Potent hyperglycemic and hyperinsulinemic effects of thyrotropin-releasing hormone microinjected into the rostroventrolateral medulla and abnormal responses in type 2 diabetic rats

Abstract

We identified ventrolateral medullary nuclei in which thyrotropin-releasing hormone (TRH) regulates glucose metabolism by modulating autonomic activity. Immunolabeling revealed dense prepro-TRH-containing fibers innervating the rostroventrolateral medulla (RVLM) and nucleus ambiguus (Amb), which contain, respectively, pre-sympathetic motor neurons and vagal motor neurons. In anesthetized Wistar rats, microinjection of the stable TRH analog RX77368 (38-150 pmol) into the RVLM dose-dependently and site-specifically induced hyperglycemia and hyperinsulinemia. At 150 pmol, blood glucose reached a peak of 180+/-18 mg% and insulin increased 4-fold. The strongest hyperglycemic effect was induced when RX77368 was microinjected into C1 area containing adrenalin cells. Spinal cord transection at cervical-7 abolished the hyperglycemia induced by RVLM RX77368, but not the hyperinsulinemic effect. Bilateral vagotomy prevented the rise in insulin, resulting in a prolonged hyperglycemic response. The hyperglycemic and hyperinsulinemic effects of the TRH analog in the RVLM was peptide specific, since angiotensin II or a substance P analog at the same dose had weak or no effects. Microinjection of RX77368 into the Amb stimulated insulin secretion without influencing glucose levels. In conscious type 2 diabetic Goto-Kakizaki (GK) rats, intracisternal injection of RX77368 induced a remarkably amplified hyperglycemic effect with suppressed insulin response compared to Wistar rats. RX77368 microinjected into the RVLM of anesthetized GK rats induced a significantly potentiated hyperglycemic response and an impaired insulin response, compared to Wistar rats. These results indicate that the RVLM is a site at which TRH induces sympathetically-mediated hyperglycemia and vagally-mediated hyperinsulinemia, whereas the Amb is mainly a vagal activating site for TRH. Hyperinsulinemia induced by TRH in the RVLM is not secondary to the hyperglycemic response. The potentiated hyperglycemic and suppressed hyperinsulinemic responses in diabetic GK rats indicate that an unbalanced "sympathetic-over-vagal" activation by TRH in brainstem RVLM contributes to the pathophysiology of impaired glucose homeostasis in type 2 diabetes.

Fig. 1

Confocal images of brainstem sections double-labeled for prepro-TRH_160-169 (ppTRH) and tyrosine hydroxylase (TH) or ppTRH and choline acetyltransferase (ChAT). Transverse sections of the brainstem at interaural levels of −4.24 mm (left panels) or −4.68 mm (right panels) were labeled with antibodies against ppTRH (green, all panels), TH (red, panels A–D) and ChAT (red, panels E–H). The location of the illustrated fields is indicated by the rectangles in the diagrams in the middle panels, which were reproduced from the Paxinos & Watson Atlas of the Rat Brain (Paxinos et al., 1997): RVLM, rostroventrolateral medulla (A, C); NTS, nucleus tractus solitarius (B, C); Amb, nucleus ambiguus (E, G); DMV, dorsal motor nucleus of the vagus (F, H). Images in panels C, D, E and F are 3 optical sections acquired with a 10x objective (scale bars are 100 μm). Images in panels A, B, G and H are 13–29 optical sections acquired with a 63x objective (scale bars are 10 μm). In the RVLM (A, C) and the NTS (B, D), strong ppTRH immunoreactivity was found in fibers and puncta, which represent axons and presynaptic terminals containing ppTRH. The ppTRH staining was also detected inside neurons, both TH-positive and TH-negative (yellow arrows in A), indicating that the RVLM contains neurons synthesizing TRH. In the Amb-RVLM (E, G) and the DVM (F, H), ppTRH-immunoreactivity was found in fibers surrounding ChAT-positive and ChAT-negative neurons, and also inside the ChAT-immunoreactive neurons. The white arrowheads in panels A and G indicate examples of putative axodendritic and axosomatic interactions between TRH axons/dendrites and TH (A) or ChAT (G) neurons/axons. The nature of these structures was inferred by close examination and rotation of the whole confocal stack in three dimensions. cc: central canal.

