Enhanced insulin clearance in mice lacking TRPM8 channels

Abstract

Blood glucose concentration is tightly regulated by the rate of insulin secretion and clearance, a process partially controlled by sensory neurons serving as metabolic sensors in relevant tissues. The activity of these neurons is regulated by the products of metabolism which regulate transmitter release, and recent evidence suggests that neuronally expressed ion channels of the transient receptor potential (TRP) family function in this critical process. Here, we report the novel finding that the cold and menthol-gated channel TRPM8 is necessary for proper insulin homeostasis. Mice lacking TRPM8 respond normally to a glucose challenge while exhibiting prolonged hypoglycemia in response to insulin. Additionally, Trpm8-/- mice have increased rates of insulin clearance compared with wild-type animals and increased expression of insulin-degrading enzyme in the liver. TRPM8 channels are not expressed in the liver, but TRPM8-expressing sensory afferents innervate the hepatic portal vein, suggesting a TRPM8-mediated neuronal control of liver insulin clearance. These results demonstrate that TRPM8 is a novel regulator of serum insulin and support the role of sensory innervation in metabolic homeostasis.

Keywords: TRPM8; clearance; insulin; neuronal; sensitivity.

Fig. 1.

Streptozotocin (STZ) sensitivity in Trpm8^-/- mice. A: body weight tracking in wild-type and Trpm8^-/- mice injected with 180 mg/kg STZ or vehicle (veh: n = 4–6, STZ: n = 4). B: representative picture of wild-type and Trpm8^-/- mice 1 wk following STZ administration. C: weight tracking in male littermates (male and female) from 4–16 wk of age fed normal chow (wt: 24.1 ± 1.0; Trpm8^-/-: 21.1 ± 0.3 g by 12 wk, P < 0.001, n = 6 each genotype). D: body length measurements of Trpm8^-/- and wild-type mice of both sexes. E: weight tracking in mice from D; P > 0.05 NS, *P < 0.05, **P < 0.01, n = 6–8 mice. Food intake [wt: 3.68 ± 0.11; Trpm8^-/-: 3.72 ± 0.09 g/day, P = 0.75 (F); wt: 0.17 ± 0.004, Trpm8^-/-: 0.19 ± 0.01 g/day/g body wt, P < 0.01, n = 11–12 (G)].

Fig. 2.

Metabolic characterization of Trpm8^-/- mice. A: telemetric monitoring of core body temperature in wild-type and Trpm8^-/- mice. Scatter plot and averaged date are shown over a 48-h period (n = 2). B: mean core body temperature during light (day: 6 AM-6 PM) and dark cycles (night: 6 PM-6 AM) for wild-type and Trpm8^-/- mice (n = 4). Blood glucose (C) and serum insulin (D) levels under fed and fasting conditions (n = 7–16). Values are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's unpaired t-test.

Fig. 3.

Pancreatic β-cell function in Trpm8^-/- mice. A: representative immunostaining for insulin in mouse pancreatic islets. B: distribution of pancreatic islet sizes in Trpm8^-/- compared with wild-type mice (n = 2,076 islets measured from 3 mice of each genotype). C: whole pancreatic insulin content expressed as ng insulin/μg total protein (wt: 3.5 ± 1.3ng/μg, Trpm8^-/-: 4.8 ± 1.3 ng/μg protein, P = 0.51, n = 3). D: gene expression analysis of common β-cell-specific transcripts in isolated pancreatic islets by qPCR. Values expressed as ΔC_T relative to GAPDH expression (n = 3). E: insulin secretion stimulated by 16.8 mM glucose (wt: 0.58 ± 0.02, Trpm8^-/-: 0.63 ± 0.06 ng/islet/15 min), or 40 mM KCl (wt: 0.72 ± 0.03, Trpm8^-/-: 0.75 ± 0.06 ng/islet/15 min, P > 0.05, n = 3) from isolated islets (baseline: 2.8 mM glucose). All experiments were carried out on adult male mice aged 8–12 wk old unless otherwise noted. Values are expressed as means ± SE; P > 0.05 NS by Student's unpaired t-test.

