Electric stimulation of ears accelerates body weight loss mediated by high-fat to low-fat diet switch accompanied by increased white adipose tissue browning in C57BL/6 J mice

Twenty four male C57BL/6JNarl mice were purchased from the National Laboratory Animal Center of the National Applied Research Laboratories, Taipei, Taiwan. At 6 wk. of age, all mice were fed a butter-based high-fat diet (30% dietary fat comprised of 29% butter and 1% soybean oil) for 5 wk. to induce obesity [indicated as wk. − 5〜0 in Fig. 1b]. C57BL/6 J mouse is prone to obesity and diabetes in response to a high-fat diet [28, 29]. The diet composition as reported in Chen et al. [30] has been verified to induce obesity and associated metabolic disorders in mice [30, 31]. Their body weight reached 31.3 ± 2.4 g at wk0 in contrast to 24.0 ± 1.6 g of chow-diet fed peers (> 20% higher). After DIO, mice were weighed and allocated by body weight into 3 groups (n = 8 for each group), i.e. AES (+/+ for electricity/clamps), Sham (−/+ for electricity/clamps), and Ctrl (−/− for electricity/clamps). They were fed a low-fat non-purified diet (Altromin 1320 Rat & Mouse Maintenance diet, Fwusow Industry Co. Ltd., Taiwan; containing 6% water, 51% crude carbohydrate, 23.5% crude protein, 4.5% crude lipid, 6% crude fiber, and 9% ash) onward for 5 wk. and concomitantly, AES or sham operation were applied during this period [indicated as wk. 0〜5 in Fig. 1b]. The body weight of Ctrl, Sham and AES group at wk. 0 was not different (i.e. 30.8 ± 2.6, 31.3 ± 2.4 and 31.5 ± 1.8 g, respectively). All mice were housed in polypropylene cages in groups of four mice per cage and were kept in a room maintained at 23 ± 2 °C, with a controlled 12-h-light:-dark cycle with ad libitum access to feed and drinking water. Body weight and feed intake were recorded twice per week. Cumulative body weight loss (from baseline, i.e. switching point of diet) was calculated each week. Animal care and research protocols were based on principles and guidelines approved by the Guide for the Care and Use of Laboratory Animals [32]. Protocols for animal care and handling were approved by the Institutional Animal Care and Use Committee of China Medical University (Protocol 103–69-N).

AES was applied under anesthesia by isoflurane administered through a vaporizing system (MATRX VIP 3000, Midmark, USA). Mice in the AES group received electrical stimulation (frequency, 2 Hz; intensity, 2 mA; visual ear twitch) using clip electrodes (ES apparatus Trio 300, Ito, Japan) with the anode placed at the ear lobe and cathode at the ear apex [23‐25], as shown in Fig. 1a. Stimulation was done 20 min/d (with each ear receiving the stimulus for 10 min) and 3 d each week (13:00–16:00 on Monday, Wednesday, and Friday) for 5 wk. consecutively. For the Sham group, anesthesia and clip electrodes were applied but no electricity was given and Ctrl mice were only anesthetized.

At the end of the study, feed was withheld overnight and the mice were killed by carbon dioxide asphyxiation. Blood was collected from the abdominal vena cava, allowed to clot and serum separated. WAT (mesenteric, retroperitoneal, epididymal, and inguinal fat) were excised and weighed. Aliquots of inguinal fat and hypothalamus were quick-frozen in liquid nitrogen and stored at − 80 °C for RNA or protein extraction. Concentrations of catecholamines (epinephrine and norepinephrine) in serum and inguinal fat (RIPA buffer extract) were measured using commercial kits (R&D, Minneapolis, MN), following manufacturer’s instructions.

The mRNA levels of Npy, AgRP and Pomc (encoding neuropeptide Y, agouti-related peptide and pro-opiomelanocortin, markers for appetite regulation) in hypothalamus, as well as Ucp1 (markers for WAT browning) in inguinal fat were measured by qRT-PCR. Total RNA was extracted from homogenized tissue using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and 1 mg total RNA was reverse-transcribed into first-strand cDNA using 200 units of MMLV-RT (Promega, Madison, WI, USA) in a total volume of 20 mL. For real-time PCR, a TaqMan system with inventory primers and probes (Applied Biosystems, Foster City, CA, USA) or a SYBR system with self-designed primers was used (Table 1). Amplification using 40 cycles of 2 steps (95 °C for 15 s and 60 °C for 1 min) was done with an ABI Prism 7900HT sequence detection system.

