TRPC5 controls the adrenaline-mediated counter regulation of hypoglycemia

Figures

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  Figure 1. Deletion of TRPC channels aggravates insulin-induced hypoglycemia and impairs the associated plasma adrenaline rise. (A) Schematic representation of the experimental procedure: Diabetes is induced by i.p injections of streptozotocin (STZ) on five consecutive days. The resultant diabetic hyperglycemia is controlled by s.c. injections of insulin glargin (Lantus®), according to the current blood glucose levels. (B) Survival rate of STZ-induced diabetic mice during the long-term regulation of the blood glucose levels with insulin (t = 0: n = 10 for Trpc1/4/5/6–/–; n = 10 for wild-type). After 2 weeks, the insulin dose was lowered for the Trpc1/4/5/6–/– animals (for details see Methods). (C) Insulin tolerance test (ITT): Time course of the blood glucose levels after the i.p. injection of insulin or saline in non-diabetic wild-type or Trpc1/4/5/6–/– mice. Wild-Type vs. Trpc1/4/5/6–/– during insulin treatment at 15 min, p = 6.55 × 10−5; at 30 min p = 6.93 × 10−6; at 45 min p = 6.08 × 10−7; at 60 min p = 3.72 × 10−5. (D) The incremental integrated area under the curve from the ITT in C (Trpc1/4/5/6–/–: n = 11 for insulin, n = 9 for saline; wild-type: n = 9 for insulin, n = 8 for saline;…

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  Figure 2. Aggravated insulin-induced hypoglycemia in mice lacking TRPC5 due to impaired adrenaline response. (A) Long-term Insulin tolerance test (ITT). Blood glucose levels of Trpc5–/0 mice, measured over 150 min after insulin injection (Trpc5–/0 insulin: n = 19 (0 min), n = 7 (15 min) n = 12 (30, 60, 90 min), n = 6 (120, 150 min); saline: n = 19 (0 min), n = 7 (15 min), n = 8 (30, 60, 90 min), n = 4 (120, 150 min). wild-type insulin: n = 18 (0 min), n = 7 (15 min), n = 11 (30, 60, 90 min), n = 6 (120, 150 min); Saline: n = 13 (0 min), n = 5 (15 min), n = 8 (30, 60, 90 min), n = 4 (120, 150 min). Wild-type insulin: 0 vs. 15 min p = 3.31 × 10−10, 0 vs. 30 min p = 1.45 × 10−14, 0 vs. 60 min p = 2.07 × 10−16, 0 vs. 90 min p = 3.36 × 10−16, 0 vs. 120 min p = 1.28 × 10−10, 0 vs. 150 min p = 1.85 × 10−7, 60 vs. 120 min p = 0.000487, 60 vs. 150 min p = 1.70 × 10−14. Trpc5–/0 insulin: 0 vs. 15 min p = 1.75 × 10−10, 0 vs. 30 min p = 2.13 × 10−13, 0 vs. 60 min p = 8.67 × 10−17, 0 vs. 90 min p = 3.99 × 10−17, 0 vs. 120 min p = 1.48 × 10−12, 0 vs. 150 min p = 1.37 × 10−11. Insulin, wild-type vs. Trpc5–/0: 15 min p = 0.00253, 30 min p = 0.0102, 60 min p = 0.000533, 90 min p = 0.000277, 120 min p =…

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  Figure 3. Tissue-specific Trpc5-deletion and its effect on autonomic counter regulation. (A) Schematic representation of the processes underlying the central stress perception and stress response to hypoglycemia. IML intermediolateral nucleus, Th thoracic vertebrae, RVLM ventrolateral medulla. (B) Time course of blood glucose levels during an insulin tolerance test in a mouse line with Trpc5 deletion in cholinergic neurons (male Trpc5fx/0;ChAT-Cre+: n = 11; Trpc5fx/0;ChAT-Cre–: n = 9). (C) Incremental integrated area under the curve during the ITT. (D) Plasma adrenaline levels 30 min after insulin injection (male Trpc5fx/0;ChAT-Cre+: p = 8.19 × 10−7, n = 11 for insulin, n = 11 for saline; Trpc5fx/0;ChAT-Cre–: p = 0.000217, n = 10 for insulin, n = 13 for saline). ***p < 0.001, two sample t-test. (B–D) Mean ± s.e.m. all indicated n-values are biological replicates. Source data are available online for this figure.

