Figure 7

Lateral PBN IL-6-Expressing Neurons Innervate the Paraventricular Hypothalamus (A) Retrograde viral tracer was unilaterally injected into the paraventricular hypothalamus of two 6-week-old male rats at a volume of 0.5 μL. Tissues were harvested 3 weeks later. (B) Representative confocal image of the retrograde viral tracer injection site along with the corresponding rat brain atlas slide to show the confinement of the viral infection to the paraventricular hypothalamus. (C and D) The densest PBN area of green fluorescent protein (EGFP)-labeled neurons was located in the lPBN (C) and the corresponding rat brain atlas slide (D). (E and F) Lateral PBN-focused images show cell bodies labeled with EGFP retrogradely carried from the paraventricular hypothalamus without (E) and with (F) DAPI. Interleukin-6 mRNA expression, detected by RNAscope in situ hybridization and shown in red, throughout the same area of lPBN. (G–J) Four examples of high-resolution cell images to show IL-6 mRNA (red) in the lPBN neurons projecting to the PVH (green). The green serrated line represents a trace of the green EGFP label (to outline the cell body and fibers in the image) and is superimposed on the RNAscope image in order to reveal the signal in the cell that is otherwise made less visible by the strong EGFP label. Cell nuclei are labeled in blue with DAPI. 4V, fourth ventricle; PVN, paraventricular nucleus; PaDC, paraventricular hypothalamic nucleus, dorsal cap; PaV, paraventricular hypothalamic nucleus, ventral part; PaMP, paraventricular hypothalamic nucleus, medial parvicellular part; PaLM, paraventricular hypothalamic nucleus, lateral magnocellular part; LPBI, lateral parabrachial nucleus, internal part; LPBD, lateral parabrachial nucleus, dorsal part; LPBV, lateral parabrachial nucleus, ventral part; LPBC, lateral parabrachial nucleus, central part; LPBE, lateral parabrachial nucleus, external part.

Similar articles

Cited by

References

    1. Alhadeff A.L., Baird J.P., Swick J.C., Hayes M.R., Grill H.J. Glucagon-like Peptide-1 receptor signaling in the lateral parabrachial nucleus contributes to the control of food intake and motivation to feed. Neuropsychopharmacology. 2014;39:2233–2243. - PMC - PubMed
    1. Alhadeff A.L., Hayes M.R., Grill H.J. Leptin receptor signaling in the lateral parabrachial nucleus contributes to the control of food intake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2014;307:R1338–R1344. - PMC - PubMed
    1. Anesten F., Santos C., Gidestrand E., Schéle E., Pálsdóttir V., Swedung-Wettervik T., Meister B., Patrycja Skibicka K., Jansson J.O. Functional interleukin-6 receptor-α is located in tanycytes at the base of the third ventricle. J. Neuroendocrinol. 2017;29 - PMC - PubMed
    1. Bastard J.P., Jardel C., Bruckert E., Blondy P., Capeau J., Laville M., Vidal H., Hainque B. Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese women after weight loss. J. Clin. Endocrinol. Metab. 2000;85:3338–3342. - PubMed
    1. Benrick A., Schéle E., Pinnock S.B., Wernstedt-Asterholm I., Dickson S.L., Karlsson-Lindahl L., Jansson J.O. Interleukin-6 gene knockout influences energy balance regulating peptides in the hypothalamic paraventricular and supraoptic nuclei. J. Neuroendocrinol. 2009;21:620–628. - PubMed

Publication types

MeSH terms

Substances