(a) Extracts of whole brain, hypothalamus and microdissected ARC, VMH, DMH, LH, cortex and PVN were processed for immunoblot analysis. (b) Hypothalamic extracts from 8 week old chow-fed versus 12 week HFF C57BL/6 male mice, or chow-fed Ob/+ lean versus leptin-deficient Ob/Ob obese mice were processed for immunoblot analysis. (c) Hypothalami were extracted from chow-fed versus 12 week HFF C57BL/6 mice and processed for quantitative real time PCR to measure Ptpn2 expression. (d–e) CHO-LEPR-B cells were serum starved for 12 h and left untreated or pre-treated with 10 μg/ml cycloheximide (CHX) for 4 h and stimulated with 10 ng/ml murine leptin for the indicated times and processed for immunoblot analysis. 8 week old C57BL/6 mice were fasted for 18 h and (f) administered leptin [5 μg/g intraperitoneal (IP)] and hypothalami extracted at the indicated times and processed for real time PCR to measure ptpn2 expression, or (g) administered leptin (every 2 h for 16 h) and hypothalami extracted for immunoblot analysis. Results in a are representative of two and b–g of at least three independent experiments; quantified results are means ± SEM.

(a) TCPTP protein levels in Ptpn2^lox/lox control (lox/lox) and Nes-Cre;Ptpn2^lox/lox neuronal cell TCPTP knockout (NTKO) mice. (b–c) Body weights in 3–20 week old male lox/lox, Nes-Cre;Ptpn2^lox/+ (Cre;lox/+), NTKO, Ptpn2^+/+ (+/+) and Nes-Cre;Ptpn2^+/+ (Cre;+/+) mice. (d) Nose to tail body lengths in 8 week old and (e) tissue weights in 8–10 week old male lox/lox and NTKO mice. (f) Body composition [lean, fat, bone mineral content (BMC) and bone mineral density (BMD)] measured by DEXA and normalised to total body weight and (g) light and dark cycle oxygen consumption, energy expenditure, ambulatory activity (x and z axes) and daily food intake in 8–10 week old lox/lox and NTKO mice. (h) Fed serum GH and IGF-1 levels (collected at 8 am) from 8 week old male lox/lox and NTKO mice. (i) 8 week old male lox/lox and NTKO mice were fasted for 18 h and injected with saline or GH (0.5 μg/g IP) and hypothalami extracted and processed for immunoblot analysis with antibodies to Y1007/Y1008 phosphorylated JAK2 (p-JAK2) and phosphorylated (Y694) STAT5 (p-STAT5) and reprobed for STAT5 and actin. (j) Hypothalami were extracted from 8 week old male lox/lox and NTKO mice (at 8 am) and processed for real time PCR to measure Ghrh, Ghr and Sst1 expression. Results are representative of at least three independent experiments; quantified results are means ± SEM for the indicated number of mice.

(a) Fed plasma leptin and adiponectin in 8–10 week old male lox/lox and NTKO mice. (b–d) 8–10 week old male lox/lox, NTKO, Ptpn2^lox/+ (lox/+) and Nes-Cre;Ptpn2^lox/+ (Cre;lox/+) mice were administered leptin and body weight and food intake monitored as indicated; b and d were undertaken at the same time and the NTKO data set is the same in both. (e–f) 8–10 week old male lox/lox and NTKO mice were fasted for 18 h and injected with saline or leptin and (e) hypothalami extracted and processed for immunoblot analysis with antibodies to p-STAT3, p-JAK2, or Ser-473 phosphorylated Akt (p-Akt) and reprobed as indicated, or (f) paraformaldehyde (PFA)-perfused brains extracted and processed for immunohistochemistry with antibodies to p-STAT3, or (g) hypothalami extracted after 2 h and processed for real time PCR to measure Pomc, Agrp and Npy expression. In (f) p-STAT3 positive nuclei in the ARC, DMH or VMH were counted in serial sections. (h) Hypothalamic slices from 8–10 week old male lox/lox and NTKO mice were incubated in aCSF for 45 min (basal) and then stimulated with leptin (20–100 nM) for 45 min. Secreted NPY and a-MSH were measured by radioimmunoassay. Results in a–e and h are representative of three independent experiments; quantified results are means ± SEM for the indicated number of mice.

