Hypothalamic AMPK-ER Stress-JNK1 Axis Mediates the Central Hypothalamic AMPK-ER Stress-JNK1 Axis Mediates the Central Actions of Thyroid Hormones on Energy Balance Actions of Thyroid Hormones on Energy Balance

We that lipogenesis in and in respectively. Central T3 promotes hepatic lipogenesis with parallel stimulation of the thermogenic program in The action of T3 on AMP-activated protein (AMPK)-induced of two in the ventromedial the hypothalamus (ER) thermogenesis, N-terminal kinase (JNK) activation, hepatic lipid of a factor (SF1) neurons of effect of on Grass Instruments, Warwick, RI, USA) and the nerve signal was ampliﬁed 10 5 times with a Grass P5 AC pre-ampliﬁer (Grass Instruments, Warwick, RI, USA). After ampliﬁcation, the nerve signal was ﬁltered at a 100- and 1000-Hz cutoff with a nerve trafﬁc analysis system (Model 706C, University of Iowa Bioengineering; Iowa City, IA, USA). The nerve signal was then routed to an oscilloscope (Model 54501A, Hewlett-Packard; Palo Alto, CA, USA) for monitoring the quality of the sympathetic nerve recording and to a resetting voltage integrator (Model B600c, University of Iowa Bioengineering, Iowa City, IA, USA). BAT SNA measurements were made every basal measurements were To ensure that electrical noise was excluded in assessment of sympathetic outﬂow, we corrected each recording post-mortem background activity. 2014), a rabbit anti-c-FOS Cruz Biotechnology, CA, USA). After perfusion of the animals the ascending aorta with 150 mL 0.9% NaCl, followed by 500 mL of 4% formaldehyde in 100 mM phosphate buffer (PB), pH under deep anesthesia with ketamine-xylazine. The were ﬁxed in 10% buffered formaldehyde and subsequently treated for histological study by dehydration (increasing alcohol concentrations), xylene and immersion blocks were 3 m were processed, deparafﬁnized rehydrated rinsed with speciﬁc primary antibody incubated washed incubated secondary antibody EnVision system, peroxidase; Glostrup, Denmark). c-FOS positive MD, bilateral image Direct detection ﬂuorescence animals; by Oil O ﬁxed were sequentially washed in 1X SSC at room temperature, four times in 1X SSC at 42 (cid:1) C (30 min per wash), one time in 1X SSC at room temperature (1 h), and then rinsed in water and ethanol. Finally, the sections were air-dried and exposed to Hyperﬁlm b -Max (KODAK; Rochester, NY, USA) at room temperature for 7 days for TRH and 14 days for AMPK a 1. The slides from all experimental groups were exposed to the same autoradiographic ﬁlm and then they were developed in developer/replenisher (Developer G150, AGFA HealthCare: Mortsel, Belgium) and ﬁxator (Manual Fixing G354; AGFA HealthCare: Mortsel, Belgium). Sections were scanned and the hybridization signal was quantiﬁed by densitometry using ImageJ-1.33 software (NIH; Bethesda, MD, USA). The optical density of the hybridization signal was determined and subsequently corrected by the optical density of its adjacent background value. A rectangle, with the same dimensions in each case, was drawn enclosing the hybridization signal over each nucleus and over adjacent brain areas of each section (background) (Lo´pez et al., 2010; Martı ´ nez de Morentin et al., 2014). Sixteen-20 sections for each animal (4-5 slides with 4 sections/slide) were used. The mean of these 16-20 values was used as densitometry value for each animal.