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  Figure 4. TRPC5 channels in catecholaminergic cells are relevant for autonomic counter regulation. (A) Time course of blood glucose levels during insulin tolerance tests in a mouse line with Trpc5 deleted in catecholaminergic cells. Male Trpc5fx/0;DBH-Cre+: n = 11 for insulin, n = 5 for saline; Trpc5fx/0;DBH-Cre–: n = 12 for insulin, n = 6 for saline. Trpc5fx/0;DBH-Cre+ vs. Trpc5fx/0;DBH-Cre– after insulin injection, p = 0.0194 (15 min), p = 3.12 × 10−6 (30 min), p = 1.05 × 10−7 (45 min), p = 1.11 × 10−5 (60 min). (B) The incremental integrated area under the curve from (A). Male Trpc5fx/0;DBH-Cre+: n = 11 for insulin, n = 5 for saline; Trpc5fx/0;DBH-Cre–: n = 12 for insulin, n = 6 for saline. Trpc5fx/0;DBH-Cre+ vs. Trpc5fx/0;DBH-Cre– after insulin injection, p = 0.00219. (C) The plasma adrenaline levels 30 min after insulin injection. Male Trpc5fx/0;DBH-Cre+: p = 6.72 × 10−5, n = 14 for insulin, n = 11 for saline; Trpc5fx/0;DBH-Cre–: p = 0.000966, n = 10 for insulin, n = 4 for saline; insulin treatment between genotypes p = 0.00205. (D) As (A) but in female mice. Female Trpc5fx/fx; DBH-Cre+: n = 9; Trpc5fx/fx; DBH-Cre–: n = 8, p = 0.000792 (15 min), p = 2.50 × 10−9 (30 min), p = 4.20 ×…

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  Figure 5. Genetic loss or pharmacological inhibition of TRPC5 strongly reduces muscarine and PACAP-evoked catecholamine secretion from chromaffin cells. (A) Average time course of the intracellular calcium concentration in Fura-2-AM-loaded chromaffin cells stimulated with 1 µM PACAP. (B) The increase in [Ca2+]i is significantly reduced in Trpc1/4/5–/– (red, p = 0.0306) and TRPC5-deficient (Trpc5–/0, green, p = 0.0395) cells when cells were perfused with 1 µM PACAP. Wt, n = 12; Trpc5–/0, n = 10; Trpc1/4/5–/–, n = 15 from 4 independent preparations. (C) As (A) but with 30 µM muscarine. (D) The increase in [Ca2+]i is significantly reduced in Trpc1/4/5–/– (red, p = 4.22 × 10−5) and TRPC5-deficient (Trpc5–/0, green, p = 3.70 × 10−4) cells when cells were perfused with 30 µM muscarine. wt, n = 15; Trpc5–/0, n = 13; Trpc1/4/5–/–, n = 15 from 3 independent preparations. (E) As (A) but with 30 nM Englerin A (EA). (F) The increase in [Ca2+]i is significantly reduced in Trpc1/4/5–/– (red, p < 2 × 10−16) and TRPC5-deficient (Trpc5–/0, green, p < 2 × 10−16) cells when cells were perfused with 30 nM EA. wt, n = 15; Trpc5–/0, n = 15; Trpc1/4/5–/–, n = 19 from 3 independent preparations. (G) As (A) but…

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  Figure 6. PACAP evokes TRPC channel mediated inward currents and secretion in mouse chromaffin cells. (A) Exemplary recordings (perforated-patch configuration) of a PACAP-evoked inward current at a holding potential of −70 mV in a wt cell (upper panel) and its cumulative charge plot (lower panel). Note the burst-like current deflections. (B) Repeated application of PACAP to the same cell shown in (A). (C, D) No significant conductance changes could be evoked in Trpc1/4/5–/– cells (1st, (C) and 2nd application (D). (E, F) Time course of mean charge transfer for the 1st (E) and 2nd application of PACAP (F). (G) The PACAP-evoked charge transfer (left panel p = 6.43 × 10−6) and increase in membrane capacitance (right panel p = 2.75 × 10−5) are strongly reduced in Trpc1/4/5–/– cells. (H) Changes in membrane capacitance (indicative of exocytosis) correlate well with the charge transfer evoked by PACAP. (E–H) Data were collected from 12 WT and 15 Trpc1/4/5–/– cells from 3 independent preparations, mean ± s.e.m., ***p < 0.001, Mann–Whitney U test. Source data are available online for this figure.

