PMC

Representative photomicrographs of brain slices from Pomc-Cre; Z/EG (control) or Pomc-Cre; Sirt1^loxP/loxP; Z/EG mice stained for SIRT1 and GFP (the Cre-conditional expression of GFP is restricted only to POMC neurons in these mice ()). Dark-brown staining and green fluorescence represent SIRT1 and GFP immunoreactivity, respectively. Higher magnification of the boxed-region is in the top-left corner of the photomicrograph. Arrows indicate POMC neurons. Note the co-localization between SIRT1 and POMC in Pomc-Cre; Z/EG control brain. This co-localization is absent in almost all of POMC neurons in Pomc-Cre; Sirt1^loxP/loxP; Z/EG brain. Dash lines indicate ARH boundaries. Abbreviations: third ventricle (3V), hypothalamic arcuate nucleus (ARH), immunohistochemistry (IHC). Scale bar = 50 μm.

(A) Relative body weights of high-calorie (HC)-fed Sirt1^loxP/loxP and Pomc-Cre, Sirt1^loxP/loxP mice during the 10 days of intracerebroventricular delivery of either placebo or leptin and (B) food intake from the second to the sixth and from the sixth to the tenth day into the treatment. (C) Ucp1 and Cidea mRNA levels in perigonadal WAT at the end of the treatment (n=8–10). Individual mRNA levels were normalized to β-actin mRNA contents. (D) Representative photomicrographs of brain slices from Pomc-Cre; FoxO1-GFP (control) or Pomc-Cre; Sirt1^loxP/loxP; FoxO1-GFP mice stained for GFP (the Cre-conditional expression of FoxO1-GFP is restricted only to POMC neurons in these mice). Scale bar = 20 μm. Error bars represent s.e.m. Statistical analyses were done using one-way ANOVA (Tukey’s post test). *P<0.05; **P<0.01; **P<0.001 Sirt1^loxP/loxP leptin vs. Sirt1^loxP/loxP placebo. ^#P<0.05; ^{# #}P<0.01; Pomc-Cre, Sirt1^loxP/loxP leptin vs. Pomc-Cre, Sirt1^loxP/loxP placebo.

O₂ consumption, CO₂ and heat production, respiratory quotient (RQ), (B) ambulatory movements, and (C) food intake were measured before body weight diverged in 12-week-old Sirt1^loxP/loxP and Pomc-Cre, Sirt1^loxP/loxP females fed on a high-calorie diet for 4 weeks (n=12 in each group). Data were collected using the Columbus Instruments Comprehensive Lab Animal Monitoring System (CLAMS). Time 0 represents the beginning of the dark cycle in a 12-hour dark/light cycle environment. Black and white boxes represent dark and light cycles, respectively. Error bars represent s.e.m. Statistical analyses were done using two-tailed unpaired Student’s t test. *P<0.05.

(A) Body weight curves of standard chow (SC)-fed Sirt1^loxP/loxP (n=17) and Pomc-Cre, Sirt1^loxP/loxP (n=20) males, high-calorie (HC)-fed Sirt1^loxP/loxP (n=15) and Pomc-Cre, Sirt1^loxP/loxP (n=20) males, (B) SC-fed Sirt1^loxP/loxP (n=33) and Pomc-Cre, Sirt1^loxP/loxP (n=37) females, HC-fed Sirt1^loxP/loxP (n=14) and Pomc-Cre, Sirt1^loxP/loxP (n=19) females. (C) Body composition, (D) representative microcomputed tomography images, (E) mass of hemi-perigonadal fat, and (F) serum leptin levels of Sirt1^loxP/loxP and Pomc-Cre, Sirt1^loxP/loxP females after 20 weeks on the HC diet (n=10–19). Mass of hemi-perigonadal fat of 28-week-old SC-fed Sirt1^loxP/loxP (n=12) and Pomc-Cre, Sirt1^loxP/loxP (n=12) females is shown in (E). In all figures, HC-fed mice were fed on a SC diet up to 8 weeks of age and then switched and maintained on a HC diet. In (D) red and yellow/green colors represent lean and fat mass, respectively. Arrows indicate visceral adipose tissue. Error bars represent s.e.m. Statistical analyses were done using two-tailed unpaired Student’s t test. *P<0.05.

