Tyrosine Hydroxylase Knockdown at the Hypothalamic Supramammillary Nucleus Area Induces Obesity and Glucose Intolerance

Figures

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  Fig. 1.. A Verification of Th gene expression knockdown at the SuMN area with TH-immunopositive DAB staining. The extent of Th gene expression knockdown via a virus-mediated shRNA was evaluated by TH immunohistochemistry using DAB as a chromogen. The images represent the TH-immunopositive DAB staining [TH(+)-DAB] of Scr-shRNA versus Th-shRNA brain sections at the rostral (bregma −4.52), middle (bregma −4.70), and caudal (bregma −4.80) levels of the SuMN. In Scr-shRNA brain sections (low magnification (a, c, e) and high magnification (b, d, f)), the DAB staining revealed TH(+)- cell bodies and processes clustered at the medial SuMN and scattered around the adjacent medial posterior hypothalamic area dorsal to the medial SuMN. Dense TH(+)- immunoreactivity at the VTA was detected bilaterally. In Th-shRNA brain sections (low magnification (g, i, k) and high magnification (h, j, l)), TH(+)- cell bodies were no longer detectable in the medial SuMN and dramatically reduced in the adjacent medial PH (within ∼400 μm from dorsal SuMN) leaving TH(+)- processes readily visible. Strong and comparable TH(+)- immunoreactivity was bilaterally detected at the VTA on Th-shRNA and Scr-shRNA brain sections.…

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  Fig. 2.. THx at the SuMN area induces obesity, increased feeding and feeding efficiency while decreasing FAO in f-HFDr-RC rats (Study 2Ai). f-HFDr-RC rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA-GFP to knockdown Th gene expression (THx) or control AAV5-Scr-shRNA-GFP (Ctr). Compared with Ctr, THx treatment increased body weight gain by 42% (p = 0.003) (a, b) and food consumption by 15% (p = 0.0001) (c, d) with a concurrent increase in feed efficiency (25%, p = 0.048) over 72 days (e) post-virus inoculation. When sacrificed at 17 weeks post-virus inoculation, THx rats relative to Ctr rats had an increase in body weight gain (48%, p = 0.001) (a) and WAT weight (parametrial pWAT by 45%, p = 0.008; retroperitoneal rWAT by 37%, p = 0.041; total tl WAT by 42%, p = 0.012) (f). Fatty acid oxidation (FAO) of pWAT (8 mg) was reduced by THx treatment versus Ctr (basal FAO, 51%, p < 0.0001; endogenous FAO [Endo-FAO], 58%, p < 0.0001; exogenous FAO [Exo-FAO], 54%, p = 0.024) (g). *p < 0.05, two-tailed Student’s t test.

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  Fig. 3.. THx at the SuMN area induces glucose intolerance in f-HFDr-RC rats (Study 2Ai). f-HFDr-RC rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA-GFP to knockdown Th gene expression (THx) or control AAV5-Scr-shRNA-GFP (Ctr). At 11 weeks post-virus inoculation, compared with Ctr, THx treatment increased basal blood glucose (p = 0.024) and GTT blood glucose level at 30, 60, 90, and 120 min following glucose challenge (a) with an increase in GTT glucose AUC (27%, p = 0.009) (b). GTT insulin AUC was not increased by THx treatment (c, d). The reduction in Belfiore ISI did not reach significance in THx rats versus Ctr rats (17% reduction, p = 0.258) (e). *p < 0.05, two-tailed Student’s t test.

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  Fig. 4.. THx at the SuMN area alters mRNA expression of metabolic relevant genes in the liver of f-HFDr-RC rats (Study 2Aii). f-HFDr-RC rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA-GFP to knockdown Th gene expression (THx) or control AAV5-Scr-shRNA-GFP (Ctr). The relative mRNA expression levels of metabolic relevant genes were analyzed by RT-qPCR in the livers of THx and Ctr rats collected during the fasting period of the day (ZT 6–8). a Gluconeogenic genes: compared with Ctr, THx treatment increased relative mRNA expression of phosphoenolpyruvate carboxylase 1 (Pck1) by 2.3-fold (p = 0.0013) and glucose-6-phosphatase, catalytic subunit (G6pc) by 1.6-fold (p = 0.046). b Lipogenic genes: compared with Ctr, THx increased the relative mRNA expression of lipogenic transcriptional coregulator Ppargc1β by 1.4-fold (p = 0.037 Ɨ) with no detectable increase in fatty acid synthase (Fasn) (p = 0.6) and lipogenic transcriptional regulators (Srebf1, p = 0.9; Mtor, p = 0.9). c Lipid oxidation genes: no significant increase was detected in the mRNA expression levels of the key transcriptional factors/coactivators regulating FAO in the livers of THx rats versus Ctr rats, Pparα…