Fig. 4.

Trpm8^-/- mice exhibit heightened insulin sensitivity in vivo. Intraperitoneal glucose tolerance test (IPGTT) performed on acute- (5.5 h; A) and overnight-fasted (14–16 h; B) mice by injecting 2.0 g/kg body wt ip glucose (n = 14–16 for acute and n = 18–19 for overnight). C: blood glucose concentrations in an insulin tolerance (IPITT) test performed on acute-fasted mice by injecting 0.75 U/kg body wt ip insulin (n = 17–18). D: insulin tolerance test performed on fed mice by injecting 0.7 5 U/kg insulin ip (n = 5–6). E: serum insulin (basal: wt: 0.42 ± 0.04, Trpm8^-/-: 0.24 ± 0.01 ng/ml, P < 0.01; at 60 min: wt: 0.59 ± 0.07, Trpm8^-/-: 0.55 ± 0.06 ng/ml, P = 0.68, n = 14–17) following 2.0 g/kg body wt ip glucose to overnight-fasted mice. Values are expressed as means ± SE. **P < 0.01 by Student's unpaired t-test.

Fig. 5.

Enhanced insulin clearance in Trpm8^-/- mice. A: resting serum C-peptide levels in fed and fasted mice (n = 7–10). B: serum C-peptide following 2.0 g/kg body wt ip glucose to overnight-fasted mice (n = 6). C: serum insulin-to-C-peptide ratios during different intervals following glucose injection. D: incremental increase of insulin (wt: 116.1 ± 2.6, Trpm8^-/-: 52.4 ± 3.5 pmol/l) compared with C-peptide (wt: 249.1 ± 38.2, Trpm8^-/-: 251.1 ± 46.0 pmol/l) from 0–15 min following glucose challenge. Values are expressed as means ± SE. *P < 0.05, ***P < 0.001, NS P > 0.05 by Student's unpaired t-test.

Fig. 6.

Innervation of TRPM8-expressing afferent fibers in hepatic portal vein (HPV). A: RT-PCR from cDNA from trigeminal/dorsal root ganglia, whole pancreas, isolated islets, or liver. Two primer sets were used to amplify both 5′- (M8 5′) and 3′- (M8 3′) regions of TRPM8. GAPDH (GAP) was used as a universal control for sample integrity, and insulin (INS) and α1-anti-trypsin (α-AT) were used as tissue-specific controls for pancreas and liver, respectively. All experiments were carried out on male mice age 8–12 wk. Values are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's unpaired t-test. B and C: Whole-mount staining shows TRPM8-expressing afferents (green) in the HPV of Trpm8^GFP reporter mice vs. all afferent fibers labeled with the pan-neuronal marker PGP9.5 (red). Arrowheads demarcate axons, and all experiments were carried out on male mice aged 8–12 wk. D: immunoreactivity to TRPM8 (green) and PGP9.5 (red) from whole-mount rat HPV. E: TRPM8 immunoreacitivity was absent when tissue was probed with TRPM8 antibodies pretreated with the antigenic peptide.

Fig. 7.

Insulin-degrading enzyme expression is increased in Trpm8^-/- mouse livers. Semiquantitative Western blotting on whole tissue lysates isolated from overnight-fasted wild-type and Trpm8^-/- mice. A–C: insulin-degrading enzyme (IDE) protein expression is quantified in liver (A), kidney (B), and muscle (C). Expression levels are shown by animal (left) and means ± SE (right) for each genotype relative to β-actin (liver and kidney) or GAPDH (muscle). D: insulin receptor (InsR) expression levels in the 3 aforementioned tissues expressed as means ± SE (n = 3). *P < 0.05, by Student's unpaired t-test.