(A) Representative photomicrographs of rostral to caudal (top to bottom) brain sections from either Sirt1^loxP/loxP (control) or Pomc-Cre, Sirt1^loxP/loxP mice stained for β-endorphin (a product of POMC). Dark-brown staining represents β-endorphin immunoreactivity. Scale bar = 200 μm. (B) POMC neurons were counted in several sections that contained similar regions of the hypothalamus or hindbrain in both genotypes. No difference in the estimated number of POMC neurons per section was noted between genotypes in 4-week-old males (4-wks M), 4-week-old females (4-wks F), 20-week-old males (20-wks M), and 12-week-old females fed on the high-calorie diet for 4 weeks (12-wks F HC). (C), Representative photomicrographs of brain sections from either Sirt1^loxP/loxP (control) or Pomc-Cre, Sirt1^loxP/loxP mice stained for β-endorphin. Dark-brown staining represents β-endorphin immunoreactive fibers. Both genotypes display similar POMC neurons fibers density in dorsomedial hypothalamic nucleus (DMH), paraventricular hypothalamic nucleus (PVH), and paraventricular thalamic nucleus (PVT). Scale bar = 100 μm. Other abbreviations: third ventricle (3V); immunohistochemistry (IHC). n=2–3 in each group. Error bars represent s.e.m. Statistical analyses were done using two-tailed unpaired Student’s t test.

(A) Hypothalamic Pomc mRNA level in 4-week-old and in 28-week-old Sirt1^loxP/loxP and Pomc-Cre; Sirt1^loxP/loxP females fed on a high calorie (HC) diet for 20 weeks (n=9–11). Pomc mRNA levels were normalized to β-actin mRNA contents. (B) Schematic diagram depicting POMC processing in hypothalamus. (C) Hypothalamus was collected from 12-week-old Sirt1^loxP/loxP and Pomc-Cre; Sirt1^loxP/loxP females fed on a HC diet for 4 weeks (n=9–11). Acid-extracted peptides were HPLC-fractionated and quantified for ACTH or α-MSH using specific radioactive immunoassays. Representative HPLC profile for ACTH-related peptides and values of ACTH peptide for each group are shown (upper panel). Representative HPLC profile for α-MSH-related peptides and values of desacetyl-α-MSH peptide for each group are shown (lower panel). Each HPLC profile displays the elution position of respective synthetic standards. Values are the mean ± s.e.m. Statistical analyses were done using two-tailed unpaired Student’s t test and no differences were noted between groups. Abbreviations: ACTH (adrenocorticotropic hormone); CLIP (corticotropin-like intermediate lobe peptide); α-MSH (alpha melanocyte-stimulating hormone), and β-LPH (beta lipotropin hormone).

(A) Ucp1 and Pgc1α mRNA levels in interscapular brown adipose tissue (IBAT) of 28-week-old Sirt1^loxP/loxP and Pomc-Cre; Sirt1^loxP/loxP females fed on a high calorie (HC) diet for 20 weeks. (B) Ucp1 and Cidea mRNA levels in visceral (perigonadal, perirenal, mesenteric) and subcutaneous (inguinal and mammary) white adipose tissue (WAT) of same mice as in A (n=7–11). Individual mRNA levels were normalized to β-actin mRNA contents. (C) UCP1 and β-actin (used as loading control) protein levels were assessed in the perigonadal fat of same mice as in in A (UCP1/β-actin content is reduced in mutants; P=0.021; n=3–4) by western blot. (D) Representative photomicrographs of paraffin-embedded perigonadal WAT sections stained with hematoxylin and eosin (H&E) or treated for UCP1 immunohistochemistry (IHC). Tissues were collected from same mice as in A. Dark-brown staining represents UCP1-expressing brown adipocytes. Higher magnification of the boxed-region is in the top-right corner. Scale bar = 100 μm. (E) Quantification of SNA in perigonadal WAT of 12-week-old Sirt1^loxP/loxP and Pomc-Cre; Sirt1^loxP/loxP females fed on a HC diet for 4 weeks (n=8). (F) Body weights before and after one week of treatment with either placebo or the selective β3-adrenergic receptor agonist CL316,243 (n=15–16). Error bars represent s.e.m. Statistical analyses were done using two-tailed unpaired Student’s t test. ^† P=0.05; *P<0.05; **P<0.01; **P<0.001.