(a) CHO-LEPR-B cells transfected with vector control or constructs for the expression of 45 kDa TCPTP (TC45) were serum starved and stimulated with murine leptin and processed for immunoblot analysis. (b) CHO-LEPR-B cells transfected with constructs for the expression of wildtype TC45, or TC45-D182A ‘substrate-trapping’ mutant, were serum starved and stimulated with leptin for the indicated times and processed for immunofluorescence microscopy with antibodies to p-STAT3 and TCPTP and stained with Hoechst to visualise nuclei. SK-N-SH-SY5Y cells transduced with control or TCPTP shRNA lentiviral particles were serum starved, stimulated with leptin and processed for (c) immunoblot analysis, or (d) immunofluorescence microscopy. Results are representative of at least three independent experiments; quantified results are means ± SEM.

(a) 12 week old C57BL/6 male mice were fasted for 18 h and aCSF or TCPTP inhibitor compound 8 (Comp. 8) administered icv and then injected with saline or leptin as indicated. (a) Hypothalami were extracted and processed for immunoblotting, or (b–c) body weights, oxygen consumption and energy expenditure monitored. (d) 12 week old male lox/lox and NTKO mice were icv administered aCSF or Comp. 8 and injected with leptin as indicated and body weights monitored. In (b) significance values correspond to aCSF + Leptin versus Comp. 8 + Leptin treated mice and in (d) lox/lox (Leptin + aCSF) versus NTKO (Leptin + aCSF) mice. Results are representative of three independent experiments; quantified results are means ± SEM for the indicated number of mice.

8–10 week old male lox/lox and NTKO mice were HFF for 12 wk. (a) Body weights were measured on a weekly basis and the incremental increase in body weight determined. (b) Body composition measured by DEXA. (c) Mice were administered leptin as indicated and body weights monitored. (d) Mice were fasted for 18 h and injected with saline or leptin and hypothalami extracted and processed for immunoblot analysis. (e) Oxygen consumption, energy expenditure and ambulatory activity were determined and daily food intake measured. (f) Mice were fasted and ITTs performed; areas under ITT curves were determined. (g) Fed and fasted blood glucose and plasma insulin levels and fed plasma leptin, adiponectin and TNF levels were measured. Results are representative of three independent experiments; quantified results are means ± SEM for the indicated number of mice.

8 week old C57BL/6 male mice were HFF for 0–9 weeks and (a) hypothalami isolated and processed for immunoblot analysis for an assessment of PTP1B, TCPTP and SOCS3 levels, or (b–c) mice were bled to measure plasma leptin, TNF and IL-6 levels. (d) Ptpn1^lox/loxPtpn2^loxlox and neuronal cell TCPTP and PTP1B double KO mice (Nes-Cre;Ptpn1^lox/loxPtpn2^loxlox; DKO) were generated and whole brains or hypothalami (Hypo) extracted for an assessment of PTP1B and TCPTP protein levels. (e) Body weights (f) or body lengths (nose to tail) in male Ptpn1^lox/lox, Ptpn2^loxlox, Ptpn1^lox/loxPtpn2^loxlox, Nes-Cre;Ptpn1^lox/lox, Nes-Cre;Ptpn2^loxlox and DKO mice. (g) Body composition in 8–10 week old male Ptpn1^lox/loxPtpn2^loxlox and DKO measured by DEXA and normalised to total body weight. (h) 8 week old male mice were administered leptin as indicated and body weights monitored. Fed plasma leptin levels were determined. (i) ITTs in 8 week old male mice; areas under ITT curves were determined. Fed and fasted blood glucose levels were measured. (j) The indicated 8–10 week old male mice were HFF and the incremental increase in body weight determined. (k) Relative lean and fat masses in HFF Ptpn1^lox/loxPtpn2^loxlox and DKO measured by DEXA. (l) Leptin sensitivity was assessed and fed plasma leptin levels determined in HFF mice. (m) ITTs were performed and fed and fasted blood glucose levels were determined in HFF mice. Results are representative of three independent experiments; quantified results are means ± SEM for the indicated number of mice; n.s.: not significant. In (j and l) * corresponds to Ptpn1^lox/loxPtpn2^loxlox versus DKO mice, # to Ptpn1^loxlox versus Nes-Cre;Ptpn1^lox/lox mice and θ Nes-Cre;Ptpn1^lox/lox versus DKO mice.