Samples of inguinal fat (0.1 g) were homogenized in RIPA buffer which contained 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Appropriate amounts of homogenate containing 50 mg of protein were electrophoresed on 10% SDS gels, transferred to a PVDF transfer membrane, and immunoblotted. Primary antibodies used were mouse antibodies against β-actin (St John’s Laboratory, London, UK), tyrosine hydroxylase (TH; Millipore, California) and rabbit antibodies against human UCP-1 (Abcam, Cambridge, UK) (diluted 1:1000 in PBS). In addition, HRP-labeled goat anti-mouse IgG antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and goat anti-rabbit IgG antibodies (Abcam) at a dilution of 1:5000 in PBS were used as a secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence Western blotting kit (Amersham International, Uppsala, Sweden) and images quantified by densitometric analysis (Multimage Light Cabinet, Alpha Innotech Corporation, San Leandro, CA, USA).

A portion of inguinal fat was fixed in 10% formalin, dehydrated through a graded ethanol series, embedded in paraffin, and cut into 5 μm sections. Sections were incubated with 5% goat serum in PBS after deparaffinization and rehydration. The primary antibody was a rabbit antibody against human UCP-1 (Abcam) (diluted 1:100 in PBS), whereas the secondary antibody was biotinylated goat anti-rabbit IgG antibodies (Dako, Carpinteria, CA, USA) (diluted 1:250 in PBS). Sections for UCP-1 staining were processed using a Dako kit (Dako REALTM envision TM detection system) according to the manufacturer’s instructions and examined on a Primo Star microscope (Zeiss, Oberkochen, Germany). Adiposoft software (ImageJ; National Institutes of Health, Bethesda, MD, USA) was used to calculate adipocyte cell diameter.

As expected, body weight was linearly increased by a high-fat diet, whereas switching to a low-fat diet caused a rapid decrease in body weight at the 2nd wk., followed by less rapid declines, irrespective of AES or not (Fig. 1b). However, when body weight change (Δ) were calculated and expressed as cumulative weight loss from baseline (switching point of diet), there was a trend of AES > Sham>Ctrl across the 5-wk weight reduction period, with a difference (AES vs. Ctrl, P < 0.05) at the last week (Fig. 1c). Although daily feed intake was not significantly different among groups, there was an opposite trend to weight loss, i.e. AES < Sham<Ctrl (Fig. 1d).

There were no significant differences among groups for body fat percentage in mesenteric, retroperitoneal, epididymal, and inguinal fat pads (Table 2). However, adipocyte diameter in inguinal fat (representative of subcutaneous fat) in the AES group was significantly smaller than in Ctrl, with an intermediate value for Sham (Table 2).

Serum concentrations of epinephrine and norepinephrine were elevated by auricular stimulation, regardless of whether electricity was applied (Fig. 2a), although concentrations were highest in the AES group. For norepinephrine, concentrations in AES were significantly greater than Sham, and the values in both groups were significantly greater than that of Ctrl.

Hypothalamic transcripts for neuropeptides associated with appetite regulation did not differ among groups, though the orexigenic Npy and AgRP tended to be lowered, while anorexigenic Pomc tended to be elevated in the AES group (Fig. 2b).

The mRNA levels of Ucp1 in inguinal fat were significantly greater in group AES than Ctrl, with an intermediate value for Sham (Fig. 2c). Protein levels of UCP-1 and TH in inguinal fat were greatest for AES, followed by Sham, and lowest for Ctrl (AES vs. Ctrl, P < 0.05; Fig. 3a). Norepinephrine concentrations in inguinal fat did not differ between groups Sham and Ctrl, with both significantly lower than in AES. In the latter group, there was morphological and immunohistochemical evidence of WAT browning, with adipocytes with multilocular and UCP-1⁺ staining characteristics (Fig. 3b), along with a smaller cell size relative to the other 2 groups.