Botulinum Injection Into the Proximal Intestinal Wall of Diet-Induced Obese Mice Leads to Weight Loss and Improves Glucose and Fat Tolerance

Abstract

Botulinum neurotoxin (available commercially as BOTOX) has been used successfully for treatment of several neuromuscular disorders, including blepharospasm, dystonia, spasticity, and cerebral palsy in children. Our data demonstrate that injection of Botox into the proximal intestinal wall of diet-induced obese (DIO) mice induces weight loss and reduces food intake. This was associated with amelioration of hyperglycemia, hyperlipidemia, and significant improvement of glucose tolerance without alteration of energy expenditure. We also observed accelerated gastrointestinal transit and significant reductions in glucose and lipid absorption, which may account, at least in part, for the observed weight loss and robust metabolic benefits, although possible systemic effects occurring as a consequence of central and/or peripheral signaling cannot be ignored. The observed metabolic benefits were found to be largely independent of weight loss, as demonstrated by pair-feeding experiments. Effects lasted ∼8 weeks, for as long as the half-life of Botox as reported in prior rodent studies. These results have valuable clinical implications. If the observed effects are translatable in humans, this approach could lay the foundation for therapeutic approaches geared toward robust and sustained weight loss, mimicking some of the benefits of bariatric operations without its cost and complications.

Figure 1

A: Representative Western blot showing SNAP-25 expression in mice duodenums injected with saline or Botox, measured 6 weeks later. B: Weight loss in DIO mice injected with saline or Botox (0.1–5 units/kg body wt [BW]) in the duodenal wall (n = 7–8 mice). C: Body composition analyses of DIO mice injected with saline or Botox (3 units/kg body wt) before (week 0) and 4 and 10 weeks after injections. D: Food intake in DIO mice injected with saline or Botox (3 units/kg body wt). E: Weight loss in pair-fed saline- or Botox-injected DIO mice (n = 6–8 pairs) *P < 0.05; **P < 0.01.

Figure 2

Average EE in saline- or Botox-injected mice, measured 1 (A), 4 (B and C), and 6 weeks (D and E) after injections (n = 6–8 mice). Multiple regression analyses were performed to determine possible effects of differences in body weight on EE during 4 (C) and 6 weeks (E).

Figure 3

Fasting plasma glucose (A), insulin levels (B), and HOMA-IR scores (C) in saline- or Botox-injected (3 units/kg) DIO mice after 1, 4, 7, and 10 weeks of injections (n = 7–8 mice). Fasting plasma glucose (D), insulin levels (E), and HOMA-IR scores (F) in saline- or Botox-injected mice pair-fed for 4 weeks (n = 6–8 pairs). *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 4

Blood glucose levels and AUCs after oral glucose gavage measured in mice with or without pair-feeding at week 0 (before pair-feeding and saline or Botox injections) (A and B), week 1 (C and D) and week 4 (E and F) (n = 6–8 pairs for pair-fed mice; n = 6–7 mice for non–pair-fed mice). Bars with different letters are significantly different. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Fasting plasma TGs (A), FFA (B), and cholesterol levels (C) in saline- or Botox-injected (3 units/kg) DIO mice after 1, 4, 7, and 10 weeks of injections (n = 6–8 mice). Blood TG levels (D) and TG AUC (E) in saline- or Botox-treated DIO mice after oral gavage of lipid emulsion (10 µL/g body wt), measured 10 days after injections (n = 5–7 mice). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Fasting plasma TGs (A), FFA (B), and cholesterol levels (C) in Botox-injected mice and in saline-injected mice pair-fed for 4 weeks (n = 6–8 pairs). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Plasma ³H recovery in saline- or Botox-treated mice at 5, 15, 30, 45, 60, 90, and 120 min after an oral gavage of 20% dextrose (2 mg/g body wt [BW]) spiked with 3-[³H]OMG (∼5 µCi/mouse) (A) and 3-[³H]OMG AUC (B). dpm, disintegrations per minute. Fecal fat (C), TG (D), FFA (E), and cholesterol (F) in saline- or Botox-injected mice measured after 1, 4, 7, and 10 weeks of injections (n = 6 mice). Plasma [³H]OA) recovery in saline- or Botox-treated mice at 30, 60, 120, 180, and 240 min after an oral gavage of olive oil spiked with [³H]OA (∼5 µCi/mouse) (G) and [³H]OA AUC (H) (n = 6–8 mice). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8

A: Total GI transit time in saline- or Botox-injected mice by carmine dye method (n = 6 mice/group). Percentage of FITC-dextran recovered during 0–90 min of gavage in intestinal segment S1 (B), S2 (C), S3 (D), and S4 (E). F: Percentage of FITC-dextran recovered in S4 at 2–6 h after gavage. Saline- or Botox-treated mice (n = 5–8/group) were fasted overnight, and 5 mmol/L FITC-dextran was administered by gavage. Mice were euthanized 0, 15, 30, 45, 60, 90, 120, 180, 240, and 360 min later, and the small intestines were collected for analyses. Intestines were sectioned into four 8-cm segments (S1-S4 from duodenum to cecum). All four segments for every time point were collected from the same mouse (n = 4–8 mice).