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  Fig. 5.. THx at the SuMN area induces hyperleptinemia, hypertriglycemia and alters plasma corticosterone in f-HFDr-RC rats (Study 2Aiii). f-HFDr-RC rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA-GFP to knockdown Th gene expression (THx) or control AAV5-Scr-shRNA-GFP (Ctr). Compared with Ctr, THx treatment increased plasma leptin by 46% (p = 0.0382 Ɨ) (a) and plasma triglyceride by 86% (p = 0.008) (b) with no increase in liver triglyceride content (p = 0.235) (c) or plasma level of FFAs (p = 0.153) (d). Plasma corticosterone measured during fasting period of the day (ZT 6–8) was reduced by 68% (p = 0.007) (e) in THx rats versus control rats. *p < 0.05, two-tailed Student’s t test. ƚp < 0.05, one-tailed Student’s t test.

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  Fig. 6.. Effect of THx at the SuMN area on circadian feeding rhythm, food preference, hunger/satiety balance, and responses to systemic ghrelin and GLP-1 agonist administration in f-HFDr-RC rats (Study 2Bi-v). f-HFDr-RC rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA-GFP to knockdown Th gene expression (THx) or control AAV5-Scr-shRNA-GFP (Ctr). SuMN-THx increased food consumption during the light (fasting/resting) period of the day (a1–3). Regular chow consumption was monitored throughout light and dark periods of the day in THx and Ctr rats over two consecutive days. THx rats, like Ctr rats, maintained a circadian feeding rhythm with peak food intake during the dark photoperiod (a1). Nonetheless, THx rats ate 14% more food per day than Ctr rats (p = 0.014) (a2) which was consumed particularly during the light period. THx rats as compared to Ctr rats consumed 75% more food (p < 0.001) during the light period with no difference in food consumption during the dark period (p = 0.523) (a3). SuMN-THx did not alter food preference of HFD over RC, but increased HFD consumption (b1–2). THx rats and Ctr rats were exposed to RC and HFD simultaneously, and food consumption…

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  Fig. 7.. THx at the SuMN area induces glucose intolerance/IR and increases body weight gain in f-HFDr-HFD rats (Study 3). f-HFDr-HFD rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA-GFP to knockdown Th gene expression (THx) or control AAV5-Scr-shRNA-GFP (Ctr). Compared with Ctr, THx treatment increased GTT glucose AUC (16%, p = 0.043) (a, b) and insulin AUC (62%, p = 0.001) (c, d) resulting in a reduction in Belfiore ISI (39%, p < 0.0001) (e). THx treatment increased body weight gain by 23% (p = 0.06) (f, g) and increased food consumption by 7% (p = 0.024) (h, i) and feed efficiency by 14% (p = 0.008) (j) relative to Ctr. Compared with Ctr, THx treatment reduced FAO in pWAT (8 mg) (basal FAO by 29%, p < 0.0001; endogenous FAO [Endo-FAO] by 39%, p < 0.0001; exogenous FAO [Exo-FAO] by 53%, p = 0.029) (k). The plasma triglyceride level was increased by 15% in THx rats relative to Ctr rats (p = 0.033 Ɨ) (l). *p < 0.05, two-tailed Student’s t test. Ɨp < 0.05, one-tailed Student’s t test.

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  Fig. 8.. THx at the SuMN area induces glucose intolerance and increases body weight gain in m-HFDr-RC rats (Study 4). m-HFDr-RC rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA-GFP to knockdown Th gene expression (THx) or control AAV5-Scr-shRNA-GFP (Ctr). Compared with Ctr, THx treatment increased GTT glucose AUC by 38% (p = 0.014) (a, b) with no change in insulin AUC (c, d) resulting in 28% reduction in Belfiore ISI (p = 0.015) (e). The THx rats exhibited an increase in body weight gain by 12% (p = 0.02) (f, g) and 7% increase in food consumption (p = 0.04) (h, i) relative to Ctr rats. There was no significant increase in feed efficiency (p = 0.137) (j) in THx rats compared to Ctr rats. *p < 0.05, two-tailed Student’s t test.

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  Fig. 9.. Long-lasting effect of THx at the SuMN area on body weight gain and glucose metabolism in f-HFDs-RC rats (Study 5). f-HFDs-RC rats were locally inoculated at the medial SuMN area with AAV5-Th-shRNA to knockdown Th gene expression (THx) or control AAV-Scr-shRNA-GFP (Ctr). THx treatment increased % body weight gain by 1.5-fold (96% vs. 65% increase in Ctr, p = 0.012) after 12 months post-virus inoculation (a, b). Compared with Ctr, THx treatment increased the plasma levels of fasting glucose (18%, p = 0.015) (c) and insulin (78%, p = 0.031) (d) resulting in an increase in HOMA-IR (120%, p = 0.027) (e) indicating IR. *p < 0.05, two-tailed Student’s t test.