Leptin Increases Hepatic Insulin Sensitivity and Protein Tyrosine Phosphatase 1B Expression

Abstract

Leptin has been shown to improve insulin sensitivity and glucose metabolism in obese diabetic ob/ob mice, yet the mechanisms remain poorly defined. We found that 2 d of leptin treatment improved fasting but not postprandial glucose homeostasis, suggesting enhanced hepatic insulin sensitivity. Consistent with this hypothesis, leptin improved in vivo insulin receptor (IR) activation in liver, but not in skeletal muscle or fat. To explore the cellular mechanism by which leptin up-regulates hepatic IR activation, we examined the expression of the protein tyrosine phosphatase PTP1B, recently implicated as an important negative regulator of insulin signaling. Unexpectedly, liver PTP1B protein abundance was increased by leptin to levels similar to lean controls, whereas levels in muscle and fat remained unchanged. The ability of leptin to augment liver IR activation and PTP1B expression was also observed in vitro in human hepatoma cells (HepG2). However, overexpression of PTP1B in HepG2 cells led to diminished insulin-induced IR phosphorylation, supporting the role of PTP1B as a negative regulator of IR activation in hepatocytes. Collectively, our results suggest that leptin acutely improves hepatic insulin sensitivity in vivo with concomitant increases in PTP1B expression possibly serving to counterregulate insulin action and to maintain insulin signaling in proper balance.

Leptin resistance following over-expression of protein tyrosine phosphatase 1B in liver

“…The lower dosage of leptin in this study relative to the study with the C57BL/6 mice was because of the hypersensitivity of ob/ob mice to leptin. We have previously found that this dose of leptin does not affect body weight after 2 days of administration but does affect food consumption (Lam et al 2004). Body weight, food consumption, plasma glucose and plasma insulin levels were measured daily for the next 2 days.…”

“…Protein lysates were prepared and immunoblotting was performed as previously described (Lam et al 2004). For analysis of tyrosine phosphorylation of the -subunit of IR (IR ), 2 mg protein lysate was immunoprecipitated with rabbit polyclonal anti-IR (sc-71; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and protein A-conjugated beads (Pierce Chemical Company, Rockford, IL, USA).…”

“…The total amount of IR chain was determined by stripping the membranes and re-blotting with rabbit polyclonal anti-IR (sc-711; Santa Cruz Biotechnology, Inc.) and donkey polyclonal anti-rabbit antibody conjugated to HRP (NA934; Amersham Pharmacia) and proteins were visualized as described above. Analysis of tyrosine phosphorylation of STAT3 was performed as previously described (Lam et al 2004) by immunoprecipitating 750 µg protein lysate with polyclonal rabbit anti-STAT3 antibodies (sc-482; Santa Cruz Biotechnology, Inc.) and immunoblotting with monoclonal mouse anti-phosphotyrosine (sc-7020; Santa Cruz Biotechnology, Inc.). Proteins were visualized as described above.…”

“…The recombinant adenoviruses containing PTP1B or -gal cDNA, generously provided by Drs J Wang, J K Kolls and M Bryer-Ash (Egawa et al 2001, were propagated and titered as previously described (Lam et al 2004). The adenovirus has a high degree of hepatotropism, and when delivered intravenously results in highly specific liver expression.…”

“…CHO cells over-expressing the leptin receptor b (OBRb) were a kind gift from Dr T Murakami (Murakami et al 1997), and were cultured as previously described (Lam et al 2004). For infection with adenovirus, cells in 100 mm plates at 60% confluence were incubated for 15 min in 2 ml Dulbecco's PBS (D-PBS; Life Technologies Inc., Burlington, Ontario, Canada) with 4·3 mM calcium (D-PBS-Ca 2+ ) containing 6·7 10 9 PFU of either Ad -gal or AdPTP1B.…”