Fig. 2

Left panel: Dose-related hyperglycemic effect of the stable TRH analog RX77368 microinjected into the RVLM of overnight-fasted Wistar rats. Each point represents the mean ± SEM of the number of rats indicated in the parenthesis. * P < 0.05 compared with the effect of microinjection into sites outside of the RVLM. # P< 0.05 compared with the basal levels of the same group. Right panel: Effective and ineffective microinjection sites of the TRH analog RX77368 to induce hyperglycemia, marked on plates reproduced from the atlas of Paxinos & Watson (Paxinos et al., 1997).

Fig. 3

Examples of glucose and insulin responses to microinjection of normal saline (100 nl) or TRH analog RX77368 (150 pmol/100 nl) into the RVLM in individual Wistar rats.

Fig. 4

The hyperglycemic and hyperinsulinemic effects of TRH analog RX77368 (150 pmol) microinjected into the RVLM in sham-operated, spinal cord-transected, and bilateral cervical-vagotomized Wistar rats. Each point represents mean ± SEM of the number of rats indicated at the top of each graph in the lower panel. * P < 0.05 compared with the value of the time point in sham operated rats with saline microinjection into the RVLM (group A). # P< 0.05 compared with the value of the time point in sham operated rats with TRH analog RX77368 microinjection into the RVLM (group B).

Fig. 5

The hyperglycemic and hyperinsulinemic effects of RVLM microinjection of the TRH analog RX77368, the substance P analog DiMe-C7 [pGlu5, Mephe8, MeGly9]substance P_5-11, and angiotensin II (all at 150 pmol, data of the TRH analog are adapted from Fig. 2) in Wistar rats. Each point represents mean ± SEM of the number of rats indicated in the parenthesis. * P < 0.05 compared with the value of the same time point in TRH analog RX77368 injected rats. # P< 0.05 compared with the basal levels of the same group.

Fig. 6

The potentiated hyperglycemic and insulin-suppressive responses to intracisternal injection (ic) of the stable TRH analog RX77368 in type 2 diabetic (T2D) Goto-Kakizaki (GK) rats, compared to Wistar rats. Each point represents mean ± SEM of the number of rats indicated in the parenthesis. * P < 0.05 compared with the response of Wistar rats receiving ic saline. # P< 0.05 compared with the response of Wistar rats receiving ic RX77368 (25 ng).

Fig. 7

Top panel: A. Potentiated hyperglycemic response to TRH analog RX77368 (150 pmol) microinjected into the RVLM in GK rats compared to Wistar rats (data of the Wistar rats are adapted from Fig. 2). B, Unchanged blood glucose levels after RX77368 (150 pmol) microinjected into the nucleus ambiguus (Amb) in Wistar and GK rats. Each point represents mean ± SEM of the number of rats indicated near the line. * P < 0.05 compared with the value of the same time point in Wistar rats. # P< 0.05 compared with the basal levels of the same group. Lower panel: Coronal sections showing the microinjection sites in the RVLM (A) and Amb (B). Black circles are the microinjection sites in Wistar rats and the gray circles are those in GK rats.

Fig. 8

Top panel: A, Impaired first-hour insulin response to TRH analog RX77368 (150 pmol) microinjection into the RVLM in GK rats compared to Wistar rats. B. Similar insulin response to RX77368 (150 pmol) microinjected into the Amb in Wistar and GK rats. Lower panel: The corresponding changes in blood glucose levels in the same rats. Each point represents mean ± SEM of the number of rats indicated near the line. * P < 0.05 compared with the value of the same time point in Wistar rats. # P< 0.05 compared with the basal levels of the same group.