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  Figure 7. PACAP evokes TRPC5 channel mediated inward currents in a PLC-dependent manner. (A) Exemplary recordings (perforated-patch configuration) of a PACAP-evoked inward current in a wt cell (upper panel), the corresponding cumulative charge plot (middle panel) and membrane capacitance measurement (lower panel). The capacitance increase (ΔCM) in response to the stimulation reflects dense core vesicle exocytosis. (B) PACAP fails to evoke any inward current or secretion in Trpc5–/0 cells. (C) The PLC–inhibitor U-73122 (10 µM) blocks the PACAP induced current and any secretion in wt cells. (D) Genetic loss of TRPC5 or inhibition of the PLC prevents any change in CM (ΔCM) seen in wt cells upon PACAP application. Data were collected from wt, n = 17, Trpc5–/0, n = 15 p = 0.0377 and wt+U-73122, n = 21 p = 0.0026 from 3 independent preparations. (E) The cumulative charge transfer is abolished in the absence of TRPC5 or after PLC inhibition, data were collected from wt, n = 17, Trpc5–/0, n = 15, p = 0.00002 and wt+U-73122, n = 20, p = 0.000011 from 3 independent preparations. (F) Changes in membrane capacitance (indicative of exocytosis) correlate with the charge influx evoked by PACAP-38, data…

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  Figure 8. PACAP induces long lasting TRPC5 mediated depolarization of the membrane potential. (A) Exemplary recordings (perforated-patch and current-clamp configuration) of a PACAP-evoked membrane depolarization in wt cells (left panel). No changes in VM could be detected in Trpc5–/0 cells (right panel). (B) Time course of the average PACAP-evoked depolarization of the membrane potential, mean ± s.e.m. (C) PACAP leads to a significant increase in the VM in wt cells but not in Trpc5–/0 cells. Data were collected from wt, n = 13 and Trpc5–/0, n = 14 cells from 3 independent preparations. One-way ANOVA Kruskal Wallis for statistical testing within the group (wt, baseline vs. PACAP p = 0.001; wt baseline vs. washout p = 0.0001) and Mann–Whitney U-test between wt and Trpc5–/0 (wt vs. Trpc5–/0 PACAP, p = 0.00275; Washout, p = 0.000528; **p < 0.01, ***p < 0.001, bar graphs are presented as mean ±s.e.m. Source data are available online for this figure.

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  Figure 9. Metabolic parallels between TRPC5 deficiency and HAAF. (A) Plasma levels of lysine, aspartic acid, and taurine (from left to right) for diabetic patients diagnosed with HAAF before (basal) and during controlled hypoglycemia (HG; 60–70 mg/dL). n = 7 for HAAF patients, n = 8 for diabetes patients without HAAF. Lysine, basal, p = 0.0230; Aspartic acid, HG, p = 0.00169; Taurine, HG, p = 0.0166. (B) Plasma levels of lysine, aspartic acid, and taurine 30 min after the injection of saline (basal) or insulin (HG; 2.25 U/kg insulin, i.p.) for mice with Trpc5 deletion in catecholaminergic cells. Trpc5fx/0;DBH-Cre+: n = 13 for insulin, n = 7 for saline; Trpc5fx/0;DBH-Cre–: n = 13 for insulin, n = 7 for saline). Lysine, HG, p = 2.08 × 10−5; Lysine, Trpc5fx/0;DBH-Cre+, p = 2.24 × 10−6; Taurine, HG, p = 0.000715. HG, hypoglycemia. (A, B) Mean ± s.e.m., *p < 0.05, **p < 0.01, ***p < 0.001, two sample t test. Source data are available online for this figure.