Leptin Production by Encapsulated Adipocytes Increases Brown Fat, Decreases Resistin, and Improves Glucose Intolerance in Obese Mice

Abstract

The neuroendocrine effects of leptin on metabolism hold promise to be translated into a complementary therapy to traditional insulin therapy for diabetes and obesity. However, injections of leptin can provoke inflammation. We tested the effects of leptin, produced in the physiological adipocyte location, on metabolism in mouse models of genetic and dietary obesity. We generated 3T3-L1 adipocytes constitutively secreting leptin and encapsulated them in a poly-L-lysine membrane, which protects the cells from immune rejection. Ob/ob mice (OB) were injected with capsules containing no cells (empty, OB[Emp]), adipocytes (OB[3T3]), or adipocytes overexpressing leptin (OB[Lep]) into both visceral fat depots. Leptin was found in the plasma of OB[Lep], but not OB[Emp] and OB[3T3] mice at the end of treatment (72 days). The OB[Lep] and OB[3T3] mice have transiently suppressed appetite and weight loss compared to OB[Emp]. Only OB[Lep] mice have greater brown fat mass, metabolic rate, and reduced resistin plasma levels compared to OB[Emp]. Glucose tolerance was markedly better in OB[Lep] vs. OB[Emp] and OB[3T3] mice as well as in wild type mice with high-fat diet-induced obesity and insulin resistance treated with encapsulated leptin-producing adipocytes. Our proof-of-principle study provides evidence of long-term improvement of glucose tolerance with encapsulated adipocytes producing leptin.

Fig 1. Engineered 3T3-L1 adipocytes express and constitutively secrete leptin.

3T3-L1 cells were stably transduced to overexpress Lep (3T3^Lep) (A, B) Lep expression in non-differentiated preadipocytes (ND = non-differentiated) (A) and differentiated preadipocytes (D = differentiated) (B) is shown for 3T3-L1 (white bar) and 3T3^Lep (black bar) (both n = 4, P<0.001). All gene expression was measured by qRT-PCR and normalized to TATA box binding protein (TBP). (C) The ratio of Pref1 to Pparg expression non-differentiated (ND, white bar) and differentiated (D, black bar) and 3T3-L1 (white bar) (n = 3, P<0.02). (D, E) Pref1 (D) and Pparg (E) expression in differentiated (both n = 4) 3T3-L1 (white bar) and 3T3^Lep (black bar, P<0.001). (F) A mouse leptin ELISA was performed on media collected from confluent differentiated 3T3-L1 (white bar) and 3T3^Lep (black bar) adipocytes that were incubated in this media for 48 hours (n = 4, P<0.001). (G) Leptin concentration was measured by ELISA in media collected from a monolayer of encapsulated 3T3-L1 ([3T3-L1], white bar) and 3T3^Lep ([3T3^Lep], black bar) adipocytes cultured overnight. All data are expressed as means ± SD (n = 4, P<0.001).

Fig 2. Treatment with leptin-producing encapsulated adipocytes increases plasma leptin and transiently influences food intake and weight.

Ob/ob mice (n = 7 per group) were injected with encapsulated acellular capsules (OB^[Emp]), encapsulated 3T3-L1 preadipocytes (OB^[3T3]), and encapsulated 3T3^Lep (OB^[Lep]) in both visceral fat pads (total 0.6*10⁶ adipocytes for each cell type). (A) Leptin concentrations in plasma from OB^[Emp] (n = 4), OB^[3T3] (n = 4), and OB^[Lep] (n = 7) mice. The leptin ELISA was performed on non-hemolyzed plasma samples. All data are expressed as means ± SD. Significant differences are shown with a black bar and determined by one-way ANOVA followed by Tukey’s post hoc test (P<0.01). (B) Comparison of leptin concentrations in plasma from OB^[Lep] (n = 7) and WT mice fed a high fat (HF) diet for 60 days (n = 5). Leptin was analyzed by ELISA, (P<0.01, Student’s t-test). (C) Average food intake kinetics in OB^[Emp] (open circles, n = 6) and OB^[Lep] (closed circles, n = 7) mice are shown as percent of intake prior to injection. Asterisks show P<0.05, Student’s t-test. Data for OB^[3T3] mice is in S1 Fig. (D) Average weight kinetics in same groups of mice. Weight is shown as percent of weight prior to injection (g/g prior to injection x 100). Asterisks show P<0.05, Student’s t-test. (E) Weights at sacrifice (72 days post-treatment, mean ± SD) in same group of mice.

Fig 3. Treatment with leptin-producing encapsulated adipocytes increases BAT but not WAT weight.

(A) Average percent body fat for OB^[Emp] (white bar, n = 5), OB^[3T3] (gray bar, n = 5), and OB^[Lep] (black bar, n = 7) was measured using DEXA. Representative DEXA images are shown for each group. (B-D) Average normalized weight of subcutaneous fat (SF, B), visceral (VF, C), and brown (BAT, D) to body weight in same mouse groups. (E) The average ratio of BAT to VF in same mouse groups. All data are represented as mean ± SD. Significant differences are shown by black bar and determined by one-way ANOVA followed by Tukey’s test.

Fig 4. Leptin increases metabolic rate and raises RER.

(A-D) Metabolic measurements were performed in OB^[Emp] (white bar, n = 5) and OB^[Lep] (black bar, n = 6) mouse groups in CLAMS metabolic cages at 28 days post injection. Metabolic data for OB^[3T3] group are shown in S3 Fig. The average (mean ± SD) x, y, and z activity (A), average VO₂ (mL/kg/h) (B), respiratory exchange ratio (RER) (C), and average metabolic rate (kcal/h/kg) (D) are shown for both the dark (D) and light (L) cycles. (E, F) Kinetic data for RER (E) and metabolic rate (kcal/h/kg) (F) are shown as mean for each time point in OB^[Emp] (open black circles) and OB^[Lep] (filled red circles) groups. Significant differences are shown by black bar and determined by one-way ANOVA followed by Tukey’s test.

Fig 5. Leptin improves glucose tolerance and reduces resistin.

(A,B) Glucose tolerance tests (GTT) (A) or insulin tolerance tests (ITT) (B) were performed in fasting overnight mice OB^[Emp] (open circles, n = 5) and OB^[Lep] (filled circles, n = 5). GTT and ITT tests for OB^[3T3] group are shown in S4 Fig. Average blood glucose (mg/dL) concentrations are displayed for each time point. Asterisks indicate significant differences P<0.05, Student’s t-test. The area under the curve (AUC) for each GTT was calculated using a trapezoidal approximation and displayed as percent of the control OB^[Emp] group (100%). (C-E) Plasma insulin (C), glucagon (D), and resistin (E) concentrations were measured by ELISA in OB^[Emp] (white bar, n = 4), OB^[3T3] (gray bar, n = 3), and OB^[Lep] (black bar, n = 7). Statistical difference was examined by one-way ANOVA followed by Tukey’s post hoc test. (F, G) WT mice with diet-induced obesity (Study 2) were injected with encapsulated acellular capsules (WT^[Emp]) and encapsulated 3T3^Lep (WT^[Lep]) in both visceral fat pads (total 0.6*10⁶ adipocytes for each cell type). GTT (F) or ITT (G) were performed in fasting overnight mice WT^[Emp] (open circles, n = 3) and WT^[Lep] (filled circles, n = 5). All data are represented as mean ± SD.